US20190153465A1

US20190153465A1 - Genes and uses for plant improvement

Info

Publication number: US20190153465A1
Application number: US16/350,019
Authority: US
Inventors: Mark Scott Abad; Barry S. Goldman; Jaishree M. Chittoor-Vijayanath; Ronald C. Rich; Faten Shaikh; Joseph C. Mitchell; Diana Wray
Original assignee: Monsanto Technology LLC
Current assignee: Monsanto Technology LLC
Priority date: 2006-12-06
Filing date: 2018-09-12
Publication date: 2019-05-23
Also published as: US20120167245A1; US20130269056A1; US20080295196A1; WO2008070179A2; US9315822B2; WO2008070179A3; US20160272993A1; US10093943B2

Abstract

Transgenic seed for crops with improved traits are provided by trait-improving recombinant DNA in the nucleus of cells of the seed where plants grown from such transgenic seed exhibit one or more improved traits as compared to a control plant. Of particular interest are transgenic plants that have increased yield. The present invention also provides recombinant DNA molecules for expression of a protein, and recombinant DNA molecules for suppression of a protein.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/998,939, filed Mar. 8, 2016, which application is a continuation of U.S. application Ser. No. 13/914,701, filed Jun. 11, 2013, which application is a continuation of U.S. application Ser. No. 13/372,542, filed Feb. 14, 2012 (now abandoned), which application is a continuation of U.S. application Ser. No. 12/001,025, filed Dec. 6, 2007 (now abandoned), which application claims benefit of priority under 35 USC § 119(e) of U.S. provisional application Ser. No. 60/873,247, filed Dec. 6, 2006, herein incorporated by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 Sep. 12, 2018 and Copy 2 Sep. 12, 2018) and a computer readable form (CRF Sep. 12, 2018) of the sequence listing, all on CD-Rs, each containing the file named 3126005US5.TXT, which is 67,123,200 bytes (measured in MS-WINDOWS) and was created on Sep. 12, 2018, are incorporated herein by reference in their entirety.

INCORPORATION OF COMPUTER PROGRAM LISTING

Two copies of Computer Program Listing (Copy 1 Sep. 12, 2018 and Copy 2 Sep. 12, 2018) containing folders “hmmer-2.3.2” and “161pfamDir” are provided on separate CD-ROMs and are incorporated herein by reference in their entirety. Folder hmmer-2.3.2 contains the source code and other associated file for implementing the HMMer software for Pfam analysis. Folder 161pfamDir contains 161 profile Hidden Markov Models. Both folders were created on the disk on Sep. 12, 2018 having a total size of 15,534,080 bytes when measured in MS-WINDOWS® operating system.

FIELD OF THE INVENTION

Disclosed herein are transgenic plant cells, plants and seeds comprising recombinant DNA and methods of making and using such plant cells, plants and seeds

BACKGROUND OF THE INVENTION

Transgenic plants with enhanced traits such as improved yield, environmental stress tolerance, pest resistance, herbicide tolerance, modified seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired traits, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique traits. The ability to develop transgenic plants with improved traits depends in part on the identification of useful recombinant DNA for production of transformed plants with improved properties, e.g. by actually selecting a transgenic plant from a screen for such improved property. An object of this invention is to provide transgenic plant cell nuclei, plant cells, plants and seeds by screening transgenic crop plants for one of more enhanced agronomic traits where the nucleus in cells of the plant or seed has recombinant DNA that was identified as imparting an improved trait in a model plant, e.g. Arabidopsis thaliana. In some cases the model plant may exhibit an improved trait that corresponds to an enhanced agronomic trait, e.g. cold stress tolerance, water deficit stress tolerance, low nitrogen stress tolerance and the like. In other cases the model plant may exhibit an improved trait that is a surrogate to an enhanced agronomic trait, e.g. salinity stress tolerance being a surrogate to drought tolerance or improvement in plant growth and development being a surrogate to enhanced yield. A further object of the invention is to provide screening methods requiring routine experimentation by which such transgenic plant cell nuclei, cells, plants and seeds can be identified by making a reasonable number of transgenic events and engaging in screening identified in this specification and illustrated in the examples.

SUMMARY OF THE INVENTION

This invention provides plant cell nuclei with recombinant DNA that imparts enhanced agronomic traits in transgenic plants having the nuclei in their cells. Recombinant DNA in this invention is provided in a construct comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein having at least one amino acid domain in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam domain names identified in Table 17. In more specific embodiments of the invention plant cells are provided which express a protein having amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 198 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO: 394 and homologs thereof listed in Table 2. Amino acid sequences of homologs are SEQ ID NO: 395 through 19,938. In even more specific embodiments of the invention the protein expressed in plant cells is a protein selected from the group of proteins identified in Table 1 by annotation to a related protein in Genbank and alternatively identified in Table 16 by identification of protein domain family.
Other aspects of the invention are specifically directed to transgenic plant cells, and transgenic plants comprising a plurality of plant cells with such nuclei, progeny transgenic seed, embryo and transgenic pollen from such plants. Such plant cell nuclei are selected from a population of transgenic plants regenerated from plant cells with a nucleus transformed with recombinant DNA by screening the transgenic plants in the population for an enhanced trait as compared to control plants that do not have the recombinant DNA in their nucleus, where the enhanced trait is enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced shade tolerance, enhanced tolerance to salt exposure, increased yield, enhanced nitrogen use efficiency, enhanced seed protein or enhanced seed oil. In some aspects of the invention the recombinant DNA expresses a protein that imparts the enhanced trait; in other aspects of the invention the recombinant DNA expresses RNA for suppressing the level of an endogenous protein. In yet another aspect of the invention the nucleus of plant cells in plants, seeds, embryo and pollen further comprise DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type plant cell. Such tolerance is especially useful not only as an advantageous trait in such plants but is also useful in a selection step in the methods of the invention. In aspects of the invention the agent of such herbicide is a glyphosate, dicamba, or glufosinate compound.
Yet other aspects of the invention provide nuclei in cells of transgenic plants which are homozygous for the recombinant DNA and transgenic seed of the invention from corn, soybean, cotton, canola, alfalfa, wheat or rice plants.
In other embodiments for practice of various aspects of the invention in Argentina the recombinant DNA in the nucleus is provided in plant cells derived from corn lines that are and maintain resistance to a virus such as the Mal de Rio Cuarto virus or a fungus such as the Puccina sorghi fungus or to both.
This invention also provides methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA in the nucleus of the plant cells. In some aspects of the invention the recombinant DNA can express a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names identified in Table 17; in other aspects the recombinant DNA suppresses the level of such a protein. More specifically the method comprises (a) screening a population of plants for an enhanced trait and recombinant DNA, where individual plants in the population can exhibit the trait at a level less than, essentially the same as or greater than the level that the trait is exhibited in control plants which do not express the recombinant DNA; (b) selecting from the population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants; (c) verifying that the recombinant DNA is stably integrated in said selected plants; (d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NO: 1-197; and (e) collecting seed from a selected plant. In one aspect of the invention the plants in the population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells and where the selecting is effected by treating the population with the herbicide, e.g. a glyphosate, dicamba, or glufosinate compound. In another aspect of the invention the plants are selected by identifying plants with the enhanced trait. The methods are especially useful for manufacturing corn, soybean, cotton, alfalfa, wheat or rice seed selected as having at least one of the enhanced traits described above.
Another aspect of the invention provides a method of producing hybrid corn seed comprising acquiring hybrid corn seed from a herbicide tolerant corn plant which also has a nucleus of this invention with stably-integrated, recombinant DNA The method further comprises producing corn plants from said hybrid corn seed, where a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; repeating the selecting and collecting steps at least once to produce an inbred corn line; and crossing the inbred corn line with a second corn line to produce hybrid seed.
Another aspect of the invention provides a method of selecting a plant comprising a nucleus of this invention in its plant cells by using an immunoreactive antibody to detect the presence of protein expressed by recombinant DNA in seed or plant tissue. Another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, a nucleus of this invention with unique recombinant DNA.
Aspects of the invention relating to nucleus in plant cells having recombinant DNA for suppressing the expression of a protein are identified in Table 1 and Table 16. More specific aspects of the invention provide plant cells having recombinant DNA for suppressing the expression of a protein having the function in a plant of the protein with amino acid sequence of SEQ ID NO: 200, 201, 205, 207, 211 and 394 or the corresponding Pfam identified in Table 16, i.e. SNF5, LMBR1, TFIIS_M, TFIIS_C, Glyco_transf_8, respectively. Such suppression can be effected by any of a number of ways known in the art, e.g. anti-sense suppression, RNAi or mutation knockout and the like.
Another aspect of this invention relates to growing transgenic plants with enhanced water use efficiency or enhanced nitrogen use efficiency. For instance, this invention provides methods of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of the invention which are selected for enhanced water use efficiency. Alternatively methods comprise applying reduced irrigation water, e.g. providing up to 300 millimeters of ground water during the production of a corn crop. This invention also provides methods of growing a corn, cotton or soybean crop without added nitrogen fertilizer comprising planting seed having plant cells of the invention which are selected for enhanced nitrogen use efficiency. Alternatively methods comprise applying reduced amount of nitrogen input as compared to the conventional input during the production of a corn crop.
The various aspects of this invention are especially useful for transgenic plant cells in seeds and transgenic plants having any of the above-described enhanced traits in crop plants such as corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
The invention also provides recombinant DNA constructs comprising the DNA useful in the nuclei in plant cells for imparting enhanced traits in plants having those cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1AA illustrate a consensus amino acid sequence of SEQ ID NO: 227 and its homologs.

FIGS. 2, 3 and 4 illustrate plasmid maps.

DETAILED DESCRIPTION OF THE INVENTION

In the attached sequence listing:
SEQ ID NO: 1-197 are nucleotide sequences of the coding strand of DNA for “genes” used in the recombinant DNA imparting an enhanced trait in plant cells, i.e. each represents a coding sequence for a protein;
SEQ ID NO: 198-394 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequence 1-197;
SEQ ID NO: 395-19938 are amino acid sequences of homologous proteins;
SEQ ID NO: 19939 is a consensus amino acid sequence.
SEQ ID NO: 19940 is a nucleotide sequence of a plasmid base vector useful for corn transformation; and
SEQ ID NO: 19941 is a DNA sequence of a plasmid base vector useful for soybean or canola transformation.
SEQ ID NO: 19942 is a DNA sequence of a plasmid base vector useful for cotton transformation.
The nuclei of this invention are identified by screening transgenic plants for one or more traits including improved drought stress tolerance, improved heat stress tolerance, improved cold stress tolerance, improved high salinity stress tolerance, improved low nitrogen availability stress tolerance, improved shade stress tolerance, improved plant growth and development at the stages of seed imbibition through early vegetative phase, and improved plant growth and development at the stages of leaf development, flower production and seed maturity.
“Gene” refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequences involved in the regulation of expression. In aspects of the invention where an improved trait is provided by expression of a protein, “gene” refers at least to coding nucleotide sequence for a protein or a functional polypeptide fragment of a protein that imparts the trait. In aspects of the invention where an improved trait is provided by suppression of expression of an endogenous protein, “gene” refers to any part of the gene that can be a target for suppression.
“Transgenic seed” means a plant seed whose nucleus has been altered by the incorporation of recombinant DNA, e.g., by transformation as described herein. The term “transgenic plant” is used to refer to the plant produced from an original transformation event, or progeny from later generations or crosses of a plant to a transformed plant, so long as the progeny contains a nucleus with the recombinant DNA in its genome.
“Recombinant DNA” means a polynucleotide having a genetically engineered modification introduced through combination of endogenous and/or exogenous elements in a transcription unit, manipulation via mutagenesis, restriction enzymes, and the like or simply by inserting multiple copies of a native transcription unit. Recombinant DNA may comprise DNA segments obtained from different sources, or DNA segments obtained from the same source, but which have been manipulated to join DNA segments which do not naturally exist in the joined form. A recombinant polynucleotide may exist outside of the cell, for example as a PCR fragment, or integrated into a genome, such as a plant genome.
“Trait” means a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance.
A “control plant” is a plant without trait-improving recombinant DNA in its nucleus. A control plant is used to measure and compare trait improvement in a transgenic plant with such trait-improving recombinant DNA. A suitable control plant may be a nontransgenic plant of the parental line used to generate a transgenic plant herein. Alternatively, a control plant may be a transgenic plant that comprises an empty vector or marker gene, but does not contain the recombinant DNA that produces the trait improvement. A control plant may also be a negative segregant progeny of hemizygous transgenic plant. In certain demonstrations of trait improvement, the use of a limited number of control plants can cause a wide variation in the control dataset. To minimize the effect of the variation within the control dataset, a “reference” is used. As use herein a “reference” is a trimmed mean of all data from both transgenic and control plants grown under the same conditions and at the same developmental stage. The trimmed mean is calculated by eliminating a specific percentage, e.g., 20%, of the smallest and largest observation from the data set and then calculating the average of the remaining observation.
“Trait enhancement” means a detectable and desirable difference in a characteristic in a transgenic plant relative to a control plant or a reference. In some cases, the trait enhancement can be measured quantitatively. For example, the trait improvement can entail at least a 2% desirable difference in an observed trait, at least a 5% desirable difference, at least about a 10% desirable difference, at least about a 20% desirable difference, at least about a 30% desirable difference, at least about a 50% desirable difference, at least about a 70% desirable difference, or at least about a 100% difference, or an even greater desirable difference. In other cases, the trait enhancement is only measured qualitatively. It is known that there can be a natural variation in a trait. Therefore, the trait enhancement observed entails a change of the normal distribution of the trait in the transgenic plant compared with the trait distribution observed in a control plant or a reference, which is evaluated by statistical methods provided herein. Trait enhancement includes, but is not limited to, yield increase, including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density.
Many agronomic traits can affect “yield”, including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Other traits that can affect yield include, efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill. Also of interest is the generation of transgenic plants that demonstrate desirable phenotypic properties that may or may not confer an increase in overall plant yield. Such properties include enhanced plant morphology, plant physiology or improved components of the mature seed harvested from the transgenic plant.
“Yield-limiting environment” means the condition under which a plant would have the limitation on yield including environmental stress conditions.
“Stress condition” means a condition unfavorable for a plant, which adversely affect plant metabolism, growth and/or development. A plant under the stress condition typically shows reduced germination rate, retarded growth and development, reduced photosynthesis rate, and eventually leading to reduction in yield. Specifically, “water deficit stress” used herein preferably refers to the sub-optimal conditions for water and humidity needed for normal growth of natural plants. Relative water content (RWC) can be used as a physiological measure of plant water deficit. It measures the effect of osmotic adjustment in plant water status, when a plant is under stress conditions. Conditions that may result in water deficit stress include heat, drought, high salinity and PEG induced osmotic stress.
“Cold stress” means the exposure of a plant to a temperature below (two or more degrees Celsius below) those normal for a particular species or particular strain of plant.
“Nitrogen nutrient” means any one or any mix of the nitrate salts commonly used as plant nitrogen fertilizer, including, but not limited to, potassium nitrate, calcium nitrate, sodium nitrate, ammonium nitrate. The term ammonium as used herein means any one or any mix of the ammonium salts commonly used as plant nitrogen fertilizer, e.g., ammonium nitrate, ammonium chloride, ammonium sulfate, etc.
“Low nitrogen availability stress” means a plant growth condition that does not contain sufficient nitrogen nutrient to maintain a healthy plant growth and/or for a plant to reach its typical yield under a sufficient nitrogen growth condition. For example, a low nitrogen condition can refer to a growth condition with 50% or less of the conventional nitrogen inputs. “Sufficient nitrogen growth condition” means a growth condition where the soil or growth medium contains or receives optimal amounts of nitrogen nutrient to sustain a healthy plant growth and/or for a plant to reach its typical yield for a particular plant species or a particular strain. One skilled in the art would recognize what constitute such soil, media and fertilizer inputs for most plant species.
“Shade stress” means a growth condition that has limited light availability that triggers the shade avoidance response in plant. Plants are subject to shade stress when localized at lower part of the canopy, or in close proximity of neighboring vegetation. Shade stress may become exacerbated when the planting density exceeds the average prevailing density for a particular plant species. The average prevailing densities per acre of a few examples of crop plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000 plants per acre (Cheikh, e.g., (2003) U.S. Patent Application No. 20030101479).
“Increased yield” of a transgenic plant of the present invention is evidenced and measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e., seeds, or weight of seeds, per acre), bushels per acre, tons per acre, tons per acre, kilo per hectare. For example, maize yield can be measured as production of shelled corn kernels per unit of production area, e.g., in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, e.g., at 15.5% moisture. Increased yield can result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved tolerance to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Trait-improving recombinant DNA can also be used to provide transgenic plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways.
“Expression” means transcription of DNA to produce RNA. The resulting RNA may be without limitation mRNA encoding a protein, antisense RNA, or a double-stranded RNA for use in RNAi technology. Expression also refers to production of encoded protein from mRNA.
A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissues are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most conditions. As used herein, “antisense orientation” includes reference to a polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.
As used herein, “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
A “consensus sequence” refers to an artificial, amino acid sequence of conserved parts of the proteins encoded by homologous genes, e.g., as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.
Homologous genes are genes which encode proteins with the same or similar biological function to the protein encoded by the second gene. Homologous genes may be generated by the event of speciation (see ortholog) or by the event of genetic duplication (see paralog). “Orthologs” refer to a set of homologous genes in different species that evolved from a common ancestral gene by specification. Normally, orthologs retain the same function in the course of evolution; and “paralogs” refer to a set of homologous genes in the same species that have diverged from each other as a consequence of genetic duplication. Thus, homologous genes can be from the same or a different organism. As used herein, “homolog” means a protein that performs the same biological function as a second protein including those identified by sequence identity search.
Percent identity refers to the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, e.g., nucleotide sequence or amino acid sequence. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by sequences of the two aligned segments divided by the total number of sequence components in the reference segment over a window of alignment which is the smaller of the full test sequence or the full reference sequence. “Percent identity” (“% identity”) is the identity fraction times 100. “% identity to a consensus amino acid sequence” is 100 times the identity fraction in a window of alignment of an amino acid sequence of a test protein optimally aligned to consensus amino acid sequence of this invention.
“Arabidopsis” means plants of Arabidopsis thaliana.
“Pfam” refers to a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 18.0 (August 2005) contains alignments and models for 7973 protein families and is based on the Swissprot 47.0 and SP-TREMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. Pfam is currently maintained and updated by a Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the Pfam alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low. Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins in the same protein family are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Model which characterizes the Pfam domain using HMMER software, a current version of which is provided in the appended computer listing. Candidate proteins meeting the gathering cutoff for the alignment of a particular Pfam are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this invention. Hidden Markov Model databases for use with HMMER software in identifying DNA expressing protein in a common Pfam for recombinant DNA in the plant cells of this invention are also included in the appended computer listing. The HMMER software and Pfam databases are version 18.0 and were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO: 198 through SEQ ID NO: 394. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 17 by Pfam analysis disclosed herein can be used in recombinant DNA of the plant cells of this invention, e.g. for selecting transgenic plants having enhanced agronomic traits. The relevant Pfams for use in this invention, as more specifically disclosed below, are L51_S25_CI-B8, iPGM_N, WD40, BPL_LipA_LipB, DUF676, AAA, S_locus_glycop, ArfGap, Rotamase, Metallophos, CMAS, Sugar_tr, LMBR1, RrnaAD, NAF, BolA, Pkinase, C2, FA_hydroxylase, p450, Complex1_30 kDa, Histone, DUF822, PEP-utilizers, PCI, ETC_C1_NDUFA5, 2-Hacid_dh, Tryp_alpha_amyl, PK_C, MAP65_ASE 1, FBPase, SWIB, Ank, Ribosomal_S8e, 2-Hacid_dh_C, SMC_N, GTP_cyclohydro2, PfkB, ORMDL, ADH_zinc_N, SWIM, TrkA_N, HLH, GH3, SNF5, Ceramidase_alk, Ribonuclease_T2, Complex1_49 kDa, Gp_dh_C, Aldo_ket_red, zf-AN1, TFIIS_C, MFS_1, Thioredoxin, DUF1005, LEA_3, Sterol_MT_C, Gp_dh_N, TFIIS_M, PAN_2, BPL_C, DUF26, Aa_trans, ACT, ADH_N, NAD_binding_1, Auxin_inducible, B_lectin, Anti-silence, Response_reg, 14-3-3, LRRNT_2, GDC-P, zf-CCHC, NPH3, TPR_1, TFIIA, DHBP_synthase, UQ_con, TPR_2, TPT, F-box, adh_short, Cyclin_C, Na_H_Exchanger, AA_permease, MtN3_slv, TIM, NDK, Pantoate_transf, Allene_ox_cyc, Cyclin_N, Methyltransf_11, CBM_20, Methyltransf_12, Rhodanese, Glycolytic, Actin, Usp, eIF-4B, Glyco_transf_8, BURP, Alpha-amylase, F420_oxidored, EGF_CA, Kelch_1, PGAM, Aminotran_1_2, Kelch_2, UPF0261, CoA_binding, DUF868, Peptidase_S10, Lung_7-TM_R, Oleosin, Sad1_UNC, Gln-synt_C, LSM, NTP_transferase, Metalloenzyme, Prenylcys_lyase, Subtilisin_N, SAM_1, DUF298, ESCRT-III, DNA_pol_E_B, Aminotran_3, NAD_Gly3P_dh_N, Gln-synt_N, MMR_HSR1, DUF588, zf-CCCH, DnaJ, Pkinase_Tyr, Cupin_2, LRR_1, Cupin_3, zf-CSL, FAR1, HD, FH2, APC8, PTR2, MannoseP_isomer, Rib_5-P_isom_A, DUF1336, Phosphorylase, DUF1191, Asp, Mit_rib_S27, PAP_fibrillin, DUF1195, Aldedh, zf-C3HC4, PPR, PK, PurA, RMMBL, HTH_11, Tim 17, and PBD.

Recombinant DNA Constructs

The present invention provides recombinant DNA constructs comprising one or more polynucleotides disclosed herein for imparting one or more improved traits to transgenic plant when incorporated into the nucleus of the plant cells. Such constructs also typically comprise a promoter operatively linked to said polynucleotide to provide for expression in the plant cells. Other construct components may include additional regulatory elements, such as 5′ or 3′ untranslated regions (such as polyadenylation sites), intron regions, and transit or signal peptides. Such recombinant DNA constructs can be assembled using methods known to those of ordinary skill in the art.
In a preferred embodiment, a polynucleotide of the present invention is operatively linked in a recombinant DNA construct to a promoter functional in a plant to provide for expression of the polynucleotide in the sense orientation such that a desired protein or polypeptide fragment of a protein is produced. Also provided are embodiments wherein a polynucleotide is operatively linked to a promoter functional in a plant to provide for expression of gene suppression RNA to suppress the level of an endogenous protein.
Recombinant constructs prepared in accordance with the present invention also generally include a 3′ untranslated DNA region (UTR) that typically contains a polyadenylation sequence following the polynucleotide coding region. Examples of useful 3′ UTRs include those from the nopaline synthase gene of Agrobacterium tumefaciens (nos), a gene encoding the small subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS), and the T7 transcript of Agrobacterium tumefaciens.
Constructs and vectors may also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides, see U.S. Pat. Nos. 5,188,642 and 5,728,925, incorporated herein by reference.
Table 1 provides a list of genes that provided recombinant DNA that was expressed in a model plant and identified from screening as imparting an improved trait. When the stated orientation is “sense”, the expression of the gene or a homolog in a crop plant provides the means to identify transgenic events that provide an enhanced trait in the crop plant. When the stated orientation is “antisense”, the suppression of the native homolog in a crop plant provides the means to identify transgenic events that provide an enhanced trait in the crop plant. In some cases the expression/suppression in the model plant exhibited an improved trait that corresponds to an enhanced agronomic trait, e.g. cold stress tolerance, water deficit stress tolerance, low nitrogen stress tolerance and the like. In other cases the expression/suppression in the model plant exhibited an improved trait that is a surrogate to an enhance agronomic trait, e.g. salinity stress tolerance being a surrogate to drought tolerance or improvement in plant growth and development being a surrogate to enhanced yield. Even when expression of a transgene or suppression of a native gene imparts an improved trait in a model plant, not every crop plant expressing the same transgene or suppressing the same native gene will necessarily demonstrate an indicated enhanced agronomic trait. For instance, it is well known that multiple transgenic events are required to identify a transgenic plant that can exhibit an enhanced agronomic trait. However, by with routine experimentation a transgenic plant cell nuclei, cell, plant or seed of this invention can be identified by making a reasonable number of transgenic events and engaging in screening process identified in this specification and illustrated in the examples. An understanding of Table 1 is facilitated by the following description of the headings:
“NUC SEQ ID NO” refers to a SEQ ID NO. for particular DNA sequence in the Sequence Listing.
“PEP SEQ ID NO” refers to a SEQ ID NO. in the Sequence Listing for the amino acid sequence of a protein cognate to a particular DNA
“construct_id” refers to an arbitrary number used to identify a particular recombinant DNA construct comprising the particular DNA.
“Gene ID” refers to an arbitrary name used to identify the particular DNA.
“orientation” refers to the orientation of the particular DNA in a recombinant DNA construct relative to the promoter.

TABLE 1

NUC	PEP
Seq	SEQ
ID	ID		Construct
No.	No	Gene ID	ID	Orientation

1	198	CGPG106	11029	SENSE
2	199	CGPG1133	12223	SENSE
3	200	CGPG117	10422	ANTI-SENSE
4	201	CGPG1226	13485	ANTI-SENSE
5	202	CGPG1288	13235	SENSE
6	203	CGPG1301	13411	SENSE
7	204	CGPG1458	73944	SENSE
8	205	CGPG1542	13846	ANTI-SENSE
9	206	CGPG170	12602	SENSE
10	207	CGPG1828	74065	ANTI-SENSE
11	208	CGPG2206	72783	SENSE
12	209	CGPG2217	17210	SENSE
13	210	CGPG2292	72724	SENSE
14	211	CGPG2457	17805	ANTI-SENSE
15	212	CGPG2499	16610	SENSE
16	213	CGPG2653	76602	SENSE
17	214	CGPG2813	18456	SENSE
18	215	CGPG3002	18414	SENSE
19	216	CGPG3154	71538	SENSE
20	217	CGPG3235	76532	SENSE
21	218	CGPG3274	18231	SENSE
22	219	CGPG3275	18232	SENSE
23	220	CGPG3363	18256	SENSE
24	221	CGPG3367	18258	SENSE
25	222	CGPG3375	19193	SENSE
26	223	CGPG3528	71301	SENSE
27	224	CGPG3534	18354	SENSE
28	225	CGPG3638	77334	SENSE
29	226	CGPG3918	19767	SENSE
30	227	CGPG3920	19774	SENSE
31	228	CGPG3962	70992	SENSE
32	229	CGPG3972	19956	SENSE
33	230	CGPG3990	70948	SENSE
34	231	CGPG3994	70201	SENSE
35	232	CGPG4026	19973	SENSE
36	233	CGPG4048	70987	SENSE
37	234	CGPG4052	70950	SENSE
38	235	CGPG4057	70962	SENSE
39	236	CGPG4058	70915	SENSE
40	237	CGPG4069	19947	SENSE
41	238	CGPG4087	70969	SENSE
42	239	CGPG4088	70985	SENSE
43	240	CGPG4102	70971	SENSE
44	241	CGPG4121	70963	SENSE
45	242	CGPG4122	70994	SENSE
46	243	CGPG4140	70956	SENSE
47	244	CGPG4154	70995	SENSE
48	245	CGPG4311	73306	SENSE
49	246	CGPG4363	70657	SENSE
50	247	CGPG4369	70660	SENSE
51	248	CGPG442	74536	SENSE
52	249	CGPG4454	71328	SENSE
53	250	CGPG4456	71329	SENSE
54	251	CGPG4473	70755	SENSE
55	252	CGPG4588	70684	SENSE
56	253	CGPG4765	73330	SENSE
57	254	CGPG4788	76202	SENSE
58	255	CGPG4912	72807	SENSE
59	256	CGPG4926	72811	SENSE
60	257	CGPG4967	73235	SENSE
61	258	CGPG4977	72813	SENSE
62	259	CGPG5001	72825	SENSE
63	260	CGPG5025	73628	SENSE
64	261	CGPG5041	76105	SENSE
65	262	CGPG5116	73242	SENSE
66	263	CGPG5144	74217	SENSE
67	264	CGPG5171	73735	SENSE
68	265	CGPG5194	73256	SENSE
69	266	CGPG5200	73260	SENSE
70	267	CGPG5210	75822	SENSE
71	268	CGPG5221	72001	SENSE
72	269	CGPG5269	72056	SENSE
73	270	CGPG5404	77308	SENSE
74	271	CGPG5432	73766	SENSE
75	272	CGPG5518	72774	SENSE
76	273	CGPG5535	72788	SENSE
77	274	CGPG5540	72753	SENSE
78	275	CGPG5568	72709	SENSE
79	276	CGPG5577	73954	SENSE
80	277	CGPG5587	73137	SENSE
81	278	CGPG5594	73161	SENSE
82	279	CGPG5633	73057	SENSE
83	280	CGPG5640	73127	SENSE
84	281	CGPG5646	73033	SENSE
85	282	CGPG5656	73105	SENSE
86	283	CGPG5659	73141	SENSE
87	284	CGPG5661	73165	SENSE
88	285	CGPG5684	73155	SENSE
89	286	CGPG5694	73026	SENSE
90	287	CGPG5704	73120	SENSE
91	288	CGPG5714	73133	SENSE
92	289	CGPG5721	73134	SENSE
93	290	CGPG5728	73123	SENSE
94	291	CGPG5757	73981	SENSE
95	292	CGPG5764	73136	SENSE
96	293	CGPG5783	73172	SENSE
97	294	CGPG5791	73020	SENSE
98	295	CGPG5799	72946	SENSE
99	296	CGPG5856	74746	SENSE
100	297	CGPG5927	77312	SENSE
101	298	CGPG5941	75237	SENSE
102	299	CGPG5957	75240	SENSE
103	300	CGPG5967	74349	SENSE
104	301	CGPG6040	76422	SENSE
105	302	CGPG607	70812	SENSE
106	303	CGPG6178	77322	SENSE
107	304	CGPG6185	74662	SENSE
108	305	CGPG6306	76527	SENSE
109	306	CGPG6318	77020	SENSE
110	307	CGPG6326	77609	SENSE
111	308	CGPG6370	73485	SENSE
112	309	CGPG6429	73433	SENSE
113	310	CGPG6440	73411	SENSE
114	311	CGPG6516	73568	SENSE
115	312	CGPG6653	74688	SENSE
116	313	CGPG6712	74420	SENSE
117	314	CGPG6737	74435	SENSE
118	315	CGPG6747	74460	SENSE
119	316	CGPG6796	74566	SENSE
120	317	CGPG6805	77610	SENSE
121	318	CGPG6810	77618	SENSE
122	319	CGPG6952	77517	SENSE
123	320	CGPG6953	77518	SENSE
124	321	CGPG7121	76460	SENSE
125	322	CGPG7163	77069	SENSE
126	323	CGPG7168	76161	SENSE
127	324	CGPG7206	76171	SENSE
128	325	CGPG7225	76178	SENSE
129	326	CGPG7267	76467	SENSE
130	327	CGPG7272	77536	SENSE
131	328	CGPG7281	76576	SENSE
132	329	CGPG7308	74862	SENSE
133	330	CGPG7316	74863	SENSE
134	331	CGPG7371	74858	SENSE
135	332	CGPG7457	74933	SENSE
136	333	CGPG7520	75379	SENSE
137	334	CGPG7529	77816	SENSE
138	335	CGPG7636	75434	SENSE
139	336	CGPG7737	77821	SENSE
140	337	CGPG7767	75685	SENSE
141	338	CGPG7804	75654	SENSE
142	339	CGPG7823	75692	SENSE
143	340	CGPG7828	75657	SENSE
144	341	CGPG7833	75622	SENSE
145	342	CGPG7933	77549	SENSE
146	343	CGPG7986	77917	SENSE
147	344	CGPG8012	77568	SENSE
148	345	CGPG8015	77570	SENSE
149	346	CGPG8055	77338	SENSE
150	347	CGPG8062	77580	SENSE
151	348	CGPG8082	77928	SENSE
152	349	CGPG8083	77349	SENSE
153	350	CGPG8106	77357	SENSE
154	351	CGPG8107	77587	SENSE
155	352	CGPG8136	77933	SENSE
156	353	CGPG8152	77619	SENSE
157	354	CGPG8166	77621	SENSE
158	355	CGPG8377	77629	SENSE
159	356	CGPG8976	77832	SENSE
160	357	CGPG8987	76802	SENSE
161	358	CGPG9013	76829	SENSE
162	359	CGPG9080	76961	SENSE
163	360	CGPG9081	76973	SENSE
164	361	CGPG9130	77150	SENSE
165	362	CGPG9133	77186	SENSE
166	363	CGPG9134	77103	SENSE
167	364	CGPG9137	77139	SENSE
168	365	CGPG9141	77187	SENSE
169	366	CGPG9145	77140	SENSE
170	367	CGPG9147	77164	SENSE
171	368	CGPG9148	77176	SENSE
172	369	CGPG9155	77165	SENSE
173	370	CGPG9163	77166	SENSE
174	371	CGPG9170	77155	SENSE
175	372	CGPG9180	77180	SENSE
176	373	CGPG9183	77121	SENSE
177	374	CGPG9186	77157	SENSE
178	375	CGPG9205	77195	SENSE
179	376	CGPG9207	77124	SENSE
180	377	CGPG9219	77261	SENSE
181	378	CGPG9220	77273	SENSE
182	379	CGPG9230	77203	SENSE
183	380	CGPG9236	77275	SENSE
184	381	CGPG9238	77204	SENSE
185	382	CGPG9259	77266	SENSE
186	383	CGPG9271	77220	SENSE
187	384	CGPG9275	77268	SENSE
188	385	CGPG9278	77209	SENSE
189	386	CGPG9283	77269	SENSE
190	387	CGPG9309	77451	SENSE
191	388	CGPG9311	77452	SENSE
192	389	CGPG9322	77430	SENSE
193	390	CGPG9335	77432	SENSE
194	391	CGPG9341	77433	SENSE
195	392	CGPG9344	77444	SENSE
196	393	CGPG9345	77409	SENSE
197	394	CGPG976	12313	ANTI-SENSE
10	207	CGPG1828	16322	SENSE

Recombinant DNA

DNA for use in the present invention to improve traits in plants have a nucleotide sequence of SEQ ID NO:1 through SEQ ID NO:197, as well as the homologs of such DNA molecules. A subset of the DNA for gene suppression aspects of the invention includes fragments of the disclosed full polynucleotides consisting of oligonucleotides of 21 or more consecutive nucleotides. Oligonucleotides the larger molecules having a sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO: 197 are useful as probes and primers for detection of the polynucleotides used in the invention. Also useful in this invention are variants of the DNA. Such variants may be naturally occurring, including DNA from homologous genes from the same or a different species, or may be non-natural variants, for example DNA synthesized using chemical synthesis methods, or generated using recombinant DNA techniques. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, a DNA useful in the present invention may have any base sequence that has been changed from the sequences provided herein by substitution in accordance with degeneracy of the genetic code.
Homologs of the genes providing DNA demonstrated as useful in improving traits in model plants disclosed herein will generally have significant identity with the DNA disclosed herein. DNA is substantially identical to a reference DNA if, when the sequences of the polynucleotides are optimally aligned there is about 60% nucleotide equivalence; more preferably 70%; more preferably 800% equivalence; more preferably 85% equivalence; more preferably 90%; more preferably 95%; and/or more preferably 98% or 99% equivalence over a comparison window. A comparison window is preferably at least 50-100 nucleotides, and more preferably is the entire length of the polynucleotide provided herein. Optimal alignment of sequences for aligning a comparison window may be conducted by algorithms; preferably by computerized implementations of these algorithms (for example, the Wisconsin Genetics Software Package Release 7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). The reference polynucleotide may be a full-length molecule or a portion of a longer molecule. Preferentially, the window of comparison for determining polynucleotide identity of protein encoding sequences is the entire coding region.
Proteins useful for imparting improved traits are entire proteins or at least a sufficient portion of the entire protein to impart the relevant biological activity of the protein. Proteins useful for generation of transgenic plants having improved traits include the proteins with an amino acid sequence provided herein as SEQ ID NO: 198 through SEQ ID NO: 394, as well as homologs of such proteins.
Homologs of the proteins useful in the invention are identified by comparison of the amino acid sequence of the protein to amino acid sequences of proteins from the same or different plant sources, e.g., manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. As used herein, a homolog is a protein from the same or a different organism that performs the same biological function as the polypeptide to which it is compared. An orthologous relation between two organisms is not necessarily manifest as a one-to-one correspondence between two genes, because a gene can be duplicated or deleted after organism phylogenetic separation, such as speciation. For a given protein, there may be no ortholog or more than one ortholog. Other complicating factors include alternatively spliced transcripts from the same gene, limited gene identification, redundant copies of the same gene with different sequence lengths or corrected sequence. A local sequence alignment program, e.g., BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity. As a protein hit with the best E-value for a particular organism may not necessarily be an ortholog or the only ortholog, a reciprocal BLAST search is used in the present invention to filter hit sequences with significant E-values for ortholog identification. The reciprocal BLAST entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit is a likely ortholog, when the reciprocal BLAST's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. Thus, homolog is used herein to describe proteins that are assumed to have functional similarity by inference from sequence base similarity. The relationship of homologs with amino acid sequences of SEQ ID NO: 395 to SEQ ID NO: 19,938 to the proteins with amino acid sequences of SEQ ID NO: to 198 to SEQ ID NO: 394 are found in the listing of Table 2.
Other functional homolog proteins differ in one or more amino acids from those of a trait-improving protein disclosed herein as the result of one or more of the well-known conservative amino acid substitutions, e.g., valine is a conservative substitute for alanine and threonine is a conservative substitute for serine. Conservative substitutions for an amino acid within the native sequence can be selected from other members of a class to which the naturally occurring amino acid belongs. Representative amino acids within these various classes include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conserved substitutes for an amino acid within a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the invention comprises proteins that differ in one or more amino acids from those of a described protein sequence as the result of deletion or insertion of one or more amino acids in a native sequence.
Homologs of the trait-improving proteins provided herein will generally demonstrate significant sequence identity. Of particular interest are proteins having at least 50% sequence identity, more preferably at least about 70% sequence identity or higher, e.g., at least about 80% sequence identity with an amino acid sequence of SEQ ID NO:198 through SEQ ID NO: 394. Of course useful proteins also include those with higher identity, e.g., 90% to 99% identity. Identity of protein homologs is determined by optimally aligning the amino acid sequence of a putative protein homolog with a defined amino acid sequence and by calculating the percentage of identical and conservatively substituted amino acids over the window of comparison. The window of comparison for determining identity can be the entire amino acid sequence disclosed herein, e.g., the full sequence of any of SEQ ID NO: 198 through SEQ ID NO: 394.
Genes that are homologous to each other can be grouped into families and included in multiple sequence alignments. Then a consensus sequence for each group can be derived. This analysis enables the derivation of conserved and class-(family) specific residues or motifs that are functionally important. These conserved residues and motifs can be further validated with 3D protein structure if available. The consensus sequence can be used to define the full scope of the invention, e.g., to identify proteins with a homolog relationship. Thus, the present invention contemplates that protein homologs include proteins with an amino acid sequence that has at least 90% identity to such a consensus amino acid sequence sequences.

Promoters

Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens, caulimovirus promoters such as the cauliflower mosaic virus or Figwort mosaic virus promoters. For instance, see U.S. Pat. Nos. 5,858,742 and 5,322,938 which disclose versions of the constitutive promoter derived from cauliflower mosaic virus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a Figwort Mosaic Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which discloses a maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324 promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357 which discloses a rice actin 2 promoter and intron, U.S. Pat. No. 5,837,848 which discloses a root specific promoter, U.S. Pat. No. 6,084,089 which discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which discloses light inducible promoters, U.S. Pat. No. 6,140,078 which discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No. 6,175,060 which discloses phosphorus deficiency inducible promoters, U.S. Patent Application Publication 2002/0192813A1 which discloses 5′, 3′ and intron elements useful in the design of effective plant expression vectors, U.S. patent application Ser. No. 09/078,972 which discloses a coixin promoter, U.S. patent application Ser. No. 09/757,089 which discloses a maize chloroplast aldolase promoter, and U.S. patent application Ser. No. 10/739,565 which discloses water-deficit inducible promoters, all of which are incorporated herein by reference. These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant polynucleotides of the present invention to provide for expression of desired genes in transgenic plant cells.
Furthermore, the promoters can include multiple “enhancer sequences” to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein may be enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted in the forward or reverse orientation 5′ or 3′ to the coding sequence. In some instances, these 5′ enhancing elements are introns. Deemed to be particularly useful as enhancers are the 5′ introns of the rice actin 1 and rice actin 2 genes. Examples of other enhancers that can be used in accordance with the invention include elements from the CaMV 35S promoter, octopine synthase genes, the maize alcohol dehydrogenase gene, the maize shrunken 1 gene and promoters from non-plant eukaryotes.
In some aspects of the invention it is preferred that the promoter element in the DNA construct be capable of causing sufficient expression to result in the production of an effective amount of a polypeptide in water deficit conditions. Such promoters can be identified and isolated from the regulatory region of plant genes that are over expressed in water deficit conditions. Specific water-deficit-inducible promoters for use in this invention are derived from the 5′ regulatory region of genes identified as a heat shock protein 17.5 gene (HSP17.5), an HVA22 gene (HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea maize. Such water-deficit-inducible promoters are disclosed in U.S. application Ser. No. 10/739,565, incorporated herein by reference.
In some aspects of the invention, sufficient expression in plant seed tissues is desired to effect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al., (1997) Transgenic Res. 6(2):157-166), globulin 1 (Belanger et al., (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) supra), and peroxiredoxin antioxidant (Perl) (Stacy et al., (1996) Plant Mol Biol. 31(6):1205-1216).
In some aspects of the invention, preferential expression in plant green tissues is desired. Promoters of interest for such uses include those from genes such as SSU (Fischhoff, et al., (1992) Plant Mol Biol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi, et al., (2000) Plant Cell Physiol. 41(1):42-48).
Gene suppression includes any of the well-known methods for suppressing transcription of a gene or the accumulation of the mRNA corresponding to that gene thereby preventing translation of the transcript into protein. Posttranscriptional gene suppression is mediated by transcription of RNA that forms double-stranded RNA (dsRNA) having homology to a gene targeted for suppression. Suppression can also be achieved by insertion mutations created by transposable elements may also prevent gene function. For example, in many dicot plants, transformation with the T-DNA of Agrobacterium may be readily achieved and large numbers of transformants can be rapidly obtained. Also, some species have lines with active transposable elements that can efficiently be used for the generation of large numbers of insertion mutations, while some other species lack such options. Mutant plants produced by Agrobacterium or transposon mutagenesis and having altered expression of a polypeptide of interest can be identified using the polynucleotides of the present invention. For example, a large population of mutated plants may be screened with polynucleotides encoding the polypeptide of interest to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.

Gene Stacking

The present invention also contemplates that the trait-improving recombinant DNA provided herein can be used in combination with other recombinant DNA to create plants with multiple desired traits or a further enhanced trait. The combinations generated can include multiple copies of any one or more of the recombinant DNA constructs. These stacked combinations can be created by any method, including but not limited to cross breeding of transgenic plants, or multiple genetic transformation.
In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal or polyclonal which bind to the proteins disclosed herein. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goal. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include using glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified antifungal protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp. 75-83). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Spend virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).
Fusion procedures usually produce viable hybrids at low frequencies, about 1×10⁻⁶to 1×10⁻⁸. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azasenne blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

Transformation Methods

Numerous methods for producing plant cell nuclei with recombinant DNA are known in the art and may be used in the present invention. Two commonly used methods for plant transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. No. 5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812 (wheat) and Agrobacterium-mediated transformation is described in U.S. Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all of which are incorporated herein by reference. For Agrobacterium tumefaciens based plant transformation system, additional elements present on transformation constructs will include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.
In general it is preferred to introduce heterologous DNA randomly, i.e., at a non-specific location, in the genome of a target plant line. In special cases it may be useful to target heterologous DNA insertion in order to achieve site-specific integration, e.g., to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function implants include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.
Transformation methods of this invention are preferably practiced in tissue culture on media and in a controlled environment. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, e.g., various media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. patent application Ser. No. 09/757,089, which are incorporated herein by reference.
In practice DNA is introduced into only a small percentage of target cell nuclei in any one experiment. Marker genes are used to provide an efficient system for identification of those cells with nuclei that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide. Potentially transformed cells with a nucleus of the invention are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA in the nucleus. Useful selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such selectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Screenable markers which provide an ability to visually identify transformants can also be employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. It is also contemplated that combinations of screenable and selectable markers will be useful for identification of transformed cells. See PCT publication WO 99/61129 which discloses use of a gene fusion between a selectable marker gene and a screenable marker gene, e.g., an NPTII gene and a GFP gene.
Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in regeneration media and allowed to mature into plants. Developing plantlets can be transferred to soil less plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻²s⁻¹of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown to plants on solid media at about 19 to 28° C. After regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.
Progeny may be recovered from transformed plants and tested for expression of the exogenous recombinant polynucleotide. Useful assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCR; “biochemical” assays, such as detecting the presence of RNA, e.g., double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.

Discovery of Trait-Improving Recombinant DNA

To identify nuclei with recombinant DNA that confer improved traits to plants, Arabidopsis thaliana was transformed with a candidate recombinant DNA construct and screened for an improved trait.
Arabidopsis thaliana is used a model for genetics and metabolism in plants. Arabidopsis has a small genome, and well-documented studies are available. It is easy to grow in large numbers and mutants defining important genetically controlled mechanisms are either available, or can readily be obtained. Various methods to introduce and express isolated homologous genes are available (see Koncz, e.g., Methods in Arabidopsis Research e.g., (1992), World Scientific, New Jersey, New Jersey, in “Preface”).
A two-step screening process was employed which comprised two passes of trait characterization to ensure that the trait modification was dependent on expression of the recombinant DNA, but not due to the chromosomal location of the integration of the transgene. Twelve independent transgenic lines for each recombinant DNA construct were established and assayed for the transgene expression levels. Five transgenic lines with high transgene expression levels were used in the first pass screen to evaluate the transgene's function in T2 transgenic plants. Subsequently, three transgenic events, which had been shown to have one or more improved traits, were further evaluated in the second pass screen to confirm the transgene's ability to impart an improved trait. The following Table 3 summarizes the improved traits that have been confirmed as provided by a recombinant DNA construct.
In particular, Table 3 reports:
“PEP SEQ ID” which is the amino acid sequence of the protein cognate to the DNA in the recombinant DNA construct corresponding to a protein sequence of a SEQ ID NO. in the Sequence Listing.
“construct_id” is an arbitrary name for the recombinant DNA describe more particularly in Table 1.
“annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GenBank database of the National Center for Biotechnology Information (ncbi). More particularly, “gi” is the GenBank ID number for the top BLAST hit.
“description” refers to the description of the top BLAST hit.
“e-value” provides the expectation value for the BLAST hit.
“% id” refers to the percentage of identically matched amino acid residues along the length of the portion of the sequences which is aligned by BLAST between the sequence of interest provided herein and the hit sequence in GenBank.
“traits” identify by two letter codes the confirmed improvement in a transgenic plant provided by the recombinant DNA. The codes for improved traits are:
“CK” which indicates cold tolerance improvement identified under a cold shock tolerance screen;
“CS” which indicates cold tolerance improvement identified by a cold germination tolerance screen;
“DS” which indicates drought tolerance improvement identified by a soil drought stress tolerance screen;
“PEG” which indicates osmotic stress tolerance improvement identified by a PEG induced osmotic stress tolerance screen;
“HS” which indicates heat stress tolerance improvement identified by a heat stress tolerance screen;
“SS” which indicates high salinity stress tolerance improvement identified by a salt stress tolerance screen;
“LN” which indicates nitrogen use efficiency improvement identified by a limited nitrogen tolerance screen;
“LL” which indicates attenuated shade avoidance response identified by a shade tolerance screen under a low light condition;
“PP” which indicates improved growth and development at early stages identified by an early plant growth and development screen;
“SP” which indicates improved growth and development at late stages identified by a late plant growth and development screen provided herein.

TABLE 3

PEP		Annotation

SEQ

Construct

e

%

ID	ID	value	id	description	trait

198	11029	1.00E−170	85	gb\|AAD02882.1\|gamma-tocopherol	DS	LN
				methyltransferase [Arabidopsis
				thaliana]
199	12223	/	/	/	CK	PEG
200	10422	1.00E−133	100	gb\|AAB47766.1\|SNF5 homolog BSH	LN
				[Arabidopsis thaliana]
201	113485	4.00E−39	55	emb\|CAB79347.1\|hypothetical	LN
				protein [Arabidopsis thaliana]
202	13235	7.00E−76	100	einb\|CAB78153.1\|putative protein	LN
				[Arabidopsis thaliana]
203	13411	3.00E−29	100	ref\|NP_179638.1\|unknown protein	LN	CK
				[Arabidopsis thaliana]
204	73944	0	95	ref\|NP_189578.1\|phosphorylase]	CS
				transferase, transferring glycosyl
				groups [Arabidopsis thaliana]
205	13846	0	91	gb\|AAN-60291.1\| unknown	LN
				[Arabidopsis thaliana]
206	12602	0	100	ref\|NP_565974.1\|PP2.A-4; hydrolase/	LN
				protein phosphatase type 2A/protein
				serine/threonine phosphatase
				[Arabidopsis thaliana]
207	74065	0	94	ref\|NP_181390.1\|DNA binding/	CS	DS
				transcription factor [Arabidopsis
				thaliana] gb\|AAN13033.1\| putative
				elongation
208	72783	0	83	ref\|NP_564690.1\|unknown protein	CK	PEG	HS
				[Arabidopsis thaliana]
209	17210	1.00E−92	100	ref\|NP_563622.1\|unknown protein	CS	CK	PEG
				[Arabidopsis thaliana]
210	72724	1.00E−128	90	ref\|NP_190525.1\| protein translocase/	LL
				protein transporter [Arabidopsis
				thaliana
211	17805	1.00E−172	80	ref\|NP_563761.1\|unknown protein	LN
				[Arabidopsis thaliana]
212	16610	0	100	gb\|AAL44550.1\|fructose bisphosphate	LN	HS
				aldolase [Agrobacterium tumefaciens
				str. C58]
213	76602	0	78	ref\|NP_566515.1\|signal transducer	CS	HS
				[Arabidopsis thaliana]
214	18456	1.00E−135	81	ref\|NP_176820.1\|DNA binding/	CK	SS	PEG
				transcription factor [Arabidopsis
				thaliana]
215	18414	1.00E−169	70	ref\|NP_195583.1\| translation initiation	CK
				factor [Arabidopsis thaliana]
216	71538	1.00E−137	90	emb\|CAE85115.1\|synaptotagmin	SP
				[Arabidopsis thaliana]
217	76532	0	99	ref\|NP_564775.1\|ATP binding/	CK	CS
				carbohydrate binding/kinase/protein
				kinase/protein serine/threonine
				kinase/protein-tyrosine kinase/sugar
				binding [Arabidopsis thaliana]
218	18231	1.00E−142	100	emb\|CAC83762.1\| allene oxide	CK	LN
				cyclase [Arabidopsis thaliana]
219	18232	6.00E−85	89	ref\|NP_191628.1\|unknown protein	CS
				[Arabidopsis thaliana]
220	18256	1.00E−102	100	dbj\|BAD44508.1\| putative zinc finger	SS	CK	HS
				protein (PMZ) [Arabidopsis thaliana]
221	18258	1.00E−175	83	emb\|CAA11525.1\|transcription factor	SP
				IIA large subunit [Arabidopsis
				thaliana]
222	19193	1.00E−103	100	ref\|NP_564682.1\|lipid binding	SP	HS
				[Arabidopsis thaliana]
				dbj\|BAE73268.1\|xylogen like protein
				12 [Arabidopsis thaliana]
223	71301	0	84	ref\|NP_568393.1\|nucleic acid binding	LN
				[Arabidopsis thaliana]
224	18354	0	96	ref\|NP_567568.1\|ATP binding/	LN
				kinase/protein kinase/protein
				serine/threonine kinase/protein-
				tyrosine kinase [Arabidopsis thaliana]
225	77334	1.00E−117	100	ref\|NP_178399.1\|RNS1	LL
				(RIBONUCLEASE 1);
				endoribonuclease [Arabidopsis
				thaliana]
226	19767	1.00E−180	95	gb\|AAO69665.1\|serine threonine	CS	DS
				protein phosphatase [Phaseolus
				acutifolius]
227	19774	1.00E−100	57	ref\|NP_974271.1\|unknown protein	CS	PP
				[Arabidopsis thaliana]
228	70992	1.00E−108	65	gb\|ABE87200.1\|hypothetical protein	CS	PEG
				MtrDRAFT_AC151.668g4v1
				Medicago truncatula]
229	19956	1.00E−146	100	gb\|AAF64040.1\|14-3-3-like protein	PP
				[Glycine max]
230	70948	3.00E−59	45	ref\|NP_201297.1\|CIP8 (COP1-	PP	HS
				INTERACTING PROTEIN 8);
				protein binding/zinc ion binding
				[Arabidopsis thaliana]
231	70201	0	68	ref\|NP_567072.1\|ATP binding/	CS	SS
				kinase/protein kinase/protein
				serine/threonine kinase protein-
				tyrosine kinase [Arabidopsis thaliana]
232	19973	0	96	gb\|AAB04057.1\|S-adenosyl-L-	PP	SS
				methionine delta24-sterol-C-
				methyltransferase
233	70987	0	93	gb\|AAM83095.1\|SOS2-like protein	CK	CS	HS	SS	PEG
				kinase [Glycine max]
234	70950	1.00E−116	77	dbj\|BAD38167.1\|putative leucine	PP	PEG
				zipper protein [Oryza sativa (japonica
				cultivar-group)]
235	70962	1.00E−179	78	gb\|ABE93200.1\|conserved	PP	HS
				hypothetical protein [Medicago
				truncatula]
236	70915	0	76	dbj\|BAE711210.1\|hypothetical protein	CS	PP
				[Trifolium pratense] dbj\|BAE71208.1\|
				hypothetical protein [Trifolium
				pratense]
237	19947	1.00E−116	65	gb\|AAS38575.1\|short-chain	CS	DS	SS
				dehydrogenase Tic32 [Pisum
				sativum]
238	70969	1.00E−144	76	ref\|NP_174321.2\|unknown protein	SS
				[Arabidopsis thaliana]
239	70985	0	69	dbj\|BAB08296.1\|unnamed protein	CS	HS	PEG
				product [Arabidopsis thaliana]
				sp\|Q9FMT4\|Y541.7_ARATH Protein
				At5g14170
240	70971	1.00E−92	63	gb\|ABE80664.1\|GNS1/SUR4	SS
				membrane protein [Medicago
				truncatula] gb\|ABE78502.1\|
				GNSI/SUR4 membrane protein
				[Medicago truncatula]
241	70963	1.00E−96	56	ref\|NP_177178.1\|CYCD1; I; cyclin-	CS	PP	HS
				dependent protein kinase regulator
				[Arabidopsis thaliana]
242	70994	2.00E−33	44	ref\|NP_172962.1\|RHA2A; protein	PP	DS	HS	SS
				binding/ubiquitin-protein ligase/zinc
				ion binding [Arabidopsis thaliana]
243	70956	0	85	gb\|AAO23063.1\|ent-kaurenoic acid	CS	SS	HS
				oxidase [Pisum sativum]
244	70995	1.00E−132	66	dbj\|BAD73789.1\|putative	CK	CS	PP	LL	PEG
				uncharacterized hypothalamus protein
				HT010 [Oryza sativa (japonica
				cultivar-group)]
245	73306	0	93	ref\|NP_201243.1\|phosphoric ester	SS	PEG
				hydrolase [Arabidopsis thaliana]
246	70657	9.00E−31	73	emb\|CAB78150.1\|probable wound-	SP	LL
				induced protein [Arabidopsis
				thaliana]
247	70660	2.00E−32	79	ref\|NP_56821.7\|transcription	SP
				regulator [Arabidopsis thaliana]
248	74536	0	91	ref\|NP_001031061.1\|PGDH;	CS
				phosphoglycerate dehydrogenase
				[Arabidopsis thaliana]
249	71328	0	99	gb\|AAD46022.1\|Strong simlarity to	PP
				gb\|286426 F10M6.190 cytochrome
				p450 homolog from Arabidopsis
				thaliana BAC
250	71329	0	94	ref\|NP_177477.1\|heme binding/iron	LN
				ion binding/monooxygenase/oxygen
				binding [Arabidopsis thaliana]
251	70755	1.00E−142	100	ref\|NP_188925.1\|unknown protein	LL
				[Arabidopsis thaliana]
252	70684	3.00E−53	100	emb\|CAB80194.1\|putative protein	CS	LN	HS	PP
				[Arabidopsis thaliana]
253	73330	1.00E−158	72	emb\|CAB96855.1\|putative protein	CK	CS	SS
				[Arabidopsis thaliana]
254	76202	3.00E−83	67	emb\|CAA16586.1\|putative protein	LL
				[Arabidopsis thaliana]
255	72807	0	100	ref\|NP_173478.2\|nucleotide binding	PEG	CK	HS	SS
				[Arabidopsis thaliana]
256	72811	1.00E−176	100	ref\|NP_565782.1\|nucleotide binding	CK	SS	PEG
				[Arabidopsis thaliana]
257	73235	0	89	ref\|NP_564867.1\|pepsin A	S	HS
				[Arabidopsis thaliana]
258	72813	2.00E−79	93	ref\|NP_850506.1\|unknown protein	LL	LN
				[Arabidopsis thaliana]
259	72825	1.00E−154	95	ref\|NP_563635.1\|oxidorecluctase	CK	PEG	HS
				[Arabidopsis thaliana]
260	7 3628	1.00E−163	92	ref\|NP_849428.1\|oxidoreductase	CS	DS	LL	HS	PEG
				[Arabidopsis thaliana]
261	76105	0	93	emb\|CAB79123.1\|receptor kinase-like	DS
				protein [Arabidopsis thaliana]
262	73242	0	100	ref\|NP_567292.1\|unknown protein	SP	PEG
				[Arabidopsis thaliana]
263	74217	0	100	emb\|CAB93726.1\|cytochrome P450-	DS	CK
				like protein [Arabidopsis thaliana]
264	73735	0	97	gb\|AAU94404.1\|At3g48520	CK
				[Arabidopsis thaliana]
265	7 3256	1.00E−114	77	ref\|NP_566329.1\|unknown protein	CS	PP	PEG
				[Arabidopsis thaliana]
				gb\|AAL06975.1\|
266	73260	4.00E−37	100	ref\|NP_566488.1\|unknown protein	CS
				[Arabidopsis thaliana]
267	75822	1.00E−90	88	emb\|CAB63015.1\|putative protein	LL
				[Arabidopsis thaliana]
268	72001	0	96	ref\|NP_200045.1\|CYP96A4; heme	CS	PP	HS
				binding/iron ion binding/
				monooxygenase/oxygen binding
				[Arabidopsis thaliana]
269	72056	0	92	ref\|NP_010186.1\|Essentia1, non-	SP
				ATPase regulatory subunit of the 26S
				proteasoine lid required for the
				assembly and activity of the 26S
				proteasome;
270	77308	0	100	ref\|NP_178642.1\|SCPL38; serine	PEG
				carboxypeptidase [Arabidopsis
				thaliana]
271	73766	1.00E−163	100	emb\|CAB62363.1\|MTN3-like protein	LL
				[Arabidopsis thaliana]
272	72774	1.00E−139	95	ref\|NP_011052.1\|Constituent of 66S	LL	CK	HS
				pre-ribosomal particles, involved in
				60S ribosomal subunit biogenesis;
273	72788	0	81	ref\|NP_010007.1\|General repressor of	CK	CS	SS
				transcription, forms complex with
				Cyc8p, involved in the establishment
				of repressive chromatin structure
				through interactions with histones H3
				and H4, appears to enhance
				expression of some genes
274	72753	1.00E−105	85	ref\|NP_013125.1\|One of four subunits	CS	PP	HS	PEG
				of the endosomal sorting complex
				required for transport III (ESCRT-
				III);
275	72709	0	94	ref\|NP_011827.1\|Low affinity	LL
				methionine pennease, similar to
				Mup1p;
276	73954	0	98	ref\|NP_200954.1\|MIM; ATP binding	HS
				[Arabidopsis thaliana]
277	73137	1.00E−110	94	gb\|AAD32800.1\|putative thioredoxin	CS
				H [Arabidopsis thaliana]
278	73161	1.00E−36	65	emb\|CAB85993.1\|putative protein	CS	PEG
				[Arabidopsis thaliana]
279	73057	1.00E−133	95	ref\|NP_012770.1\|Tetrameric	SP
				phosphoglycerate mutase, mediates
				the conversion of 3-phosphoglycerate
				to 2-phosphoglycerate during
				gtycolysis and the reverse reaction
				during gluconeogenesis;
280	73127	0	99	dbj\|BAB05414.1\|aspartate	SS	HS
				aminotransferase [Bacillus halodurans
				C-125] ref\|NP_242561.1\| aspartate
				aminotransferase [Bacillus halodurans
				C-125]
281	73033	0	96	dbj\|BAB07276.1\|2,3-	CK
				bisphosphoglycerate-independent
				phosphoglycerate mutase [Bacillus
				halodurans C-125]
282	73105	0	99	emb\|CAB13630.\|glutamine	PP	PEG
				synthetase [Bacillus subtilis subsp.
				subtilis str. 168]
283	73141	0	95	ref\|NP_414696.1\|glutamate-1-	CS
				semialdehyde aminotransferase
				[Escherichia coli K12]
284	73165	0	99	ref\|NP_288110.1\|pyruvate kinase	DS	PEG
				[Escherichia coli O157:H7EDL933]
285	73155	1.00E−173	92	emb\|CAC47346.1\|PROBABLE	CS
				FRUCTOSE-BISPHOSPHATE
				ALDOLASE CLASS I PROTEIN
				[Sinorhizobium meliloti]]
286	73026	1.00E−116	87	emb\|CAG77169.1\|triophosphate	PEG
				isomerase [Erwinia carotovora subsp.
				atroseptica SCRI1043]
287	73120	8.00E−82	100	ref\|NP_441918.1\|nucleoside	CS	SS	HS
				diphosphate kinase [Synechocystis sp.
				PCC 6803]
288	73133	2.00E−63	90	gb\|ABA76342.1\|Nucleoside	LL
				diphosphate kinase [Pseudomonas
				fluorescens PfO-1]
289	73134	9.00E−98	81	emb\|CAE15979.1\|ribose 5-phosphate	SS	CK
				isomerase A (phosphoriboisomerase
				A) [Photorhabdus luminescens subsp.
				laumondii TTO1]
290	73123	0	95	ref\|NP_180412.2\|nucleic acid binding	CS	PEG
				[Arabidopsis thaliana]
291	73981	0	93	ref\|NP_014797.1\|hypothetical protein;	DS	PEG
				SlpIp [Saccharomyces cerevisiae]
292	73136	0	91	ref\|NP_012454.1\|Nuclear actin-	DS	PP
				related protein involved in chromatin
				remodeling
293	73172	0	100	ref\|NP_014952.1\|Protein involved in	CK	PEG	HS
				ER-to-Golgi transport; Sly41p
				[Saccharomyces cerevisiae]
294	7 3020	0	93	ref\|NP_014993.1[Proline permease,	LL
				required for high-affinity transport of
				proline
295	72946	0	95	ref\|NP_012534.1\|Vacuolar	PEG	LL
				transporter, imports large neutral
				amino acids into the vacuole
296	74746	0	93	ref\|NP_566298.1\|ATP binding/	CK
				kinasel protein kinase/protein
				serine/threonine kinase/protein-
				tyrosine kinase [Arabidopsis thaliana]
297	77312	0	90	ref\|NP_178463.1\|CDC4813; ATP	PP	SS	HS
				binding/ATP-dependent peptidase/
				ATPase/nucleoside-triphosphatase/
				nucleotide binding/serine-type
				endopeptidase [Arabidopsis thaliana]
298	75237	0	100	ref\|NP_173390.1\|kinase [Arabidopsis	LN
				thaliana] gb\|AAF98405.1\| Unknown
				protein [Arabidopsis thaliana]
299	75240	0	100	emb\|CAB69854.1\|putative protein	CK
				[Arabidopsis thaliana]
300	74349	1.00E−148	79	ref\|NP_565187.1\|transcription	LN
				regulator [Arabidopsis thaliana]
301	76422	5.00E−44	100	ref\|NP_171781.1\|unknown protein	LN
				[Arabidopsis thaliana]
302	70812	1.00E−152	92	ref\|NP_850182.1\|PUR ALPHA-1;	PP	PEG
				nucleic acid binding [Arabidopsis
				thaliana]
303	77322	1.00E−172	100	gb\|AAW38983.1\|At5g10750	LN	LL
				[Arabidopsis thaliana]
304	74662	4.00E−89	100	ref\|NP_001031939.1\|ubiquitin	PP	HS
				conjugating enzyme/ubiquitin-like
				activating enzyme [Arabidopsis
				thaliana]
305	76527	0	96	emb\|CAB51062.1\|cell division cycle	CS
				protein 23 homolog [Arabidopsis
				thaliana]
306	77020	0	98	emb\|CAB75781.1\|putative transporter	CS
				protein [Arabidopsis thaliana]
				ref\|NP_190154.1\| transporter
				[Arabidopsis thaliana]
307	77609	0	96	gb\|AAU95452.1\|At5g04420	LL	SS
				[Arabidopsis thaliana]
308	73485	0	100	emb\|CAD84238.1\|Glyceraldehyde 3-	PEG
				phosphate dehydrogenase
309	73433	1.00E−132	65	ref\|ZP_0081.9153.1\|putative alcohol	SP	LN	PP
				dehydrogenase [Marinobacter
				aquaeolei VT8]
310	73411	0	99	emb\|CAB14878.1\|pyruvate kinase	CS	HS	PP	PEG
				[Bacillus subtilis subsp. subtilis str.
				168]
311	73568	0	97	emb\|CAC41401.1\|PROBABLE	SP
				SUCCINAIE-SEMIALDEHYDE
				DEHYDROGENASE [NADP+]
				PROTEIN [Sinorhizobium meliloti]
312	74688	0	98	emb\|CAA16688.1\|receptor protein	CS	PP	SS	PEG
				kinase-like protein [Arabidopsis
				thaliana]
313	74420	1.00E−142	99	emb\|CAD85691.1\|Phosphoglycerate	PP	LN	SS
				mutase family [Nitrosomonas
				europaea ATCC 19718]
314	74435	1.00E−117	100	emb\|CAC41549.1\|PROBABLE	LL
				PHOSPHOGLYCERATE MUTASE

				1 PROTEIN [Sinorhizobium meliloti]
315	74460	0	95	ref\|NP_441738.1\|fructose-1,6-	CK
				bisphosphatase [Synechocystis sp.
				PCC 6803]
316	74566	0	93	emb\|CAC48499.1\|putative trehalose	SP	PP	SS	PEG	HS
				synthase protein [Sinorhizobium
				meliloti 1021]
317	77610	0	94	gb\|AAC23406.1\|hypothetical protein	CS	HS
				[Arabidopsis thaliana]
318	77618	0	100	ref\|NP_850453.1\|JAR1	PP	HS
				(JASMONAIE RESISTANT 1)
				[Arabidopsis thaliana]
319	77517	7.00E−77	100	emb\|CAB75802.1\|putative protein	CS	LL
				[Arabidopsis thaliana]
320	77518	1.00E−49	100	ref\|NP199600.1\|oxidoreductase,	S'S	LL	HS
				acting on NADH or NADPH, quinone
				or similar compound as acceptor
				[Arabidopsis thaliana]
321	76460	/	/	/	CS	PEG
322	77069	0	85	ref\|NP_201196.1\|unknown protein	PP
				[Arabidopsis thaliana]
323	76161	1.00E−161	100	ref\|NP_851282.1\|unknown protein	CK
				[Arabidopsis thaliana]
324	76171	1.00E−178	95	ref\|NP_176204.1\|oxidoreductase	LL
				[Arabidopsis thaliana]
325	76178	5.00E−60	86	ref\|NP_565029.1\|unknown protein	CS	PP	HS	LL	PEG
				[Arabidopsis thaliana]
326	76467	6.00E−86	89	ref\|NP_565671.1\|unknown protein	LL
				[Arabidopsis thaliana]
327	77536	1.00E−119	83	gb\|AAS99692.1\|At1g10020	SP	CS	DS	PP	LN
				[Arabidopsis thaliana]
328	76576	1.00E−145	100	ref\|NP_181023.1\|FAH1 (FATTY	DS
				ACID HYDROXYLASE 1); catalytic
				[Arabidopsis thaliana]
329	74862	1.00E−145	100	gb\|ABA77057.1\|Delta 1-pyrroline-5-	PP
				carboxylate reductase [Pseudomonas
				fluorescens PfO-1]
330	74863	1.00E−93	100	ref\|NP_012420.1\|Nucleosome	LL	PEG	CK	SS
				assembly factor, involved in
				chromatin assembly after DNA
				replication]
331	74858	5.00E−50	68	gb\|AAK14395.1\|response regulator	LL
				protein [Dianthus caryophyllus]
332	74933	1.00E−177	95	gb\|AAK85899.1\|AGR_C_118p	LL
				[Agrobacterium tumefaciens str. C58]
333	75379	1.00E−105	76	dbj\|BAD73205.1\|unknown protein	LN
				[Oryza sativa (japonica caltivar-
				group)]
334	77816	4.00E−41	89	gb\|ABA98984.1\|expressed protein	SP	PP	HS
				[Oryza sativa (japonica cultivar-
				group)]
335	75434	3.00E−52	79	ref\|XP_472650.1\|OSJNBa0027P08.15	LN
				[Oryza sativa (japonica cultivar-
				group)]
336	77821	0	71	gb\|ABE84883.1\|conserved	PP	SS	HS	PEG
				hypothetical protein [Medicago
				truncatula]
337	75685	0	82	ref\|XP_475937.1\|unknown protein	LL
				[Oryza sativa (japonica cultivar-
				group)]
338	75654	0	100	ref\|NP_563865.1\|unknown protein	SP	CK	HS
				[Arabidopsis thaliana]
339	75692	1.00E−119	87	dbj\|BAD30296.1\|peptidyl-prolyl cis-	CK	LN
				trans isomerase-like protein [Oryza
				sativa (japonica cultivar-group)]
340	75657	6.00E−57	67	dbj\|BAD38392.1\|DNAJ heat shock N-	CS	LL	HS
				terminal domain-containing protein-
				like [Oryza sativa (japonica cultivar-
				group)]
341	75622	1.00E−30	78	dbj\|BAD45825.1\|unknown protein	LN
				[Oryza sativa (japonica cultivar-
				gyoup)]
342	77549	1.00E−132	89	emb\|CAB80891.1\|AT400820	PP	HS
				[Arabidopsis thaliana]]
343	77917	0	91	ref\|NP_197917.1\|EBF2 (EIN3-	PP	PEG
				BINDING F BOX PROTEIN 2)
				[Arabidopsis thaliana]
344	77568	9.00E−99	76	ref\|NP_849792.1\|nucleic acid binding	PP
				[Arabidopsis thaliana]
345	77570	7.00E−54	100	gb\|AAS75309.1\|multidomin	CK	SS
				cyclophilin type peptidyl-prolyl cis-
				trans isomerase [Arabidopsis
				thaliana]]
346	77338	4.00E−24	65	gb\|AAM61454.1\|unknown	PEG
				[Arabidopsis thaliana]
347	77580	3.00E−45	100	dbj\|BAB01457.1\|unnamed protein	PEG	CS	HS	SS
				product [Arabidopsis thaliana]
348	77928	4.00E−38	100	ref\|NP_196244.1\|unknown protein	HS
				[Arabidopsis thaliana]
349	77349	1.00E−81	93	emb\|CAC05463.1\|putative lipid	LL	SS
				transfer protein [Arabidopsis thaliana]
350	77357	1.00E−155	95	ref\|NP_175357.1\|unknown protein	PEG
				[Arabidopsis thaliana]
351	77587	/	/	/	CS	PP	HS	SS
352	77933	/	/	/	SS
353	77619	3.00E−40	98	db\|BAB09403.1\|unnamed protein	CK	SS	PEG	HS
				product [Arabidopsis thaliana]
354	77621	4.00E−58	74	ref\|NP_197632.1\|Rac GTPase	CK
				activator [Arabidopsis thaliana]
				[Arabidopsis thaliana]
355	77629	5.00E−36	100	ref\|NP_196372.1\|GRP19 [Arabidopsis	SS	HS
				thaliana]
356	77832	0	83	ref\|NP_917762.1\|p050IG0I.24	CS	SS	PEG	HS	LN
				[Oryza sativa (japonica cultivar-
				group)]
357	76802	0	76	ref\|NP_181908.1\|actin binding	CK
				[Arabidopsis thaliana]
				gb\|AAB64026.1\| unknown protein
				[Arabidopsis thaliana]
358	76829	0	89	ref\|NP_909912.1\|ferredoxin-NADP+	CK
				reductase [Oryza sativa]
359	76961	0	65	ref\|XP_473189.1\|OSJNBa0073E02.11	LL	LN
				[Oryza sativa (japonica cultivar-
				group)]
360	76973	1.00E−151	76	ref\|XP_480055.1\|unknown protein	LL
				[Oryza sativa (japonica cultivar-
				group)]
361	77150	0	76	dbj\|BAD61385.1\|putative	SP	HS	PEG
				nucleostemin [Oryza sativa (japonica
				cultivar-group)]
362	77186	0	85	dbj\|BAD27898.1\|putative	PP
				pentatricopeptide (PPR) repeat-
				containing protein [Oryza sativa
				(japonica cultivar-group)]
363	77103	1.00E−174	66	ref\|NP_568580.1\|catalytic	LL	DS
				[Arabidopsis thaliana]
364	77139	2.00E−93	58	ref\|NP_171690.1\|PFC1 (PALEFACE	CS	SS
				I) [Arabidopsis thaliana]
365	77187	1.00E−160	83	ref\|NP_913437.1\|3-methyl-2 -	CS	PP	LL	PEG
				oxobutanoate hydroxy-me hyl-
				transfe rase-like protein [Oryza sativa
				(japonica cultivar-group)]
366	77140	0	77	dbj\|BAD36145.1\|membrane protein	CK	PEG	SS
				PTM1-like [Oryza sativa (japonica
				cultivar-group)]
367	77164	0	91	gb\|ABF93778.1\|DNA polymerase	PP	HS
				delta small subunit, putative,
				expressed [Oryza sativa (japonica
				cultivar-group)]
368	77176	0	76	ref\|NP_914476.1\|putative	LL
				phytochrome P450 [Oryza sativa
				(japonica cultivar-group)]
369	77165	1.00E−139	75	ref\|NP_914949.1\|serine/threonine	CK	LL	LN
				protein kinase-like protein [Oryza
				sativa (japonica cultivar-group)]
370	77166	0	85	dbj\|BAB75233.1\|all3534 [Nostoc sp.	CK	PP
				PCC 7120]
371	77155	0	97	emb\|CAA35550.1\|hycE [Escherichia	PP
				coli]
372	77180	2.00E−62	61	gb\|ABA99663.1\|expressed protein	CS	PP	HS	PEG
				[Oryza sativa (japonica cultivar-
				group)]
373	77121	1.00E−166	93	dbj\|BAB05404.1\|transcriptional	CK
				repressor of the biotin operon
				[Bacillus halodurans C-125]
374	77157	0	100	ref\|NP_53I453.1\|3,4-dihydroxy-2-	CK	LL
				butanone-4-phoshate synthase/GTP
				cyclohydrolase II [Agrobacterium
				tumefaciens str. C58]
375	77195	0	94	gb\|AAM71555.1\|mannose-6-	PP	SS	PEG	LL
				phosphate isomerase/mannose-1-
				phosphate guanylyl transferase
				[Chlorobium tepidum TLS]
376	77124	1.00E−180	100	ref\|NP_418404.1\|biotin--protein	PP	SS	PEG
				ligase [Escherichia coli K12]
377	77261	0	100	ref\|NP_416982.1\|hydrogenase 4,	PEG
				subunit [Escherichia coli K12]
378	77273	0	93	dbj\|BAB06534.1\|glycine	PP	PEG
				dehydrogenase subunit 1 [Bacillus
				halodurans C-125]
379	77203	1.00E−176	60	dbj\|BAD33942.1\|putative serine	PEG	CK
				carboxypeptidase precursor [Oryza
				sativa (japonica cultivar-group)]
380	77275	4.00E−37	55	gb\|ABA91490.1\|expressed protein	SS	HS	PEG
				[Oryza sativa (japonica cultivar-
				group)]
381	77204	2.00E−27	44	ref\|XP_469963.1\|putative protease	CK	CS
				inhibitor [Oryza sativa (japonica
				cultivar-group)]
382	77266	7.00E−25	73	gb\|AAT93978.1\|unknown protein	CS	PP	PEG
				[Oryza sativa (japonica cultivar-
				group)]
383	77220	1.00E−169	78	dbj\|BAD52854.1\|putative non-	LL
				phototropic hypocotyl 3 [Oryza sativa
				(japonica cultivar-group)]
384	77268	4.00E−71	85	gb\|ABF95596.1\|ETC complex I	PP	SS
				subunit conserved region family
				protein, expressed [Oryza sativa
				(japonica cultivar-group)]
385	77209	9.00E−86	85	ref\|XP_479456.1\|putative 60S	PP	LL
				ribosome subunit biogenesis protein
				[Oryza sativa (japonica cultivar-
				group)]
386	77269	2.00E−56	70	dbj\|BAD32031.1\|unknown protein	SS
				[Oryza sativa (japonica cultivar-
				group)]
387	77451	0	71	ref\|XP_475231.1\|putative	PP	SS
				microtubule-associated protein [Oryza
				sativa (japonica cultivar-group)]
388	77452	1.00E−106	69	dbj\|BAD69045.1\|unknown protein	PP
				[Oryza sativa (japonica cultivar-
				group)]
389	77430	1.00E−160	83	dbj\|BAD33328.1\|putative protein	CS	PP	SS
				serine/threonine kinase [Oryza sativa
				(japonica cultivar-group)]
390	77432	0	86	ref\|NP_849565.1\|carbohydrate	PP	HS	PEG
				transporter/nucleoside transporter/
				sugar porter [Arabidopsis thaliana]
				gb\|AAM19835.1\|
				AT4g35300/F23E12_140
				[Arabidopsis thaliana]
391	77433	0	100	gb\|AAK59487.1\| putative cleavage	CK	PP
				and polyadenylation specificity factor
				[Arabidopsis thaliana]
392	77444	0	96	ref\|NP_199947.1\|unknown protein	CS	PP
				[Arabidopsis thaliana]
393	77409	0	91	ref\|NP_001032163.1\|unknown protein	PP
				[Arabidopsis thaliana]
394	12313	0	94	ref\|NP_189150.1\|QUA1	SP
				(QUASIMODO1); transferase,
				transferring glycosyl groups/
				transferase, transferring hexosyl
				groups [Arabidopsis thaliana]

Trait Improvement Screens

DS—Improvement of Drought Tolerance Identified by a Soil Drought Stress Tolerance Screen:
Drought or water deficit conditions impose mainly osmotic stress on plants. Plants are particularly vulnerable to drought during the flowering stage. The drought condition in the screening process disclosed in Example 1B started from the flowering time and was sustained to the end of harvesting. The present invention provides recombinant DNA that can improve the plant survival rate under such sustained drought condition. Exemplary recombinant DNA for conferring such drought tolerance are identified as such in Table 3. Such recombinant DNA may find particular use in generating transgenic plants that are tolerant to the drought condition imposed during flowering time and in other stages of the plant life cycle. As demonstrated from the model plant screen, in some embodiments of transgenic plants with trait-improving recombinant DNA grown under such sustained drought condition can also have increased total seed weight per plant in addition to the increased survival rate within a transgenic population, providing a higher yield potential as compared to control plants.
PEG-Improvement of Drought Tolerance Identified by PEG Induced Osmotic Stress Tolerance Screen:
Various drought levels can be artificially induced by using various concentrations of polyethylene glycol (PEG) to produce different osmotic potentials (Pilon-Smits e.g., (1995) Plant Physiol. 107:125-130). Several physiological characteristics have been reported as being reliable indications for selection of plants possessing drought tolerance. These characteristics include the rate of seed germination and seedling growth. The traits can be assayed relatively easily by measuring the growth rate of seedling in PEG solution. Thus, a PEG-induced osmotic stress tolerance screen is a useful surrogate for drought tolerance screen. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the PEG-induced osmotic stress tolerance screen can survive better drought conditions providing a higher yield potential as compared to control plants.
SS-Improvement of Drought Tolerance Identified by High Salinity Stress Tolerance Screen:
Three different factors are responsible for salt damages: (1) osmotic effects, (2) disturbances in the mineralization process, and (3) toxic effects caused by the salt ions, e.g., inactivation of enzymes. While the first factor of salt stress results in the wilting of the plants that is similar to drought effect, the ionic aspect of salt stress is clearly distinct from drought. The present invention provides genes that help plants maintain biomass, root growth, and/or plant development in high salinity conditions, which are identified as such in Table 3. Since osmotic effect is one of the major components of salt stress, which is common to the drought stress, trait-improving recombinant DNA identified in a high salinity stress tolerance screen can also provide transgenic crops with improved drought tolerance. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a high salinity stress tolerance screen can survive better drought conditions and/or high salinity conditions providing a higher yield potential as compared to control plants.
HS-Improvement of Drought Tolerance Identified by Heat Stress Tolerance Screen:
Heat and drought stress often occur simultaneously, limiting plant growth. Heat stress can cause the reduction in photosynthesis rate, inhibition of leaf growth and osmotic potential in plants. Thus, genes identified by the present invention as heat stress tolerance conferring genes may also impart improved drought tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a heat stress tolerance screen can survive better heat stress conditions and/or drought conditions providing a higher yield potential as compared to control plants.
CK and CS-Improvement of Tolerance to Cold Stress:
Low temperature may immediately result in mechanical constraints, changes in activities of macromolecules, and reduced osmotic potential. In the present invention, two screening conditions, i.e., cold shock tolerance screen (CK) and cold germination tolerance screen (CS), were set up to look for transgenic plants that display visual growth advantage at lower temperature. In cold germination tolerance screen, the transgenic Arabidopsis plants were exposed to a constant temperature of 8° C. from planting until day 28 post plating. The trait-improving recombinant DNA identified by such screen are particular useful for the production of transgenic plant that can germinate more robustly in a cold temperature as compared to the wild type plants. In cold shock tolerance screen, the transgenic plants were first grown under the normal growth temperature of 22° C. until day 8 post plating, and subsequently were placed under 8° C. until day 28 post plating. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a cold shock stress tolerance screen and/or a cold germination stress tolerance screen can survive better cold conditions providing a higher yield potential as compared to control plants.
Improvement of Tolerance to Multiple Stresses:
Different kinds of stresses often lead to identical or similar reaction in the plants. Genes that are activated or inactivated as a reaction to stress can either act directly in a way the genetic product reduces a specific stress, or they can act indirectly by activating other specific stress genes. By manipulating the activity of such regulatory genes, i.e., multiple stress tolerance genes, the plant can be enabled to react to different kinds of stresses. For examples, PEP SEQ ID NO: 231 can be used to improve both salt stress tolerance and cold stress tolerance in plants. Of particular interest, plants transformed with PEP SEQ ID NO: 233 can resist heat stress, salt stress and cold stress. In addition to these multiple stress tolerance genes, the stress tolerance conferring genes provided by the present invention may be used in combinations to generate transgenic plants that can resist multiple stress conditions.
PP-Improvement of Early Plant Growth and Development:
It has been known in the art that to minimize the impact of disease on crop profitability, it is important to start the season with healthy and vigorous plants. This means avoiding seed and seedling diseases, leading to increased nutrient uptake and increased yield potential. Traditionally early planting and applying fertilizer are the methods used for promoting early seedling vigor. In early development stage, plant embryos establish only the basic root-shoot axis, a cotyledon storage organ(s), and stem cell populations, called the root and shoot apical meristems, that continuously generate new organs throughout post-embryonic development. “Early growth and development” used herein encompasses the stages of seed imbibition through the early vegetative phase. The present invention provides genes that are useful to produce transgenic plants that have advantages in one or more processes including, but not limited to, germination, seedling vigor, root growth and root morphology under non-stressed conditions. The transgenic plants starting from a more robust seedling are less susceptible to the fungal and bacterial pathogens that attach germinating seeds and seedling. Furthermore, seedlings with advantage in root growth are more resistant to drought stress due to extensive and deeper root architecture. Therefore, it can be recognized by those skilled in the art that genes conferring the growth advantage in early stages to plants may also be used to generate transgenic plants that are more resistant to various stress conditions due to improved early plant development. The present invention provides such exemplary recombinant DNA that confer both the stress tolerance and growth advantages to plants, identified as such in Table 3, e.g., PEP SEQ ID NO: 268 which can improve the plant early growth and development, and impart heat and cold tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the early plant development screen can grow better under non-stress conditions and/or stress conditions providing a higher yield potential as compared to control plants.
SP-Improvement of Late Plant Growth and Development:
“Late growth and development” used herein encompasses the stages of leaf development, flower production, and seed maturity. In certain embodiments, transgenic plants produced using genes that confer growth advantages to plants provided by the present invention, identified as such in Table 3, exhibit at least one phenotypic characteristics including, but not limited to, increased rosette radius, increased rosette dry weight, seed dry weight, silique dry weight, and silique length. On one hand, the rosette radius and rosette dry weight are used as the indexes of photosynthesis capacity, and thereby plant source strength and yield potential of a plant. On the other hand, the seed dry weight, silique dry weight and silique length are used as the indexes for plant sink strength, which are considered as the direct determinants of yield. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the late development screen can grow better and/or have improved development during leaf development and seed maturation providing a higher yield potential as compared to control plants.
LL-Improvement of Tolerance to Shade Stress Identified in a Low Light Screen:
The effects of light on plant development are especially prominent at the seedling stage. Under normal light conditions with unobstructed direct light, a plant seeding develops according to a characteristic photomorphogenic pattern, in which plants have open and expanded cotyledons and short hypocotyls. Then the plant's energy is devoted to cotyledon and leaf development while longitudinal extension growth is minimized. Under low light condition where light quality and intensity are reduced by shading, obstruction or high population density, a seedling displays a shade-avoidance pattern, in which the seedling displays a reduced cotyledon expansion, and hypocotyls extension is greatly increased. As the result, a plant under low light condition increases significantly its stem length at the expanse of leaf, seed or fruit and storage organ development, thereby adversely affecting of yield. The present invention provides recombinant DNA that enable plants to have an attenuated shade avoidance response so that the source of plant can be contributed to reproductive growth efficiently, resulting higher yield as compared to the wild type plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a shade stress tolerance screen can have attenuated shade response under shade conditions providing a higher yield potential as compared to control plants. The transgenic plants generated by the present invention may be suitable for a higher density planting, thereby resulting increased yield per unit area.
LN-Improvement of Tolerance to Low Nitrogen Availability Stress
Nitrogen is a key factor in plant growth and crop yield. The metabolism, growth and development of plants are profoundly affected by their nitrogen supply. Restricted nitrogen supply alters shoot to root ratio, root development, activity of enzymes of primary metabolism and the rate of senescence (death) of older leaves. All field crops have a fundamental dependence on inorganic nitrogenous fertilizer. Since fertilizer is rapidly depleted from most soil types, it must be supplied to growing crops two or three times during the growing season. Enhanced nitrogen use efficiency by plants should enable crops cultivated under low nitrogen availability stress condition resulted from low fertilizer input or poor soil quality.
According to the present invention, transgenic plants generated using the recombinant nucleotides, which confer enhanced nitrogen use efficiency, identified as such in Table 3, exhibit one or more desirable traits including, but not limited to, increased seedling weight, greener leaves, increased number of rosette leaves, increased or decreased root length. One skilled in the art may recognize that the transgenic plants provided by the present invention with enhanced nitrogen use efficiency may also have altered amino acid or protein compositions, increased yield and/or better seed quality. The transgenic plants of the present invention may be productively cultivated under low nitrogen growth conditions, i.e., nitrogen-poor soils and low nitrogen fertilizer inputs, which would cause the growth of wild type plants to cease or to be so diminished as to make the wild type plants practically useless. The transgenic plants also may be advantageously used to achieve earlier maturing, faster growing, and/or higher yielding crops and/or produce more nutritious foods and animal feedstocks when cultivated using nitrogen non-limiting growth conditions.
Stacked Traits:
The present invention also encompasses transgenic plants with stacked engineered traits, e.g., a crop having an improved phenotype resulting from expression of a trait-improving recombinant DNA, in combination with herbicide and/or pest resistance traits. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, for example a RoundUp Ready® trait, or insect resistance, such as using a gene from Bacillus thuringensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. Herbicides for which resistance is useful in a plant include glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and gluphosinate herbicides. To illustrate that the production of transgenic plants with herbicide resistance is a capability of those of ordinary skill in the art, reference is made to U.S. patent application publications 2003/0106096A1 and 2002/0112260A1 and U.S. Pat. Nos. 5,034,322; 5,776,760, 6,107,549 and 6,376,754, all of which are incorporated herein by reference. To illustrate that the production of transgenic plants with pest resistance is a capability of those of ordinary skill in the art reference is made to U.S. Pat. Nos. 5,250,515 and 5,880,275 which disclose plants expressing an endotoxin of Bacillus thuringiensis bacteria, to U.S. Pat. No. 6,506,599 which discloses control of invertebrates which feed on transgenic plants which express dsRNA for suppressing a target gene in the invertebrate, to U.S. Pat. No. 5,986,175 which discloses the control of viral pests by transgenic plants which express viral replicase, and to U.S. Patent Application Publication 2003/0150017 A1 which discloses control of pests by a transgenic plant which express a dsRNA targeted to suppressing a gene in the pest, all of which are incorporated herein by reference.
Once one recombinant DNA has been identified as conferring an improved trait of interest in transgenic Arabidopsis plants, several methods are available for using the sequence of that recombinant DNA and knowledge about the protein it encodes to identify homologs of that sequence from the same plant or different plant species or other organisms, e.g., bacteria and yeast. Thus, in one aspect, the invention provides methods for identifying a homologous gene with a DNA sequence homologous to any of SEQ ID NO: 1 through SEQ ID NO: 197, or a homologous protein with an amino acid sequence homologous to any of SEQ ID NO: 198 through SEQ ID NO: 394. In another aspect, the present invention provides the protein sequences of identified homologs for a sequence listed as SEQ ID NO: 395 through SEQ ID NO: 19938. In yet another aspect, the present invention also includes linking or associating one or more desired traits, or gene function with a homolog sequence provided herein.
The trait-improving recombinant DNA and methods of using such trait-improving recombinant DNA for generating transgenic plants with improved traits provided by the present invention are not limited to any particular plant species. Indeed, the plants according to the present invention may be of any plant species, i.e., may be monocotyledonous or dicotyledonous. Preferably, they will be agricultural useful plants, i.e., plants cultivated by man for purposes of food production or technical, particularly industrial applications. Of particular interest in the present invention are corn and soybean plants. The recombinant DNA constructs optimized for soybean transformation and recombinant DNA constructs optimized for corn transformation are provided by the present invention. Other plants of interest in the present invention for production of transgenic plants having improved traits include, without limitation, cotton, canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
In certain embodiments, the present invention contemplates to use an orthologous gene in generating the transgenic plants with similarly improved traits as the transgenic Arabidopsis counterpart. Improved physiological properties in transgenic plants of the present invention may be confirmed in responses to stress conditions, for example in assays using imposed stress conditions to detect improved responses to drought stress, nitrogen deficiency, cold growing conditions, or alternatively, under naturally present stress conditions, for example under field conditions. Biomass measures may be made on greenhouse or field grown plants and may include such measurements as plant height, stem diameter, root and shoot dry weights, and, for corn plants, ear length and diameter.
Trait data on morphological changes may be collected by visual observation during the process of plant regeneration as well as in regenerated plants transferred to soil. Such trait data includes characteristics such as normal plants, bushy plants, taller plants, thicker stalks, narrow leaves, striped leaves, knotted phenotype, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other enhanced traits may be identified by measurements taken under field conditions, such as days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, and pest resistance. In addition, trait characteristics of harvested grain may be confirmed, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.
To confirm hybrid yield in transgenic corn plants expressing genes of the present invention, it may be desirable to test hybrids over multiple years at multiple locations in a geographical location where maize is conventionally grown, e.g., in Iowa, Illinois or other locations in the midwestern United States, under “normal” field conditions as well as under stress conditions, e.g., under drought or population density stress.
Transgenic plants can be used to provide plant parts according to the invention for regeneration or tissue culture of cells or tissues containing the constructs described herein. Plant parts for these purposes can include leaves, stems, roots, flowers, tissues, epicotyl, meristems, hypocotyls, cotyledons, pollen, ovaries, cells and protoplasts, or any other portion of the plant which can be used to regenerate additional transgenic plants, cells, protoplasts or tissue culture. Seeds of transgenic plants are provided by this invention can be used to propagate more plants containing the trait-improving recombinant DNA constructs of this invention. These descendants are intended to be included in the scope of this invention if they contain a trait-improving recombinant DNA construct of this invention, whether or not these plants are selfed or crossed with different varieties of plants.
The various aspects of the invention are illustrated by means of the following examples which are in no way intended to limit the full breath and scope of claims.

Examples

Example 1. Identification of Recombinant DNA that Confers Improved Trait(s) to Plants

A. Plant Expression Constructs for Arabidopsis Transformation

Each gene of interest was amplified from a genomic or cDNA library using primers specific to sequences upstream and downstream of the coding region. Transformation vectors were prepared to constitutively transcribe DNA in either sense orientation (for enhanced protein expression) or anti-sense orientation (for endogenous gene suppression) under the control of an enhanced Cauliflower Mosaic Virus 35S promoter (U.S. Pat. No. 5,359,142) directly or indirectly (Moore, e.g., PNAS 95:376-381, 1998; Guyer, e.g., Genetics 149: 633-639, 1998; International patent application NO. PCT/EP98/07577). The transformation vectors also contain a bar gene as a selectable marker for resistance to glufosinate herbicide. The transformation of Arabidopsis plants was carried out using the vacuum infiltration method known in the art (Bethtold, e.g., Methods Mol. Biol. 82:259-66, 1998). Seeds harvested from the plants, named as T1 seeds, were subsequently grown in a glufosinate-containing selective medium to select for plants which were actually transformed and which produced T2 transgenic seed.

B. Soil Drought Tolerance Screen

This example describes a soil drought tolerance screen to identify Arabidopsis plants transformed with recombinant DNA that wilt less rapidly and/or produce higher seed yield when grown in soil under drought conditions
T2 seeds were sown in flats filled with Metro/Mix® 200 (The Scotts® Company, USA). Humidity domes were added to each flat and flats were assigned locations and placed in climate-controlled growth chambers. Plants were grown under a temperature regime of 22° C. at day and 20° C. at night, with a photoperiod of 16 hours and average light intensity of 170 μmol/m²/s. After the first true leaves appeared, humidity domes were removed. The plants were sprayed with glufosinate herbicide and put back in the growth chamber for 3 additional days. Flats were watered for 1 hour the week following the herbicide treatment. Watering was continued every seven days until the flower bud primordia became apparent, at which time plants were watered for the last time.
To identify drought tolerant plants, plants were evaluated for wilting response and seed yield. Beginning ten days after the last watering, plants were examined daily until 4 plants/line had wilted. In the next six days, plants were monitored for wilting response. Five drought scores were assigned according to the visual inspection of the phenotypes: 1 for healthy, 2 for dark green, 3 for wilting, 4 severe wilting, and 5 for dead. A score of 3 or higher was considered as wilted.
At the end of this assay, seed yield measured as seed weight per plant under the drought condition was characterized for the transgenic plants and their controls and analyzed as a quantitative response according to example 1M.
Two approaches were used for statistical analysis on the wilting response. First, the risk score was analyzed for wilting phenotype and treated as a qualitative response according to the example 1L. Alternatively, the survival analysis was carried out in which the proportions of wilted and non-wilted transgenic and control plants were compared over each of the six days under scoring and an overall log rank test was performed to compare the two survival curves using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). A list of recombinant DNA constructs which improve drought tolerance in transgenic plants is illustrated in Table 4.

TABLE 4

PEP	Drought	Time to wilting

SEQ

score

Seed yield

Risk

ID	Construct	Nomination		Delta	P-	Delta	P-	score	P-
NO	ID	ID	Orientation	mean	value	mean	value	mean	value

198	11029	CGPG106	SENSE	0.109	0.381	0.072	0.717	0.119	1.000
292	73136	CGPG5764	SENSE	0.030	0.090	0.136	0.067	0.104	1.000
284	73165	CGPG5661	SENSE	−0.031	0.504	0.526	0.018	−0.075	1.000
260	73628	CGPG5025	SENSE	0.342	0.038	−0.474	0.022	0.208	1.000
291	73981	CGPG5757	SENSE	0.517	0.026	−0.096	0.000	0.573	1.000
207	74065	CGPG1828	ANTI-	−0.078	0.164	0.646	0.027	0.048	1.000
			SENSE
263	74217	CGPG5144	SENSE	−0.026	0.414	0.418	0.032	0.071	1.000
261	76105	CGPG5041	SENSE	0.288	0.029	−1.780	0.027	0.173	1.000
328	76576	CGPG7281	SENSE	0.241	0.032	−0.555	0.442	0.212	1.000
327	77536	CGPG7272	SENSE	0.089	0.194	1.059	0.001	0.042	1.000
226	19767	CGPG3918	SENSE	0.117	0.038	0.164	0.296	/	/
237	19947	CGPG4069	SENSE	−0.009	0.852	−0.104	0.504	/	/
242	70994	CGPG4122	SENSE	0.040	0.021	−0.131	0.447	/	/
363	77103	CGPG9134	SENSE	0.145	0.023	−0.499	0.096	/	/

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference (p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance) If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 226, 237, 242, or 363 showed improved drought tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.

C. Heat Stress Tolerance Screen

Under high temperatures, Arabidopsis seedlings become chlorotic and root growth is inhibited. This example sets forth the heat stress tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are more resistant to heat stress based on primarily their seedling weight and root growth under high temperature.
T2 seeds were plated on ½×MS salts, 1/% phytagel, with 10 μg/ml BASTA (7 per plate with 2 control seeds; 9 seeds total per plate). Plates were placed at 4° C. for 3 days to stratify seeds. Plates were then incubated at room temperature for 3 hours and then held vertically for 11 additional days at temperature of 34° C. at day and 20° C. at night. Photoperiod was 16 h. Average light intensity was ˜140 μmol/m²/s. After 14 days of growth, plants were scored for glufosinate resistance, root length, final growth stage, visual color, and seedling fresh weight. A photograph of the whole plate was taken on day 14.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final grow stage at day 14 was scored as success if 50% of the plants had reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, e.g., (2001) The Plant Cell 13, 1499-1510). The growth stage data was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve heat tolerance in transgenic plants illustrated in Table 5.

TABLE 5

	Root length	Growth stage	Seedling weight
PEP	at day 14	at day 14	at day 14

SEQ	Construct	Nomination		Delta		Risk score		Delta
ID	ID	ID	Orientation	mean	P-value	mean	P-value	mean	P-value

239	70985	CGPG4088	SENSE	0.480	0.036	1.176	0.196	1.333	0.018
233	70987	CGPG4048	SENSE	0.228	0.021	−0.036	0.670	1.214	0.002
268	72001	CGPG5221	SENSE	0.273	0.004	0.210	0.330	1.393	0.009
274	72753	CGPG5540	SENSE	0.434	0.006	0.414	0.258	1.100	0.006
276	73954	CGPG5577	SENSE	0.406	0.002	0.455	0.282	1.274	0.000
325	76178	CGPG7225	SENSE	0.714	0.001	1.093	0.145	1.537	0.000
361	77150	CGPG9130	SENSE	0.219	0.032	0.178	0.237	1.159	0.002
367	77164	CGPG9147	SENSE	0.317	0.026	0.651	0.099	1.152	0.002
390	77432	CGPG9335	SENSE	0.581	0.001	0.457	0.124	1.551	0.000
351	77587	CGPG8107	SENSE	0.488	0.006	0.628	0.072	1.328	0.001
348	77928	CGPG8082	SENSE	0.462	0.032	0.191	0.301	1.498	0.004
212	16610	CGPG2499	SENSE	0.055	0.640	0.109	0.609	0.983	0.020
220	18256	CGPG3363	SENSE	0.070	0.138	0.071	0.541	1.021	0.004
222	19193	CGPG3375	SENSE	0.255	0.094	0.267	0.368	1.023	0.003
252	70684	CGPG4588	SENSE	0.010	0.781	0.003	0.979	1.002	0.045
230	70948	CGPG3990	SENSE	0.282	0.095	0.351	0.335	1.045	0.023
243	70956	CGPG4140	SENSE	0.155	0.313	0.091	0.320	1.129	0.018
235	70962	CGPG4057	SENSE	0.469	0.085	0.267	0.508	1.410	0.007
241	70963	CGPG4121	SENSE	−0.002	0.979	−0.131	0.311	0.976	0.005
242	70994	CGPG4122	SENSE	0.140	0.574	1.204	0.096	1.207	0.030
244	70995	CGPG4154	SENSE	0.374	0.112	1.640	0.171	1.328	0.013
208	72783	CGPG2206	SENSE	0.387	0.057	0.053	0.509	1.013	0.019
255	72807	CGPG4912	SENSE	0.357	0.071	0.443	0.020	1.099	0.009
259	72825	CGPG5001	SENSE	0.171	0.349	0.213	0.234	1.118	0.012
287	73120	CGPG5704	SENSE	0.096	0.490	0.193	0.364	1.080	0.033
280	73127	CGPG5640	SENSE	−0.220	0.077	−0.194	0.030	0.643	0.014
293	73172	CGPG5783	SENSE	0.283	0.197	0.887	0.162	1.154	0.007
310	73411	CGPG6440	SENSE	0.198	0.389	0.631	0.345	1.320	0.003
260	73628	CGPG5025	SENSE	0.406	0.149	1.475	0.365	1.169	0.016
316	74566	CGPG6796	SENSE	0.627	0.092	0.777	0.466	1.714	0.014
338	75654	CGPG7804	SENSE	0.292	0.237	0.804	0.315	0.940	0.017
340	75657	CGPG7828	SENSE	0.390	0.067	1.222	0.307	1.521	0.011
213	76602	CGPG2653	SENSE	0.036	0.748	0.092	0.670	0.752	0.025
372	77180	CGPG9180	SENSE	0.068	0.555	−0.045	0.177	0.871	0.022
380	77275	CGPG9236	SENSE	0.282	0.125	0.971	0.285	1.027	0.013
297	77312	CGPG5927	SENSE	0.201	0.344	0.710	0.372	1.039	0.032
320	77518	CGPG6953	SENSE	0.238	0.344	0.069	0.655	1.188	0.015
342	77549	CGPG7933	SENSE	0.184	0.180	−0.012	/	0.978	0.007
347	77580	CGPG8062	SENSE	0.157	0.143	−0.074	/	0.891	0.049
317	77610	CGPG6805	SENSE	0.023	0.937	−0.063	/	1.207	0.029
318	77618	CGPG6810	SENSE	0.002	0.942	−0.074	/	1.015	0.006
353	77619	CGPG8152	SENSE	0.213	0.316	−0.076	0.457	0.956	0.019
355	77629	CGPG8377	SENSE	0.095	0.124	−0.063	/	1.133	0.005
334	77816	CGPG7529	SENSE	0.033	0.705	0.856	0.385	0.611	0.038

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 237, 307, 313, 327, 330, 349, 366, or 387 showed improved heat stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.

D. Salt Stress Tolerance Screen

This example sets forth the high salinity stress screen to identify Arabidopsis plants transformed with the gene of interest that are tolerant to high levels of salt based on their rate of development, root growth and chlorophyll accumulation under high salt conditions.
T2 seeds were plated on glufosinate selection plates containing 90 mM NaCl and grown under standard light and temperature conditions. All seedlings used in the experiment were grown at a temperature of 22° C. at day and 20° C. at night, a 16-hour photoperiod, an average light intensity of approximately 120 umol/m². On day 11, plants were measured for primary root length. After 3 more days of growth (day 14), plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was also taken on day 14.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success if 50% of the plants reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, D. C., et al., (2001), The Plant Cell 13, 1499/1510). The growth stage data was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve high salinity tolerance in transgenic plants illustrated in Table 6.

TABLE 6

	Root	Root	Growth	Seedling
	length at	length	stage at	weight at
PEP	day 11	at day 14	day 14	day 14

SEQ	Construct		Delta	P-	Delta	P-	Delta	P-	Delta	P-
ID	ID	Orientation	mean	value	mean	value	mean	value	mean	value

220	18256	SENSE	0.192	0.232	0.236	0.080	1.291	0.151	0.598	0.039
214	18456	SENSE	0.213	0.089	0.209	0.197	2.062	0.197	0.476	0.046
232	19973	SENSE	0.289	0.012	0.299	0.002	0.976	0.054	1.002	0.013
231	70201	SENSE	0.363	0.030	0.403	0.052	2.083	0.229	1.057	0.014
243	70956	SENSE	0.006	0.923	0.128	0.016	−0.045	NA	−0.364	0.371
238	70969	SENSE	0.435	0.091	0.471	0.027	0.653	0.473	0.994	0.058
240	70971	SENSE	0.337	0.063	0.257	0.041	1.418	0.226	0.593	0.030
233	70987	SENSE	0.363	0.030	0.284	0.044	1.434	0.380	0.865	0.012
273	72788	SENSE	0.484	0.019	0.456	0.016	1.813	0.314	0.929	0.008
256	72811	SENSE	0.385	0.024	0.409	0.002	2.968	0.031	0.805	0.004
287	73120	SENSE	0.341	0.001	0.244	0.019	0.613	0.103	0.755	0.015
280	73127	SENSE	0.501	0.028	0.395	0.055	0.212	0.175	0.739	0.023
289	73134	SENSE	0.226	0.134	0.266	0.045	0.152	0.487	0.378	0.056
257	73235	SENSE	0.431	0.020	0.360	0.018	1.618	0.327	0.650	0.009
245	73306	SENSE	0.228	0.046	0.219	0.018	0.249	0.217	0.708	0.008
253	73330	SENSE	0.196	0.225	0.186	0.029	0.000	NA	0.543	0.045
316	74566	SENSE	0.331	0.001	0.076	0.422	0.597	0.031	0.716	0.003
312	74688	SENSE	0.480	0.076	0.415	0.031	0.000	NA	0.808	0.062
376	77124	SENSE	0.340	0.034	0.322	0.015	2.306	0.137	0.411	0.168
364	77139	SENSE	0.339	0.096	0.287	0.041	1.683	0.294	0.602	0.039
375	77195	SENSE	0.445	0.009	0.345	0.010	0.367	0.185	0.582	0.022
384	77268	SENSE	0.495	0.047	0.468	0.102	1.906	0.079	0.992	0.042
386	77269	SENSE	0.454	0.038	0.426	0.004	1.996	0.065	1.136	0.001
380	77275	SENSE	0.457	0.008	0.487	0.040	2.119	0.154	1.070	0.010
297	77312	SENSE	0.335	0.086	0.361	0.013	0.960	0.208	0.723	0.063
389	77430	SENSE	0.303	0.030	0.360	0.004	0.131	0.495	0.894	0.022
320	77518	SENSE	0.171	0.017	0.185	0.026	−0.134	0.443	0.339	0.109
345	77570	SENSE	0.326	0.091	0.241	0.134	1.396	0.090	0.862	0.026
351	77587	SENSE	0.178	0.083	0.096	0.665	0.995	0.426	0.632	0.036
353	77619	SENSE	0.389	0.058	0.398	0.011	1.913	0.257	0.857	0.040
355	77629	SENSE	0.404	0.043	0.389	0.019	2.523	0.083	0.826	0.040
336	77821	SENSE	0.393	0.063	0.444	0.033	0.196	0.228	0.619	0.015
356	77832	SENSE	0.134	0.238	0.304	0.011	0.914	0.156	0.413	0.050
352	77933	SENSE	0.112	0.044	0.165	0.009	0.749	0.482	0.517	0.171
347	77580	SENSE	−0.019	0.814	0.087	0.018	−0.158	0.151	0.124	0.204

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 237, 242, 255, 307, 313, 327, 330, 349, 366, or 387 showed improved salt stress tolerance evidenced by the second criteria as illustrated in Example 1L.

E. Polyethylene Glycol (PEG) Induced Osmotic Stress Tolerance Screen

There are numerous factors, which can influence seed germination and subsequent seedling growth, one being the availability of water. Genes, which can directly affect the success rate of germination and early seedling growth, are potentially useful agronomic traits for improving the germination and growth of crop plants under drought stress. In this assay, PEG was used to induce osmotic stress on germinating transgenic lines of Arabidopsis thaliana seeds in order to screen for osmotically resistant seed lines.
T2 seeds were plated on BASTA selection plates containing 3% PEG and grown under standard light and temperature conditions. Seeds were plated on each plate containing 3% PEG, ½×MS salts, 1% phytagel, and 10 μg/ml glufosinate. Plates were placed at 4° C. for 3 days to stratify seeds. On day 11, plants were measured for primary root length. After 3 more days of growth, i.e., at day 14, plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was taken on day 14.
Seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success or failure based on whether the plants reached 3 rosette leaves and size of leaves are greater than 1 mm. The growth stage data was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve osmotic stress tolerance in transgenic plants illustrated in Table 7.

TABLE 7

	Root	Root	Growth	Seedling
	length at	length at	stage at	weight at
PEP	day 11	day 14	day 14	day 14

SEQ	Construct		Delta	P-	Delta	P-	Delta	P-	Delta	P-
ID	ID	Orientation	mean	value	mean	value	mean	value	mean	value

199	12223	SENSE	/	/	/	/	/	/	0.353	0.024
214	18456	SENSE	0.320	0.018	0.274	0.033	3.109	0.073	0.553	0.006
302	70812	SENSE	0.195	0.078	0.078	0.296	4	0.000	0.363	0.016
234	70950	SENSE	0.322	0.056	0.427	0.016	4	0.000	0.381	0.127
239	70985	SENSE	0.270	0.066	0.221	0.005	4	0.000	0.372	0.007
233	70987	SENSE	0.227	0.205	0.128	0.470	4	0.000	0.495	0.000
228	70992	SENSE	0.369	0.002	0.370	0.013	4	0.000	0.574	0.027
244	70995	SENSE	0.379	0.049	0.320	0.065	4	0.000	0.491	0.003
208	72783	SENSE	0.154	0.084	0.043	0.553	4	0.000	0.264	0.044
255	72807	SENSE	0.165	0.080	0.158	0.150	4	0.000	0.333	0.047
256	72811	SENSE	0.489	0.052	0.491	0.050	4	0.000	0.784	0.016
259	72825	SENSE	0.423	0.055	0.388	0.080	2.741	0.161	0.608	0.020
295	72946	SENSE	0.345	0.053	0.473	0.050	0.898	0.632	0.415	0.181
286	73026	SENSE	0.636	0.021	0.619	0.028	2.741	0.161	0.752	0.045
290	73123	SENSE	0.460	0.048	0.445	0.007	NA	NA	0.609	0.069
278	73161	SENSE	0.510	0.024	0.506	0.006	2.624	0.246	0.626	0.073
284	73165	SENSE	0.304	0.101	0.285	0.044	2.499	0.270	0.643	0.077
293	73172	SENSE	0.197	0.056	0.137	0.204	2.374	0.293	0.660	0.081
262	73242	SENSE	0.305	0.029	0.332	0.022	2.249	0.317	0.677	0.085
265	73256	SENSE	0.325	0.084	0.182	0.178	2.124	0.341	0.694	0.089
308	73485	SENSE	0.133	0.070	0.039	0.583	1.999	0.365	0.711	0.093
291	73981	SENSE	0.239	0.147	0.238	0.281	1.874	0.389	0.728	0.096
316	74566	SENSE	0.451	0.004	0.248	0.118	1.749	0.412	0.745	0.100
312	74688	SENSE	0.190	0.191	0.102	0.520	1.624	0.436	0.762	0.104
330	74863	SENSE	0.174	0.164	0.216	0.109	1.499	0.460	0.779	0.108
366	77140	SENSE	0.644	0.005	0.572	0.015	1.374	0.484	0.796	0.112
361	77150	SENSE	0.313	0.047	0.359	0.044	1.249	0.508	0.813	0.116
365	77187	SENSE	0.273	0.084	0.032	0.763	1.124	0.532	0.830	0.120
375	77195	SENSE	0.319	0.021	0.256	0.041	NA	NA	0.847	0.124
379	77203	SENSE	0.113	0.485	0.019	0.829	0.999	0.555	0.864	0.127
377	77261	SENSE	0.115	0.230	0.085	0.315	0.874	0.579	0.881	0.131
270	77308	SENSE	0.174	0.321	0.074	0.700	0.749	0.603	0.898	0.135
346	77338	SENSE	0.227	0.158	0.196	0.272	0.624	0.627	0.915	0.139
350	77357	SENSE	0.169	0.300	0.259	0.066	2.715	0.169	0.303	0.020
390	77432	SENSE	0.306	0.043	0.122	0.270	2.57	0.214	0.734	0.019
347	77580	SENSE	0.300	0.107	0.240	0.009	2.288	0.119	0.500	0.179
353	77619	SENSE	0.142	0.216	0.139	0.002	4	0.000	0.129	0.582
356	77832	SENSE	0.236	0.006	0.266	0.001	1.333	0.435	0.286	0.037
343	77917	SENSE	0.174	0.163	0.160	0.006	4	0.000	0.330	0.009

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference.
If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 209, 245, 260, 274, 282, 310, 321, 325, 336, 372, 376, 378, 380, or 382 showed improved osmotic stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.

F. Cold Shock Tolerance Screen

This example set forth a screen to identify Arabidopsis plants transformed with the genes of interest that are more tolerant to cold stress subjected during day 8 to day 28 after seed planting. During these crucial early stages, seedling growth and leaf area increase were measured to assess tolerance when Arabidopsis seedlings were exposed to low temperatures. Using this screen, genetic alterations can be found that enable plants to germinate and grow better than wild type plants under sudden exposure to low temperatures.
Eleven seedlings from T2 seeds of each transgenic line plus one control line were plated together on a plate containing ½×Gamborg Salts with 0.8 Phytagel™, 1% Phytagel, and 0.3% Sucrose. Plates were then oriented horizontally and stratified for three days at 4° C. At day three, plates were removed from stratification and exposed to standard conditions (16 hr photoperiod, 22° C. at day and 20° C. at night) until day 8. At day eight, plates were removed from standard conditions and exposed to cold shock conditions (24 hr photoperiod, 8° C. at both day and night) until the final day of the assay, i.e., day 28. Rosette areas were measured at day 8 and day 28, which were analyzed as quantitative responses according to example 1M. A list of recombinant nucleotides that improve cold shock stress tolerance in plants illustrated in Table 8.

TABLE 8

Rosette area	Rosette area	Rosette area
at day 8	at day 28	difference

PEP	Construct	Nomination		Delta	P-	Risk score	P-	Delta	P-
SEQ ID	ID	ID	Orientation	mean	value	mean	value	mean	value

199	12223	CGPG1133	SENSE	0.112	0.617	0.334	0.014	0.273	0.143
218	18231	CGPG3274	SENSE	−0.131	0.269	1.144	0.002	1.179	0.003
215	18414	CGPG3002	SENSE	0.997	0.034	1.665	0.001	1.895	0.001
214	18456	CGPG2813	SENSE	0.099	0.757	0.799	0.001	0.684	0.001
233	70987	CGPG4048	SENSE	0.864	0.136	1.213	0.010	1.146	0.002
244	70995	CGPG4154	SENSE	1.088	0.008	1.262	0.023	1.407	0.025
208	72783	CGPG2206	SENSE	0.814	0.023	0.957	0.018	0.899	0.052
273	72788	CGPG5535	SENSE	0.262	0.058	1.090	0.016	1.212	0.021
256	72811	CGPG4926	SENSE	0.880	0.058	0.669	0.002	0.715	0.018
259	72825	CGPG5001	SENSE	0.032	0.887	0.338	0.071	0.357	0.027
281	73033	CGPG5646	SENSE	0.322	0.394	1.311	0.012	1.480	0.017
293	73172	CGPG5783	SENSE	0.395	0.209	1.065	0.030	1.099	0.074
253	73330	CGPG4765	SENSE	0.347	0.515	0.835	0.065	1.043	0.007
264	73735	CGPG5171	SENSE	0.624	0.076	0.655	0.001	0.718	0.005
315	74460	CGPG6747	SENSE	0.452	0.005	0.542	0.043	0.455	0.154
296	74746	CGPG5856	SENSE	0.304	0.346	0.978	0.001	1.061	0.007
299	75240	CGPG5957	SENSE	0.479	0.153	0.652	0.143	0.875	0.039
338	75654	CGPG7804	SENSE	0.531	0.140	1.712	0.010	1.979	0.015
339	75692	CGPG7823	SENSE	0.526	0.063	2.027	0.001	2.246	0.001
323	76161	CGPG7168	SENSE	0.768	0.000	1.810	0.004	2.146	0.003
217	76532	CGPG3235	SENSE	0.921	0.008	1.216	0.001	1.324	0.002
357	76802	CGPG8987	SENSE	0.468	0.097	1.578	0.000	1.872	0.000
358	76829	CGPG9013	SENSE	−0.434	0.329	0.709	0.010	0.314	0.118
373	77121	CGPG9183	SENSE	0.081	0.385	0.757	0.023	0.698	0.014
366	77140	CGPG9145	SENSE	−0.221	0.165	0.896	0.007	1.043	0.005
374	77157	CGPG9186	SENSE	0.190	0.566	1.110	0.016	1.176	0.015
369	77165	CGPG9155	SENSE	1.573	0.043	0.683	0.038	0.699	0.022
370	77166	CGPG9163	SENSE	0.777	0.011	1.432	0.016	1.570	0.025
381	77204	CGPG9238	SENSE	0.545	0.139	1.313	0.003	1.436	0.006
391	77433	CGPG9341	SENSE	1.054	0.103	0.895	0.027	0.659	0.084
345	77570	CGPG8015	SENSE	0.538	0.215	0.866	0.025	0.898	0.023
353	77619	CGPG8152	SENSE	−0.589	0.322	0.503	0.068	0.558	0.049
354	77621	CGPG8166	SENSE	0.053	0.863	0.716	0.044	0.792	0.040
203	13411	CGPG1301	SENSE	0.183	0.427	0.577	0.033	0.573	0.031
209	17210	CGPG2217	SENSE	−0.244	0.685	0.162	0.038	0.156	0.104
220	18256	CGPG3363	SENSE	0.272	0.400	0.639	0.008	0.719	0.020
289	73134	CGPG5721	SENSE	0.014	0.947	1.053	0.000	1.062	0.025
263	74217	CGPG5144	SENSE	0.168	0.390	0.261	0.043	0.206	0.075
379	77203	CGPG9230	SENSE	0.606	0.051	0.620	0.032	0.778	0.039

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference (p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance) If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 255, 272, or 330 showed improved cold stress tolerance evidenced by the second criterial as illustrated in Example 1L.

G. Cold Germination Tolerance Screen

This example sets forth a screen to identify Arabidopsis plants transformed with the genes of interests are resistant to cold stress based on their rate of development, root growth and chlorophyll accumulation under low temperature conditions.
T2 seeds were plated and all seedlings used in the experiment were grown at 8° C. Seeds were first surface disinfested using chlorine gas and then seeded on assay plates containing an aqueous solution of ½×Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo., USA G/5788), 1% Phytagel™ (Sigma-Aldrich, P-8169), and 10 ug/ml glufosinate with the final pH adjusted to 5.8 using KOH. Test plates were held vertically for 28 days at a constant temperature of 8° C., a photoperiod of 16 hr, and average light intensity of approximately 100 umol/m²/s. At 28 days post plating, root length was measured, growth stage was observed, the visual color was assessed, and a whole plate photograph was taken.
The root length at day 28 was analyzed as a quantitative response according to example 1M. The growth stage at day 7 was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve cold stress tolerance in transgenic plants illustrated in Table 9.

	TABLE 9

	Root length	Growth stage at
	at day 28	day 28

PEP	Construct	Nomination		Delta		Delta
SEQ ID	ID	ID	Orientation	mean	P-value	mean	P-value

209	17210	CGPG2217	SENSE	0.164	0.089	4.000	0.000
219	18232	CGPG3275	SENSE	−0.081	0.613	4.000	0.000
226	19767	CGPG3918	SENSE	0.212	0.007	4.000	0.000
227	19774	CGPG3920	SENSE	0.140	0.011	4.000	0.000
237	19947	CGPG4069	SENSE	/	/	4.000	0.000
231	70201	CGPG3994	SENSE	0.311	0.010	4.000	0.000
252	70684	CGPG4588	SENSE	0.335	0.209	4.000	0.000
236	70915	CGPG4058	SENSE	0.231	0.258	4.000	0.000
243	70956	CGPG4140	SENSE	0.113	0.525	4.000	0.000
241	70963	CGPG4121	SENSE	0.210	0.252	4.000	0.000
239	70985	CGPG4088	SENSE	0.283	0.063	4.000	0.000
233	70987	CGPG4048	SENSE	0.392	0.202	4.000	0.000
228	70992	CGPG3962	SENSE	0.008	0.959	4.000	0.000
244	70995	CGPG4154	SENSE	0.426	0.002	4.000	0.000
268	72001	CGPG5221	SENSE	0.070	0.728	4.000	0.000
274	72753	CGPG5540	SENSE	0.189	0.010	2.889	0.121
273	72788	CGPG5535	SENSE	0.548	0.009	4.000	0.000
287	73120	CGPG5704	SENSE	0.401	0.146	4.000	0.000
290	73123	CGPG5728	SENSE	0.171	0.166	4.000	0.000
277	73137	CGPG5587	SENSE	0.382	0.019	4.000	0.000
283	73141	CGPG5659	SENSE	0.348	0.029	1.891	0.219
285	73155	CGPG5684	SENSE	0.194	0.105	4.000	0.000
278	73161	CGPG5594	SENSE	0.327	0.018	2.889	0.121
265	73256	CGPG5194	SENSE	0.176	0.473	4.000	0.000
266	73260	CGPG5200	SENSE	0.256	0.047	4.000	0.000
253	73330	CGPG4765	SENSE	0.229	0.016	4.000	0.000
310	73411	CGPG6440	SENSE	0.232	0.213	4.000	0.000
260	73628	CGPG5025	SENSE	0.304	0.040	1.330	0.467
204	73944	CGPG1458	SENSE	0.426	0.026	4.000	0.000
207	74065	CGPG1828	ANTI-SENSE	0.215	0.026	2.599	0.205
248	74536	CGPG442	SENSE	−0.054	0.431	4.000	0.000
312	74688	CGPG6653	SENSE	0.645	0.006	4.000	0.000
340	75657	CGPG7828	SENSE	0.394	0.017	2.680	0.180
325	76178	CGPG7225	SENSE	0.334	0.107	4.000	0.000
321	76460	CGPG7121	SENSE	0.136	0.381	4.000	0.000
305	76527	CGPG6306	SENSE	0.238	0.001	0.335	0.421
217	76532	CGPG3235	SENSE	0.250	0.331	4.000	0.000
213	76602	CGPG2653	SENSE	0.283	0.387	4.000	0.000
306	77020	CGPG6318	SENSE	0.507	0.038	0.000	0.000
364	77139	CGPG9137	SENSE	0.445	0.005	0.000	0.000
372	77180	CGPG9180	SENSE	0.370	0.046	4.000	0.000
365	77187	CGPG9141	SENSE	0.456	0.036	4.000	0.000
381	77204	CGPG9238	SENSE	0.314	0.002	4.000	0.000
382	77266	CGPG9259	SENSE	0.017	0.948	4.000	0.000
389	77430	CGPG9322	SENSE	0.095	0.233	4.000	0.000
392	77444	CGPG9344	SENSE	0.171	0.292	4.000	0.000
319	77517	CGPG6952	SENSE	0.132	0.001	2.507	0.235
327	77536	CGPG7272	SENSE	−0.084	0.675	4.000	0.000
351	77587	CGPG8107	SENSE	0.415	0.112	4.000	0.000
317	77610	CGPG6805	SENSE	0.267	0.180	4.000	0.000
356	77832	CGPG8976	SENSE	0.153	0.579	4.000	0.000

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 347 showed improved cold stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.

H. Shade Tolerance Screen

Plants undergo a characteristic morphological response in shade that includes the elongation of the petiole, a change in the leaf angle, and a reduction in chlorophyll content. While these changes may confer a competitive advantage to individuals, in a monoculture the shade avoidance response is thought to reduce the overall biomass of the population. Thus, genetic alterations that prevent the shade avoidance response may be associated with higher yields. Genes that favor growth under low light conditions may also promote yield, as inadequate light levels frequently limit yield. This protocol describes a screen to look for Arabidopsis plants that show an attenuated shade avoidance response and/or grow better than control plants under low light intensity. Of particular interest, we were looking for plants that didn't extend their petiole length, had an increase in seedling weight relative to the reference and had leaves that were more close to parallel with the plate surface.
T2 seeds were plated on glufosinate selection plates with ½ MS medium. Seeds were sown on ½×MS salts, 1% Phytagel, 10 ug/ml BASTA. Plants were grown on vertical plates at a temperature of 22° C. at day, 20° C. at night and under low light (approximately 30 uE/m²/s, far/red ratio (655/665/725/735) ˜0.35 using PLAQ lights with GAM color filter #680). Twenty-three days after seedlings were sown, measurements were recorded including seedling status, number of rosette leaves, status of flower bud, petiole leaf angle, petiole length, and pooled fresh weights. A digital image of the whole plate was taken on the measurement day. Seedling weight and petiole length were analyzed as quantitative responses according to example 1M. The number of rosette leaves, flowering bud formation and leaf angel were analyzed as qualitative responses according to example 1L.
A list of recombinant DNA constructs that improve shade tolerance in plants illustrated in Table 10.

TABLE 10

Leaf	Seedling	Petiole
angle at	weight at	length at
day 23	day 23	day 23

PEP	Construct	Nomination		RS	P-	Delta	P-	Delta	P-
SEQ ID	ID	ID	Orientation	mean	value	mean	value	mean	value

251	70755	CGPG4473	SENSE	NA	NA	−0.751	0.017	−0.307	0.059
244	70995	CGPG4154	SENSE	NA	NA	0.251	0.070	0.008	0.944
275	72709	CGPG5568	SENSE	NA	NA	−0.903	0.070	−0.608	0.019
210	72724	CGPG2292	SENSE	NA	NA	−0.707	0.098	−0.638	0.020
272	72774	CGPG5518	SENSE	NA	NA	−0.268	0.110	−0.195	0.087
258	72813	CGPG4977	SENSE	NA	NA	−0.469	0.092	−0.186	0.054
294	73020	CGPG5791	SENSE	NA	NA	−1.443	0.010	−1.265	0.010
288	73133	CGPG5714	SENSE	NA	NA	−0.852	0.009	−0.896	0.012
260	73628	CGPG5025	SENSE	NA	NA	0.311	0.011	0.317	0.146
271	73766	CGPG5432	SENSE	NA	NA	−0.988	0.048	−0.671	0.088
314	74435	CGPG6737	SENSE	NA	NA	−0.735	0.042	−0.755	0.049
331	74858	CGPG7371	SENSE	NA	NA	−0.667	0.010	−0.808	0.008
330	74863	CGPG7316	SENSE	NA	NA	0.176	0.060	0.168	0.515
332	74933	CGPG7457	SENSE	NA	NA	−0.100	0.801	−0.533	0.094
340	75657	CGPG7828	SENSE	NA	NA	0.193	0.059	0.244	0.085
337	75685	CGPG7767	SENSE	NA	NA	−0.917	0.075	−0.974	0.049
267	75822	CGPG5210	SENSE	NA	NA	−0.267	0.194	−0.297	0.090
324	76171	CGPG7206	SENSE	NA	NA	−0.350	0.065	−0.570	0.049
254	76202	CGPG4788	SENSE	NA	NA	−1.153	0.009	−1.713	0.019
326	76467	CGPG7267	SENSE	NA	NA	−0.484	0.177	−0.743	0.088
359	76961	CGPG9080	SENSE	NA	NA	−0.425	0.098	−0.265	0.051
360	76973	CGPG9081	SENSE	NA	NA	0.333	0.052	0.248	0.003
363	77103	CGPG9134	SENSE	NA	NA	0.363	0.093	0.198	0.072
374	77157	CGPG9186	SENSE	NA	NA	0.287	0.044	0.107	0.086
369	77165	CGPG9155	SENSE	NA	NA	−0.081	0.413	−0.572	0.012
368	77176	CGPG9148	SENSE	NA	NA	−0.577	0.025	−0.651	0.085
365	77187	CGPG9141	SENSE	NA	NA	0.335	0.002	0.263	0.046
385	77209	CGPG9278	SENSE	NA	NA	0.671	0.027	0.451	0.011
383	77220	CGPG9271	SENSE	NA	NA	0.463	0.008	0.231	0.228
225	77334	CGPG3638	SENSE	NA	NA	0.531	0.067	0.336	0.031
349	77349	CGPG8083	SENSE	NA	NA	0.133	0.001	0.035	0.781
319	77517	CGPG6952	SENSE	NA	NA	−0.843	0.142	−1.458	0.062
320	77518	CGPG6953	SENSE	NA	NA	0.354	0.007	0.380	0.010
307	77609	CGPG6326	SENSE	NA	NA	0.252	0.087	0.211	0.145

For “seeding weight” and “leaf angle”, if p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference with p<0.2.
For “petiole length”, if p<0.05 and delta <0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta <0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in 1 to SEQ ID NO: 246, 295, 303, 325, or 375 showed enhanced shade tolerance by the second criteria as illustrated in Example 1L and 1M.

I. Early Plant Growth and Development Screen

This example sets forth a plate based phenotypic analysis platform for the rapid detection of phenotypes that are evident during the first two weeks of growth. In this screen, we were looking for genes that confer advantages in the processes of germination, seedling vigor, root growth and root morphology under non-stressed growth conditions to plants. The transgenic plants with advantages in seedling growth and development were determined by the seedling weight and root length at day 14 after seed planting.
T2 seeds were plated on glufosinate selection plates and grown under standard conditions (˜100 uE/m²/s, 16 h photoperiod, 22° C. at day, 20° C. at night). Seeds were stratified for 3 days at 4° C. Seedlings were grown vertically (at a temperature of 22° C. at day 20° C. at night). Observations were taken on day 10 and day 14. Both seedling weight and root length at day 14 were analyzed as quantitative responses according to example 1M.
A list recombinant DNA constructs that improve early plant growth and development illustrated in Table 11.

TABLE 11

Root	Root	Seedling
length at	length at	weight at
day 10	day 14	day 14

PEP	Construct	Nomination		Delta	P-	Delta	P-	Delta	P-
SEQ ID	ID	ID	Orientation	mean	value	mean	value	mean	value

227	19774	CGPG3920	SENSE	0.488	0.097	0.307	0.100	0.550	0.163
229	19956	CGPG3972	SENSE	0.248	0.062	0.154	0.214	0.284	0.001
232	19973	CGPG4026	SENSE	0.326	0.047	0.066	0.577	0.444	0.012
302	70812	CGPG607	SENSE	0.704	0.037	0.546	0.031	0.740	0.033
236	70915	CGPG4058	SENSE	0.292	0.005	0.278	0.008	0.331	0.114
230	70948	CGPG3990	SENSE	0.255	0.269	0.093	0.229	0.375	0.026
234	70950	CGPG4052	SENSE	0.106	0.577	0.124	0.374	0.239	0.041
235	70962	CGPG4057	SENSE	0.171	0.075	0.144	0.226	0.283	0.015
241	70963	CGPG4121	SENSE	0.149	0.057	0.112	0.074	0.062	0.513
242	70994	CGPG4122	SENSE	0.198	0.063	0.121	0.114	0.240	0.124
244	70995	CGPG4154	SENSE	0.132	0.052	0.101	0.006	0.198	0.045
249	71328	CGPG4454	SENSE	0.209	0.136	0.251	0.122	0.445	0.093
268	72001	CGPG5221	SENSE	0.183	0.060	0.142	0.042	0.195	0.149
274	72753	CGPG5540	SENSE	0.113	0.045	0.053	0.348	0.081	0.730
282	73105	CGPG5656	SENSE	0.046	0.577	0.138	0.057	0.298	0.142
292	73136	CGPG5764	SENSE	0.212	0.077	0.133	0.027	0.330	0.037
265	73256	CGPG5194	SENSE	0.123	0.503	0.371	0.135	0.623	0.069
313	74420	CGPG6712	SENSE	0.249	0.085	0.093	0.358	0.363	0.042
316	74566	CGPG6796	SENSE	0.169	0.003	0.147	0.114	0.173	0.171
304	74662	CGPG6185	SENSE	−0.205	0.337	0.009	0.878	−0.009	0.958
312	74688	CGPG6653	SENSE	0.221	0.083	0.059	0.358	0.295	0.163
329	74862	CGPG7308	SENSE	0.201	0.122	0.044	0.515	0.383	0.006
325	76178	CGPG7225	SENSE	0.217	0.018	0.093	0.364	0.240	0.234
322	77069	CGPG7163	SENSE	/	/	/	/	0.641	0.074
376	77124	CGPG9207	SENSE	0.337	0.121	0.256	0.045	0.762	0.034
371	77155	CGPG9170	SENSE	0.181	0.262	0.265	0.035	0.419	0.000
367	77164	CGPG9147	SENSE	0.589	0.068	0.369	0.066	0.899	0.051
370	77166	CGPG9163	SENSE	0.383	0.025	0.391	0.006	0.787	0.019
372	77180	CGPG9180	SENSE	0.442	0.058	0.267	0.031	0.402	0.339
362	77186	CGPG9133	SENSE	0.448	0.022	0.383	0.079	0.654	0.057
365	77187	CGPG9141	SENSE	0.453	0.043	0.227	0.127	0.660	0.013
375	77195	CGPG9205	SENSE	0.221	0.114	0.188	0.160	0.259	0.064
385	77209	CGPG9278	SENSE	0.360	0.092	0.181	0.203	0.594	0.047
382	77266	CGPG9259	SENSE	/	/	/	/	0.470	0.020
384	77268	CGPG9275	SENSE	0.353	0.111	0.170	0.244	0.386	0.041
378	77273	CGPG9220	SENSE	0.255	0.070	0.023	0.586	0.510	0.141
297	77312	CGPG5927	SENSE	/	/	/	/	1.006	0.065
393	77409	CGPG9345	SENSE	0.410	0.027	0.396	0.011	0.461	0.040
389	77430	CGPG9322	SENSE	0.209	0.183	0.211	0.247	0.399	0.092
390	77432	CGPG9335	SENSE	0.342	0.113	0.285	0.122	0.425	0.055
391	77433	CGPG9341	SENSE	0.204	0.120	0.222	0.041	0.382	0.085
392	77444	CGPG9344	SENSE	0.185	0.068	0.227	0.003	0.276	0.002
387	77451	CGPG9309	SENSE	0.266	0.097	0.208	0.042	0.014	0.981
388	77452	CGPG9311	SENSE	0.286	0.021	0.182	0.123	0.284	0.201
327	77536	CGPG7272	SENSE	0.121	0.389	0.147	0.026	0.384	0.027
342	77549	CGPG7933	SENSE	0.349	0.006	0.207	0.032	0.383	0.022
344	77568	CGPG8012	SENSE	0.312	0.020	0.157	0.015	0.359	0.071
351	77587	CGPG8107	SENSE	0.151	0.128	0.223	0.014	0.370	0.030
318	77618	CGPG6810	SENSE	0.179	0.018	0.104	0.276	0.154	0.388
334	77816	CGPG7529	SENSE	0.145	0.400	0.171	0.142	0.312	0.181
336	77821	CGPG7737	SENSE	0.114	0.406	0.126	0.208	0.311	0.079
343	77917	CGPG7986	SENSE	0.136	0.083	0.162	0.011	−0.242	0.458

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 252, 309, or 310 showed improved early plant growth and development evidenced by the second criteria as illustrated in Example 1L and 1M.

J. Late Plant Growth and Development Screen

This example sets forth a soil based phenotypic platform to identify genes that confer advantages in the processes of leaf development, flowering production and seed maturity to plants.
Arabidopsis plants were grown on a commercial potting mixture (Metro Mix 360, Scotts Co., Marysville, Ohio) consisting of 30-40/o medium grade horticultural vermiculite, 35-55% sphagnum peat moss, 10-20% processed bark ash, 1-15% pine bark and a starter nutrient charge. Soil was supplemented with Osmocote time-release fertilizer at a rate of 30 mg/ft³. T2 seeds were imbibed in 1% agarose solution for 3 days at 4° C. and then sown at a density of ˜5 per 2½″ pot. Thirty-two pots were ordered in a 4 by 8 grid in standard greenhouse flat. Plants were grown in environmentally controlled rooms under a 16 h day length with an average light intensity of ˜200 μmoles/m²/s. Day and night temperature set points were 22° C. and 20° C., respectively. Humidity was maintained at 65%. Plants were watered by sub-irrigation every two days on average until mid-flowering, at which point the plants were watered daily until flowering was complete.
Application of the herbicide glufosinate was performed to select T2 individuals containing the target transgene. A single application of glufosinate was applied when the first true leaves were visible. Each pot was thinned to leave a single glufosinate-resistant seedling ˜3 days after the selection was applied.
The rosette radius was measured at day 25. The silique length was measured at day 40. The plant parts were harvested at day 49 for dry weight measurements if flowering production was stopped. Otherwise, the dry weights of rosette and silique were carried out at day 53. The seeds were harvested at day 58. All measurements were analyzed as quantitative responses according to example 1M.
A list of recombinant DNA constructs that improve late plant growth and development illustrated in Table 12.

TABLE 12

	Rosette dry	Rosette	Seed net dry	Silique dry	Silique
	weight	radius at	weight	weight	length at
PEP	at day 53	day 25	at day 62	at day 53	day 40

SEQ	Construct	Delta	P-	Delta	P-	Delta	P-	Delta	P-	Delta	P-
ID	ID	mean	value	mean	value	mean	value	mean	value	mean	value

394	12313	−0.075	0.489			0.654	0.000	0.157	0.174	−0.024	0.275
221	18258	−0.494	0.017	−0.183	0.083	0.533	0.006	−0.269	0.234	0.056	0.009
246	70657	0.373	0.040	0.187	0.037	0.355	0.081	−0.187	0.222	−0.003	0.972
247	70660	−0.010	0.936	−0.072	0.431	0.445	0.026	0.116	0.369	0.145	0.045
216	71538	−0.167	0.168	0.017	0.686	0.521	0.005	−0.196	0.113	0.023	0.557
269	72056	0.563	0.008	NA	NA	−0.251	0.159	−0.289	0.314	−0.046	0.314
279	73057	−0.266	0.035	0.167	0.114	0.396	0.034	−0.304	0.085	0.023	0.543
311	73568	−0.140	0.115	−0.085	0.340	0.553	0.019	0.344	0.010	0.040	0.458
222	19193	−0.296	0.110	0.081	0.385	1.076	0.011	NA	NA	0.073	0.001
262	73242	0.159	0.132	0.133	0.050	1.074	0.009	0.652	0.002	0.045	0.048
309	73433	0.477	0.026	0.113	0.120	1.140	0.006	0.552	0.016	−0.009	0.872
316	74566	−0.387	0.023	0.104	0.047	1.213	0.003	−0.169	0.130	0.012	0.598
338	75654	0.604	0.012	−0.006	0.899	−1.193	0.004	−0.258	0.057	−0.004	0.962
361	77150	0.611	0.003	−0.098	0.115	−0.363	0.011	−0.398	0.203	0.001	0.990
327	77536	0.043	0.692	0.089	0.040	0.725	0.015	0.419	0.016	0.075	0.105
334	77816	−0.324	0.021	0.005	0.954	1.207	0.000	−0.038	0.679	0.039	0.378

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.

K. Low Nitrogen Tolerance Screen

Under low nitrogen conditions, Arabidopsis seedlings become chlorotic and have less biomass. This example sets forth the limited nitrogen tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are altered in their ability to accumulate biomass and/or retain chlorophyll under low nitrogen condition.
T2 seeds were plated on glufosinate selection plates containing 0.5×N-Free Hoagland's T 0.1 mM NH₄NO₃T 0.1% sucrose T 1% phytagel media and grown under standard light and temperature conditions. At 12 days of growth, plants were scored for seedling status (i.e., viable or non-viable) and root length. After 21 days of growth, plants were scored for BASTA resistance, visual color, seedling weight, number of green leaves, number of rosette leaves, root length and formation of flowering buds. A photograph of each plant was also taken at this time point.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The number green leaves, the number of rosette leaves and the flowerbud formation were analyzed as qualitative responses according to example 1L. The leaf color raw data were collected on each plant as the percentages of five color elements (Green, DarkGreen, LightGreen, RedPurple, YellowChlorotic) using a computer imaging system. A statistical logistic regression model was developed to predict an overall value based on five colors for each plant.
A list of recombinant DNA constructs that improve low nitrogen availability tolerance in plants illustrated in Table 13.

TABLE 13

Root length	Leaf color	Rosette weight

PEP	Construct	Nomination		Delta		Risk score	P-	Delta	P-
SEQ ID	ID	ID	Orientation	mean	P-value	mean	value	mean	value

200	10422	CGPG117	ANTI-	−0.435	0.008	1.311	0.021	0.050	0.396
			SENSE
206	12602	CGPG170	SENSE	−0.004	0.941	−0.052	0.895	0.115	0.048
202	13235	CGPG1288	SENSE	−0.047	0.267	−0.085	0.864	0.128	0.068
203	13411	CGPG1301	SENSE	0.323	0.202	−3.233	0.126	0.174	0.044
201	13485	CGPG1226	ANTI-	0.311	0.096	1.162	0.038	0.241	0.019
			SENSE
205	13846	CGPG1542	ANTI-	−0.267	0.190	1.506	0.004	0.176	0.281
			SENSE
212	16610	CGPG2499	SENSE	−0.037	0.677	1.325	0.004	−0.004	0.918
211	17805	CGPG2457	ANTI-	−0.348	0.016	3.523	0.067	−0.105	0.116
			SENSE
218	18231	CGPG3274	SENSE	−0.207	0.117	0.925	0.062	−0.093	0.094
224	18354	CGPG3534	SENSE	0.104	0.070	−0.407	0.124	0.189	0.048
252	70684	CGPG4588	SENSE	0.379	0.001	−3.676	0.003	0.328	0.030
223	71301	CGPG3528	SENSE	−0.394	0.027	1.269	0.042	−0.128	0.026
250	71329	CGPG4456	SENSE	−0.169	0.230	0.475	0.074	−0.365	0.337
258	72813	CGPG4977	SENSE	−0.361	0.193	0.521	0.076	0.013	0.120
309	73433	CGPG6429	SENSE	0.129	0.201	−0.091	0.794	0.186	0.002
300	74349	CGPG5967	SENSE	−0.139	0.019	0.592	0.080	0.045	0.080
313	74420	CGPG6712	SENSE	0.612	0.043	−3.086	0.063	0.173	0.002
298	75237	CGPG5941	SENSE	−0.097	0.237	0.948	0.031	0.074	0.136
333	75379	CGPG7520	SENSE	0.000	0.998	0.529	0.048	−0.017	0.755
335	75434	CGPG7636	SENSE	−0.336	0.039	1.208	0.076	−0.109	0.107
341	75622	CGPG7833	SENSE	−0.503	0.001	1.776	0.007	0.096	0.497
339	75692	CGPG7823	SENSE	−0.055	0.165	−0.377	0.328	0.132	0.083
301	76422	CGPG6040	SENSE	0.197	0.178	−0.263	0.734	0.162	0.094
359	76961	CGPG9080	SENSE	−0.361	0.136	1.472	0.013	−0.190	0.066
369	77165	CGPG9155	SENSE	0.366	0.041	−1.982	0.143	0.253	0.099
303	77322	CGPG6178	SENSE	/	/	1.691	0.060	−0.185	0.052
207	16322	CGPG1828	SENSE	/	/	−0.021	0.982	0.116	0.043
356	77832	CGPG8976	SENSE	/	/	2.472	0.011	−0.028	0.445

For leaf color and rosette weight, if p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference with p<0.2. For root length, if p<0.05, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 198 or 327 showed improved tolerance to low nitrogen condition evidenced by the second criteria as illustrated in Example 1L and 1M.

L. Statistic Analysis for Qualitative Responses

A list of responses that were analyzed as qualitative responses illustrated in Table 14.

TABLE 14

response	screen	categories (success vs. failure)

Wilting response Risk	Soil drought tolerance screen	non-wilted vs. wilted
Score
growth stage at day 14	heat stress tolerance screen	50% of plants reach stage1.03 vs. not
growth stage at day 14	salt stress tolerance screen	50% of plants reach stage1.03 vs. not
growth stage at day 14	PEG induced osmotic stress tolerance	50% of plants reach stage1.03 vs. not
	screen
growth stage at day 7	cold germination tolerance screen	50% of plants reach stage 0.5 vs. not
number of rosette leaves	Shade tolerance screen	5 leaves appeared vs. not
at day 23
Flower bud formation at	Shade tolerance screen	flower buds appear vs. not
day 23
leaf angle at day 23	Shade tolerance screen	>60 degree vs. <60 degree
number of green leaves at	limited nitrogen tolerance screen	6 or 7 leaves appeared vs. not
day 21
number of rosette leaves	limited nitrogen tolerance screen	6 or 7 leaves appeared vs. not
at day 21
Flower bud formation at	limited nitrogen tolerance screen	flower buds appear vs. not
day 21

Plants were grouped into transgenic and reference groups and were scored as success or failure according to Table 14. First, the risk (R) was calculated, which is the proportion of plants that were scored as of failure plants within the group. Then the relative risk (RR) was calculated as the ratio of R (transgenic) to R (reference). Risk score (RS) was calculated as −log₂ ^RR. Two criteria were used to determine a transgenic with enhanced trait(s). Transgenic plants comprising recombinant DNA disclosed herein showed trait enhancement according to either or both of the two criteria.
For the first criteria, the risk scores from multiple events of the transgene of interest were evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). RS with a value greater than 0 indicates that the transgenic plants perform better than the reference. RS with a value less than 0 indicates that the transgenic plants perform worse than the reference. The RS with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference. If p<0.05 and risk score mean >0, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference.
For the second criteria, the RS from each event was evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). The RS with a value greater than 0 indicates that the transgenic plants from this events perform better than the reference. The RS with a value less than 0 indicates that the transgenic plants from this event perform worse than the reference. The RS with a value equal to 0 indicates that the performance of the transgenic plants from this event and the reference don't show any difference. If p<0.05 and risk score mean >0, the transgenic plants from this event showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference. If two or more events of the transgene of interest showed improvement in the same response, the transgene was deemed to show trait enhancement.

M. Statistic Analysis for Quantitative Responses

A list of responses that were analyzed as quantitative responses illustrated in Table 15.

TABLE 15

response	screen

seed yield	Soil drought stress tolerance screen
seedling weight at day 14	heat stress tolerance screen
root length at day 14	heat stress tolerance screen
seedling weight at day 14	salt stress tolerance screen
root length at day 14	salt stress tolerance screen
root length at day 11	salt stress tolerance screen
seedling weight at day 14	PEG induced osmotic stress tolerance
	screen
root length at day 11	PEG induced osmotic stress tolerance
	screen
root length at day 14	PEG induced osmotic stress tolerance
	screen
rosette area at day 8	cold shock tolerance screen
rosette area at day28	cold shock tolerance screen
difference in rosette area from	cold shock tolerance screen
day 8 to day 28
root length at day 28	cold germination tolerance screen
seedling weight at day 23	Shade tolerance screen
petiole length at day 23	Shade tolerance screen
root length at day 14	Early plant growth and development
	screen
Seedling weight at day14	Early plant growth and development
	screen
Rosette dry weight at day 53	Late plant growth and development screen
rosette radius at day 25	Late plant growth and development screen
seed dry weight at day 58	Late plant growth and development screen
silique dry weight at day 53	Late plant growth and development screen
silique length at day 40	Late plant growth and development screen
Seedling weight at day 21	Limited nitrogen tolerance screen
Root length at day 21	Limited nitrogen tolerance screen

The measurements (M) of each plant were transformed by log₂calculation. The Delta was calculated as log₂M(transgenic)-log₂M(reference). Two criteria were used to determine trait enhancement. A transgene of interest could show trait enhancement according to either or both of the two criteria. The measurements (M) of each plant were transformed by log₂calculation. The Delta was calculated as log₂M(transgenic)-log₂M(reference). If the measured response was Petiole Length for the Low Light assay, Delta was subsequently multiplied by −1, to account for the fact that a shorter petiole length is considered an indication of trait enhancement.
For the first criteria, the Deltas from multiple events of the transgene of interest were evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). Delta with a value greater than 0 indicates that the transgenic plants perform better than the reference. Delta with a value less than 0 indicates that the transgenic plants perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference. If p<0.05 and risk score mean >0, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference.
For the second criteria, the delta from each event was evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). The Delta with a value greater than 0 indicates that the transgenic plants from this event performs better than the reference. The Delta with a value less than 0 indicates that the transgenic plants from this event perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of the transgenic plants from this event and the reference don't show any difference. If p<0.05 and delta mean >0, the transgenic plants from this event showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference. If two or more events of the transgene of interest showed enhancement in the same response, the transgene was deemed to show trait improvement.

Example 2 Identification of Homologs

A BLAST searchable “All Protein Database” is constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a DNA sequence provided herein was obtained, an “Organism Protein Database” is constructed of known protein sequences of the organism; the Organism Protein Database is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.
The All Protein Database is queried using amino acid sequence of cognate protein for gene DNA used in trait-improving recombinant DNA, i.e., sequences of SEQ ID NO: 198 through SEQ ID NO: 394 using “blastp” with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list is kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.
The Organism Protein Database is queried using amino acid sequences of SEQ ID NO: 198 through SEQ ID NO: 394 using “blastp” with E-value cutoff of 1e-4. Up to 1000 top hits are kept. A BLAST searchable database is constructed based on these hits, and is referred to as “SubDB”. SubDB was queried with each sequence in the Hit List using “blastp” with E-value cutoff of 1e-8. The hit with the best E-value is compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism. Likely orthologs from a large number of distinct organisms were identified and are reported by amino acid sequences of SEQ ID NO: 395 to SEQ ID NO: 19938. These orthologs are reported in Tables 2 as homologs to the proteins cognate to genes used in trait-improving recombinant DNA.

TABLE 2

SEQ ID NO:	homolog SEQ ID NOs

198:	3331	2914	8408	6836	10845	14799	13562	8848	2509	13003	875	10945
	4176	7318	4820	19156	19137	14942	17166	13809	3506	3410	1343	8019
	7882	6490	3550	4999	3130	12635	8774	10864	17857	18089	16263	7295
	10882	18252	3728	14833	11485	15463	18826	2924	18723	1446	2812	7847
	3395	4209	15768	10679	18379	14040	3974	1087	11574	6329	5482	5609
	6441	5537	17042	4716	4516	12643	11384	12736	8137	1344	19473	8452
	4094	2460	18071	16891	5620	2364	7018	5200	11131	18133	12170	2731
	1687	5014	18511	9343	19283	6682	8123	8124	10597	10622	10608	10600
	16221	4280	11494	4867	9035	9507	7969	19159	19425	5248	17367	8135
	8773	1287	2671	9365	18729	13828	8754	7170	14529	16604	8341	1135
	6224	9739	11212	12962	4331	13555	14356	14357	14783	7509	2032	4692
	5243
199:	15320
200:	7710	14519	16336	7019	7872	7719	18299	10453	4650	15008	10089	699
	15645	10533	11751	12924	10526	5313	17532	8825	6941	7743	4155	14541
	9797	16905	8304	12080	19803	3087	1397	7534	5547	2423
201:	11777	6444	1416	4353	15071	3413	14526
202:	19073	6651	15740	7399	15791	2977	7789	9248	14697	15923	12131	5470
	965	11100	11069	12624	12892	3580	5270	18663	2916	1684	8107	4240
	19240	18582	19054	15775	7301	17416	10566	16351	13846	16386	18431	4439
	4056	8159	8292	12484	7316	3763	4783	18427	15501	16164	7762	4015
	4556	18955	12061	8636	9665	9713	10212	17555	5330	12850	16436	19045
	16034	16692	2571	11676	12666	11853	5348	8771	12218	3157	10749
203:	5608	2382	17993	16598	7505	6976
204:	11369	13527	10897	6893	6962	11592	837	5116	19669	1655	7244	2623
	2591	16504	17797	4636	15133	4141	7112	7939	6898	15965	16706	16979
	11784	16115	9620	15750	14652	3008	7838	14787	4563	1873	15811	7271
	4317	16603	1715	14288	11341	6188	1546	5683	5225	3033	6069	11268
	15981	15490	4181	8633	6058	7993	6763	9671	14892	17296	990	8184
	2935	8536	11232	12598	12638	5112	10787	1107	13858	5864	16398	16380
	17071	16520	1973	5455	13852	19228	1695	17402	2865	17120	2507	5601
	14952	1462	6616	6973	3092	1382	7755	3870	9765	5711	1257	6119
	15310	3792	15970	16032	14584	7282	15140	7273	3013	15249	13253	13241
	2265	17700	18976	4009	17354	2223	17559	3151	6320	17010	8939	11258
	734	573	15015	3050	16344	3931	11238	7691	11499	1112	4825	8344
	13519	11669	5640	13588	5473	15108	7210	17617	5994	5980	16627	17906
	5876	6079	18894	6956	12887	14694	10968	1061	2778	6096	10570	680
	974	3120	16300	5885	7626	13881	4244	4233	15039	4486	3103	3115
	7654	11890	12359	10800	9409	12657	8977	9182	17818	2942	9780	9899
	18496	4118	18188	10437	13907	17312	15730	13906	2603	12770	4837	12953
	16663	9237	9546	16293	18238	6022	13908	10266	9180	5538	15536	10370
	15958	5217	8154	13095	15972	14841	15262	12702	10766	15960	18726	19395
	10345	5964	13064	3946	17225	17262	17220	17266	17224	17252	5465	5495
	12800	12814	12758	3021	12400	12492	16461	13638	5616	17449	8397	17258
	5669	5675	6852	9796	17601	17569	9775	17664
205:	14023	6117	12539	6696	18601	1463	19893	6621	9577	9166	17890	17889
	528	5413	18313	2784	8102	3988	890	19106	571	6791	2402	912
206:	17912	8799	6566	12137	4356	5803	5253	6078	9123	17523	12392	17806
	6917	19039	7350	17767	11311	17082	15535	19392	11233	470	8318	14963
	16736	8867	14135	5246	4674	2124	9901	13302	15459	18997	2954	1026
	667	7561	16841	8092	9084	4065	1836	13713	13697	17640	7422	19580
	4565	15193	526	13108	7065	11703	11362	3633	19430	6221	17433	15616
	10082	10189	3089	15238	6633	8106	16180	15317	16364
207:	14575	8831	11211	14985	17619	18829	2521	5219	15413	15835	1204	6569
	4661	4457	6066	14578	932	13694	7541	4813	15707	19563	1326	16107
	5009	7518	14825	13777	6175	15925	13617	5765	14838	9091	10776	15679
	5909	19373	756
208:	19581	18655	16310	7497	6382	8261	936	7358	8883	4866	2146	8601
	2932	1626	10354	15192	11509	9909	13503	488	17477	14483	16941	2824
	1072	4989	14179	4883	13751
209:	3493	16588	7031	5155	11150	17294	5241	10621	5786	5591	16509	2021
	1324	12454	15061	1670	15143	16506	17002	8741	18091	4013	5959	19370
	4239	12885	12174	3709	3431	9596	3378	1018	5963	13212	3150	2952
	1619	19861	19114	15446	19582	12663	19603
210:	17777	843	11020	1337	18495	1493	6396	15336	14087	2568	4517	1157
	10741	4553	14999	425	4258	17388	5327	748	11691	10174	17805	16628
	10117	7980	8902
211:	9637	17261	13232	7387	10506	19067	12021	872	11620	13603	841	1450
	2848	16911
212:	14398	1558	18339	13428	13431	4182	16260	10619	6127	12005	7704	5064
	13072	4395	9032	19907	9588	478	12782	9917	13100	5202	10562	2044
	4452	10775	3543	14840	14071	14020	10406	14218	1797	16955	3879	10312
	12432	14824	7082	14554	15744	1508	15866	16232	6797	7077	5816	3597
	3596	3311	15022	881	2638	906	12037	12039	3458	2270	8812	3217
	9466	18102	610	9042	2431	18349	16650	14418	2251	4235	6826	1244
	16651	13004	7852	14435	9553	9262	15603	4340	9235	1413	10538	8296
	17223	16048	16071	2120	19847	10420	11239	18968	2995	7551	15854	12345
	5685	18180	2381	4814	2159	16862	8677	13466	11435	6649	6015	8097
	11230	16025	9440	1353	16151	16249	15930	11226	9398	2870	12373	15043
	11765	10145	11071	285	9270	9537	9095	2350	13362	5496	7924	11756
	2624	7967	6715	15074	17068	9275	10177	11007	14857	8951	12182	11643
	13590	7684	1740	929	910	9093	10018	13344	16978	17544	6305	11378
	1393	14108	15937	16631	11803	13126	11969	18524	9644	8425	8421	8420
	10755	10757	16696	15994	1143	3549	5868	5126	7506	12547	12669	19032
	4707	4710	4708	4732	4736	4733	8772	4730	4711	3385	1831	2528
	2304	15098	13223	15197	11673	4386	11884	2336	14693	9118	9633	14548
	12725	2399	18359	11011	14692	5652	5434	9755	9513	2681	4364	12903
	19777	13525	4326	6859	6088	11611	3068	13940	3578	14440	8582	9826
	19618	18012	17233	10057	18222	15021	9371	5985	14888	14001	1279	7826
	14232	18172	7425	11502	15565	17431	8317	10294	17742	5564	5531	18144
	7400	10835	14389	13461	13857	7113	16444	4722	2115	7276	18254	1237
	5256	13951	2920	16719	1045	15371	19022	684	8004	2781	19811	19766
	3279	18712	4940	14312	6426	10076	15772	7906	19836	11219	18985	2673
	3933	14263	11860	1907	4672	15599	13737	4944	2252	15415	11543	3433
	7746	17114	4115	4881	9445	14696	9525	14484	16112	5392	10428	1400
	9349	7616	18930	2533	12585	10497	10236	18415	4874	19793	4633	12544
	1995	17255	6130	10439	15261	6768	15985	17752	8504	12863	7487	14875
	12020	2036	18127	9264	3628	5019	7498	15865	1448	9636	2840	3161
	6207	2644	1059	2065	10031	2171	16927	4747	18701	19271	4174	1389
	9893	12527	9018	10035	5039	10698	13943	7894	12671	5651	19586	12810
	1254	3519	16114	4286	13948	14493	8294	14606	2586	14069	7712	16988
	737	16797	16826	18966	5145	17610	6304	8895	7744	9280	9284	10647
	16558
213:	12662	5295	19093	10971	15359	1163	18513	889	2151	3536	7105	3292
	8466	18205	3579	1504	19486	13768	18047	6871	7174	11029	11505	17782
	5697	18822	4892	12656	1284	3062	4697	5993	18343	18790	3057	7014
	12265	10981	15204	11631	17704	17941	10633	19776	933	10363	8446	17800
	9822	2953	13693	19862	14551	15692	17673	6081	5049	12581	6910
214:	15345	19484	4871	3871	6695	3993	11459	9199	15899	13821	14206	19489
	13765	712	19072	11649
215:	1607	15669	13168	19044	790	9515	4511	13776	11573	13000
216:	4929	17143	10058	17506	15064	3332	5268	6968	16252	15949	6180	3000
	14937	18454	13071
217:	6807	19716	480	14021	4179	12236	6337	12237	6338	6336	12234	4815
	2261	18555	4706	7836	9176	18851	3443	9382	9439	8870	16691	16646
	11516	11500	11515	11498	16670	16660	16683	11513	11496	16668	16664	11519
	16686	11479	11481	7146	16643	16659	4850	17951	7144	4824	7163	4851
	11234	4860	17956	7160	17977	4827	4826	4859	7147	11475	7165	17928
	17958	4853	11473	7143	11477	5129	18893	18914	17526	17529	17548	17547
	17549	18917	13686	19089	13683	4938	16999	13666	4494	12074	17097	12093
	16874	12095	15878	17159	17154	17156	17153	5681	15126	10052	19712	4288
	15399	742	11607	15654	7006	8041	715	2713	11522	7695	3581	7865
	3776	15738	19821	17259	3948	13388	17427	15523	14216	8711	15423	19060
	16814	14298	18720	7885	3967	9710	3568	3573	3575	8503	15486	12932
	7870	989	3595	17756	9202	520	11205	14771	3574	2443	8036	19537
	14641	6739	15375	2400	6753	3336
218:	13099	6217	4454	4699	11292	4702	1554	6467	15339	15722	11035	12964
	16317	13914	17486	18809	14651	4189	9581	14957	2871	1561	17618	3835
	14955	3868	9158	13663	10042	7575	19824	6200	10522	3600	4064	14590
	12731	7201	19572	9060	4035	6255	3457	11320	7986	8818
219:	19325	10450	4487	12312	2564	10096	5641	1947	4367	6964	18031	1012
	11041	15078	12255	5761	5061	9832	16834	18348
220:	8238	19207	18657	1358	2734	9320	13526	12631	17660	16548	15528	18501
	18049	15274	19470
221:	2724	17089	9639	9208	17722	6817	11848	18904	13673	2347	11585	3054
	7486	6056	1290	14932	14930	12636	14230	1572	15708	18912	18177	1699
	10460	2090	5583	18802	606	5929	5041	19148	14224	9144	2643	17144
	14142	14674	16275
222:	14820	18626	11591	649	3005	3037	5453	5430	14248	2371	4626	11661
	9784	10560	16219	11699
223:	17739	13288	4139	15466	19773	753	1277	14333	4211	14510	662	7930
	18703	3268	19400	17533	2302	9078
224:	13859	17816	12692	10417	7051	5444	12975	1611	7050	17105	5023	3397
	13149	14751	10564	7331	7329	11868	9210	9211	18860	18879	14568	17920
	14497	12723	19352	10587
225:	11265	17061	11261	17058	11267	17064	8014	8033	8018	508	8035	8038
	8013	8017	3554	13411	12545	12528	11997	12566	11922	12520	11958	12525
	12522	12553	11961	11979	5030	3464	3910	18901	14601	14600	840	15664
	15668	15121	15142	15119	15686	15690	15657	15688	15146	15164	15716	15148
	15656	11191	13623	12732	5050	5029	5011	5055	5035	5036	5032	5081
	5058	5013	5078	5026	5059	5077	5008	5053	5076	11939	11976	12541
	2898	17207	5179	5115	13674	13423	16078	9689	11174	5252	17279	17277
	11177	1867	1865	1823	1844	1842	1793	1824	1820	10671	3139	1123
	5805	12934	12755	7504	1091	17904	17909	17926	17902	17938	17907	17929
	10598	17935	17933	5298	17980	10601	5383	14050	6585	11379	16583	17978
	17814	4838	17812	2633	3257	3189	774	12008	9988	14268	18392	12567
	14920	12980	11959	7682	7685	13235	4127	3740	3766	3770	3769	3795
	14966	457	8521	19464	4847	13386	5024	13387	13401	5957	6571	7932
	8369	10496	10492	10490	10495	10517	7943	4958	4956	4923	4986	4961
	4951	5007	4937	4934	4984	4933	4924	4921	4936	4979	4932	4981
	4988	18200	18196	7176	4739	5479	8351	7177	12886	9521	6464	17681
	14504	907	16793	1869	14509	8882	19727	8798	1197	3419	14168	12846
	15739	1115	14165	19715	492	8873	1119	497	1194	3251	1116	8876
	13982	12860	14430	451	14969	6102	4358	2650	2649	11790	12925	5882
	19696	9551	3867	19796	18933	18929	833	4908	14217	4852	4858	16064
	14208	10736	4886	14214	16062	14172	10735	14210	10720	1885	14187	14190
	14184	14207	14703	14707	14709	14706	14390	7658	7659	7661	2582	13794
	5204	17305	16541	8914	16959	17283	9043	3924	2696	8656	6648	2675
	18504	17311	6052	9425	9406	9410	9411	6051	9452	6033	15964	6267
	6031	8489	8463	8865	9427	6268	8490	8493	15602	15621	14616	15649
	15652	15606	11723	17247	9423	15595	15624	16148	2240	7428	11722	11363
	2225	2196	5480	15579	15626	16142	7413	5483	11741	16145	16162	11725
	5481	18072	10973	12451	12340	15511	12185	4348	12754	8722	14544	6171
	10732	9492	19795	12165	15082	6617	9613	739	19520	2497	14005	11128
	18795	14331	14329	14334	16439	12070	4053	3942	2387	2391	2394	755
	754	2950	752	2377	19819	17119	7118	1360	2273	11458	13758	11215
	11274	11229	13178	5110	16239	12884	18765	16637	19693	16831	17824	10810
	15395	15420	17470	10005	16397	16394	14711	14728	15503	4401	801	12244
	12292	12308	12219	9033	16763	14392	16804	16785	9566	16806	14409	14391
	16762	9027	9568	9029	16788	9009	16780	14410	16781	16782	16802	3330
	3357	3326	9001	3358	5789	14387	14412	3360	3356	1643	1805	14061
	15787	5632
226:	19514	17912	10095	10091	10252	15927	5360	18385	12313	19719	12823	5182
	1491	8631	4666	2562	9358	10461	3952	19610	9255	7782	16516	5161
	6343	4894	5577	16734	1381	16378	14552	12707	11907	7651	10404	17767
	2417	11311	17082	17741	11253	11254	7473	13718	6269	3242	7085	5224
	8787	16268	19692	10769	8867	1434	16082	19530	14789	13615	18997	2954
	9175	8092	9082	1813	1815	1817	639	3883	15427	2035	4435	3601
	414	7005	8000	1812	1131	1455	6221	11231	10817	15601	16270	6118
	2193	17500	14186	9141	3449	3089	13886	15786	15238	2001	3945	14973
	16180	15317	18083	18695
227:	17372	8093	5527	2439	13784	14768	10702	11535	7750	761	18735	5946
	17426	17988	10652	2928	18398	2009	9559	5094	12948	8522	1305	17785
	5852
228:	2790	5299	15433	5501	18128	16466	14960	6564	8782	4397	19662	16008
	10829	19118	18086	3428	17943	10935	15806	8101	1184	4968	6059	10388
229:	7539	3499	12437	19471	14158	11575	6399	15966	17187	13512	7688	2131
	10858	4169	19567	7834	7585	14073	3954	1991	12940	7756	8167	18994
	9449	15859	4341	17025	14373	12191	12982	983	13764	19774	16247	12665
	12664	2619	18875	7515	11785	2849	10913	10717	4653	11887	905	1641
	3563	6183	16765	17773	4166	4694	14328	11986	12084	1625	12695	9681
	14082	17341	15047	17498	3530	3531	7241	7972	4790	15643	14365
230:	12430	9914	10470	14976	6139	18895	8072	18544	7257	6437	15066
231:	7185	13859	12150	12692	5555	6319	14735	16754	3121	2208	14915	18531
	2991	7950	2419	4378	18922	4377	18705	17105	5023	3397	2203	5350
	895	18953	5090	9210	9211	14568	19723	12433	3877	17892	19476	11685
	9653	9066	16392	7989	5056	6545	19418
232:	10845	14019	10546	14942	7302	8774	13992	10864	7129	12564	10723	2440
	19878	3172	18252	12532	9942	2129	18826	18723	17324	19791	8580	3395
	5915	9604	7572	16359	8047	3408	5482	12915	9969	16513	1344	16044
	19210	18748	11940	14402	19311	5620	2364	19283	5249	3646	17251	17844
	14459	5315	9268	16190	19143	15124	418	3076	16204	17414	18692	13164
	8191	10636	13894	3423	2842	1173	7663	13250	18208	14090	19680	15815
	4805	10584	1287	15495	9256	2229	10166	19534	11163	2292	2687	19082
	9365	2465	5381	18660	3073	9545	17750
233:	19344	12031	1229	15123	14123	19501	7767	5944	11173	4596	11614	10841
	14129	2393	7360	621	14382	11612	8136	14668	7577	1652	2491	3392
	2160	4767	17696	6615	12377	3471	2985	17194	17177	10988	5084	9695
	16519	11445	3213	10846	16453	19346	18274	17478	17798	15663	6823	5131
	14272	3777	19911	19724	6132	19722	14803	17148	1605	11787	14359	15796
	645	10159	13264	12931	6357	4621	16774	18913	5746	7161
234:	11849	7119	1272	13625	6244	7780	7229	640	15779	14967	5388	14480
	1349	17525	12768	10327	8976	5113	5895	15006	4428	16619	4613	3393
	18954	4451	3799	17712	12488	15863	18697	18482	7844
235:	8664	9584	8904	18497	19265	8237	2617	7729	17728	8659	13842	11016
	9197	10827	5351	3224	7379
236:	12797	7459	12082	1618	7740	16856	7623	8994	7602	19549	12596	7644
	18396	13883	7266	18758	3328	18998	1660	12434	2139	19899	12252	5883
	3266	19587	11474	5870	5443
237:	14746	16460	7754	9978	16450	18053	18050	18466	8866	10337	8245	1604
	8568	13606	19627	18283	15611	10648	13871	9910	10297	3025	18486	8908
	14474	11654	2349	10867	14488	13901	7336	11511	14836	1906	13400	5568
	19527	4351	18319	6689	10519	15977	15760	7605	16652	14204	14665	727
	9225	12804	2776	11108	14258	1882	4357	1972	8324	6435	18239	6728
	18983	9555	13551	10797	18458	4622	14064	2771	1818	18410	13553	15912
	10854	9475	10873	15530	14816	6283	703	8105	16216	7038	957	11908
	13853	10718	11237	5623	10262	12248	6541	7053	4480	14011	17142	6691
	7795	17981	2822	1473	3561	4545	13347	1348	11256	6511	11356	13955
	6699	6555	6559	3185	16166	17894	9307	6011	10635	1391	13177	16458
	702	18335	18740	16787	4175	10950	3412	4193	951	13870	19183	18644
	947	16280	6184	13111	3667	10338	4125	14782	18479	18004	19084	12471
	16645	866	1895	10049	11122	12521	7132	2660	12759	14534	18429	15615
	17611	9347	12704	1525	7492	3451	18373	17125	5972	9777	16039	12350
	4073	8962	17221	1961	1958	11156	12899	3908	7429	10977	15040	16234
	17004	17293	13355	4977	260	10645	14133	9251	12220	12633	7087	9535
	8894	11452	2346	6125	2062	6710	13299	12947	12943	4422	5817	13502
	14378	13504	14379	4770	1451	19815	12842	4405	13837	9184	10051	10999
	9565	12305	11330	16546	17073	1402	5263	7189	6160	2372	6567	1248
	3858	14455	1766	2516	1656	14550	16864	14989	14368	15573	9312	7786
	13516	2969	1952	2541	11207	10625	9198	19674	10334	18065	3495	8519
	12201	3921	13249	18592	857	17545	11757	15455	2164	789	4350	3117
	8649	11708	11463
238:	9324	12427	663	6064	9836	19556	9348	1900	17219	13657	7086	7381
	6006	7357	10469	6466	8472	10588	17047	609	1637	10699	6450	14299
	15101	10536	18759	2378	18013	9269	3343	12489	11305	18178	2269	16890
	10629	18665	15962	9375	9437	16312	6501	11737	6339	10847	5290	4667
	5522	18388	1804	11566	2369	15872	12465	7687	6209	11354	15667	15938
	18971	18258	3774
239:	13622	9907	10802	1635	19607	8218	11862	18989	19820	1430	15998	4855
	18040	3455	2471	6506	7587	11932	17440	16155	13643	12011	13012	11289
	14270	17959	10271	5763	19020	5904	7809
240:	19167	14809	6116	11171	10424	1445	13848	1387	18366	9887	3732	10953
	2175
241:	939	3338	2461	7962	16158	15297	19138	7398	3035	1767	9983	16494
	10256	11165	13953	18376	4113	4116	10457	11995	19241	2063	19340	15435
	14028	14611	14366	18488	9171	5494	10438	18123	11822	13093	15526	9516
	18346	4375	8716
242:	12077	11851	16904	8919	18979	6443	17768	3571	5105
243:	12862	6311	17138	19420	12939	8147	18538	16023	6580	8709	16080	4700
	2940	9554	5556	503	12303	7072	8226	11973	19415	12551	9852	13704
	7982	1030	14887	12346	1435	1437	6242	16606	2041	5356	12706	9139
	2872	10869	9028	7957	7190	19892	16505	9383	6589	11472	8394	8396
	3626	16568	19220	18648	14425	15305	13545	8443	8427	1247	17078	14914
	5103	19922	14358	18892	8972	16582	12124	19870	9538	19845	19867	19195
	19221	19191	19323	19242	19215	19307	19275	19273	19356	19192	19173	19350
	19279	19216	19852	19223	19250	19326	19848	19330	19214	19869	19351	19332
	19276	19247	19177	19163	19305	19328	19851	19353	19197	19850	19358	19175
	11034	19871	6578	17445	3832	3831	6233	7090	9456	6970	13917	13920
	4539	4537	4542	4028
244:	1245	4324	12202	4379	6967	9036	9880	10540	13263	13138	18122	19047
	2008	19483	2468	7439	5787	10979	15035	10982	3699	11097	14482	17829
	11866	4198	14130	17239	6111	14956	17492	11385	4004	10448	9464	9894
	15398	1231	12878	12829	3833	2080
245:	11361	5661	8686	17264	8328	8330	7550	10232	18956	315	8720	1696
	5922	16632	14340	11996	8012	4522	16791	10038	5066	4969	4634	17213
	14401	7284	2099	6718	8030	14321	1903	15554	15028	12204	3060	19178
	11653	9316	17858	18132	18589	9667	17297	5418	17508	15450	6147	5012
	18588	18020	862	4920	15050	16525	3064	2884	5607	12297	11396	18052
	2912	17060	1697	17604	18870	1732	5968	18761	4044	5088	10593	5242
	4527	13682	8054	16503	1962	19380	1623	10664	4597	15319	3478	16945
	18584	3108	12724	15546	15764	15754	18354	18416	4890	19678	19130	15532
	5213	11374	9753	14702	15217	15921	5855	18752	1904	15721	18203	18156
	18864	8700	7560	16924	636	6811	16379	19046	11063	14929	16207	15425
	17368	11052	14975	6574	9560	10349	3830	8250	2015	18785	8196	7713
	11586	14490	18301	18792	15378	7699	18803	19816	16104	14468	17612	11556
	1765	5525	2247	5751	11815	17200	10136	6205	16567	10487	18141	6202
	8537	8518	7037	8133	6764	827	4941	818	8356	19365	7588	3017
	6400	5637	8416	12444	510	12026	11634	9859	1460	18194	11314	2685
	4226	984	19513	8932	12905	4112	18873
246:	13549	2112	6794	8833	12086	2984	5421	7868	9325	19092	9699	19026
	1631	4555	804	8588	19422	8685	6218	17573	17039	12147	4754	10543
	6834	6733	864	9209	4190	9301	16571	2360	4996	8081	11140	13006
	10637	5281	17394	4637	9663	1828	12569	1215	14407	13300	6644	11688
	17465	631	16880	3874	16141	975	8011	16154	18389	13579	15094	8944
	3735	8021	2288	15334	16083	15801	12760	10618	13208	19889	18999	4870
	13710	12132	15259	9267	11470	13144	17588	628	17272	14844	14000	19900
	15096	12603	13869	6532	16013	5431	1192	10395
247:	14941	2202	18124	13094	7945	3968	8551	2458	18176	15639	12789	5427
	16829	9519	1674	19789	16819	4712	18517	13990	16185	6271	15089	5575
	1227	1331	10634	4978	6366	14209	9402	12738	589	6685	16243	2070
	14026	12494
248:	4050	14993	16855	19797	5528	9097	18611	19631	18510	8376	8956	10289
	11370	13805	18078	19606	8202	15826	16478	19041	5700	6765	8134	14325
	10101	16816	4765	16287	5377	18675	7230	7964	876	9597	19802	11403
	4833	17530	18265	13417	12779	8885	11923	18564	3790	19147	1406	7066
	10573	7108	3783	4727	19785	1458	16375	8415	9522	10729	6071	9047
	9113	3248	6378	1799	14779	3630	4294	7608	13707	16321	15368	12743
	11299	2413	1584	11214	4012	2710	4268	19546	17625	6940	1273	5755
	413	5952	3098	15675	11098	3286	8068	2690	1222	13381	15257	4756
	4725	9351	7586	5770	10868	3420	12012	775	7742	8422	6103	15004
	16010	12445	13845	17033	16212	3842	19401	4100	8417	13255	1233	5307
	7664	11820	4960	4282	2333	17315	12161	4217	10485	8151	16203	1002
	11057	5823	9298	1567	411	5686	2787	6556	17045	5742	3090	12019
	12930	14231	15548	11694	8266	16248	1579	4806	2747	11349	6186	8303
	4499	14141	8094	16001	12109	10916	10761	2142	4592	5947	1880	5294
	16111	2594	9869	6575	13083	17382	14364	11324	2341	14545	1596	15428
	18963	1929	10976	3736	16709	5662	4501	19083	17865	14066	8045	18782
	2866	19741	12401	690	3507	2118	8721	10292	3096	7017	5861	9569
	15479	6800	14522	15996	3926	12508	9266	7508	3882	1770	18129	2452
	16753	7303	5038	17638	14421	11636	19188	2092	5721	781	700	15887
	8872	5320	5466	10259	7441	13041	10874	7249	4514	9725	18158	17609
	18140	18293	17059	18509	4888	9306	5484	7091	1826	19360	3124	1056
	2563	3177	16370	10946	6177	11856	17835	15766	9377	14858	9666	2881
	4713	18553	10081	3260	6030	16206	11892	10319	18672	3859	2663	15443
	5089	10992	2411	12439	17434	6053	18213	1552	16896	16303	2170	4062
	9729	5506	4195	2496	9435	18250	3643	10322	8871	11520	8307	10814
	4172	3616	467	2220	2765	10994	7181	9294	9019	14302	16474	5797
	4407	12612	9186	18928	4415	11982	14037	3959	1335	507	14948	9483
	17387	6806	8933	14886	13678	506	12462	6785	2667	19322	5005	14242
	11045	12403	2390	19573	11726	9949	5099	9853	16159	15920	13256	615
	3439	10834	8240	12330	19303	17872	12750	7494	2539	12602	5373	16910
	3341	11873	10613	7321	18101	12158	14798	8096	5837	1986	13521	5384
	7186	4164	12183	5907	11172	15553	726	4059	6816	6374	16761	7732
	6913	1868	15780	18634	16779	6640	3707	4959	10722	8632	18898
249:	8837	9354	459	481	15638	15637	10951	1014	1011	10934	460	456
	1750	19841	3504	7770	7490	2370	12273	8627	10651	13584	13749	13618
	14339	12485	7233	18749	12207	7057	408	14300	5215	19033	14163	1226
	1228	5775	5424	8505	18275	18314	10203	8674	11707	15070	19005	8386
	2693	9051	4549	8477	13649	15215	13783	10149	13027	13294	15862	674
	9926	18724	12578	8169	1419	6285	15077	2147	14574	6516	1177	13510
	9181	2520	4618	7286	7290	7135	6455	7288	9787	11002	551	2679
	8942	1701	8928	8930	1097	17269	12778	15439	17593	12347	1453	10948
	15909	19108	8061	16885	8964	5642	15311	18841	8881	9067	2902	10703
	8544	18226	18698	18197	7766	17357	6778	13002	10083	15189	7944	1067
	13701	18247	5067	8858	2375	10780	12562	6618	1888	368	13569	16443
	14088	17735	6201	1162	9317	18221	17786	9563	9562	2386	3903	5156
	18001	547	12480	13049	2495	8339	18977	12208	19466	10488	15681	11974
	4385	19012	8291	16077	16024	5878	19014	8302	17408	16026	16018	16022
	4841	9829	9828	17831	4690	2646	9830	13874	14131	9842	9827	9849
	8403	9844	9847	9865	9851	8406	8404	6995	7012	8405	4019	10234
	7448	17662	1677	19490	2475	6216	13658	5135	4005	982	15959	17990
	3508	17774	17778	15478	7855	9687	17808	10782	7592	268	18627	12803
	17776	12927
250:	12939	8147	18538	6832	13653	16376	2940	8025	9554	5556	503	12303
	11973	12551	9852	1030	10923	12346	1435	1437	2658	15612	16349	9028
	13226	19568	6589	11472	4670	17175	1302	15214	13545	992	5103	19327
	18189	7434	13340	9873	7090	16545	6970	15069	19166	16647
251:	6603	10030	13559	6740	11390	7765	3614	11615	15027	11616	14213	11329
	19168	2567	13201	10961	3308	11492	2325	6423	11527	3741	11560	8630
	7463	1786	14408	10585	15696	14167
252:	17378	17392	17375	19787	819	9037	18778	15271	2631	2615	2616	2606
	15288	2607	15269	15283	15286	15282	15287	15285	2609	2629	2613	2604
	1472	3730	6707	4595
253:	16186	14031	2169	19019	14212	16029	16170	16973	2492	10956	13426
254:	9072	16169	1130	12933	1592	17856	19381	14964	19031	3682
255:	15936	5121	14535	14860	1527	1041	997	4031	18021	12960	12659	6385
	17513	15910	6705	15441	15200	3272	16975	18974	12341	5796
256:	15904	2266	1296	6085	10365	4818	7630	6936	18652	4715	9134	15714
	2506	5391	14801	12410	18804	2831	19264	7856	15547	14814	17301	17179
	18891	11410	1475	11170	7067	17441	12196
257:	1879	1595	12797	7459	8363	12502	12756	5854	465	1425	1893	12362
	12596	7644	16328	15689	4599	17970	5443	17245	10708	17790
258:	3828	1761	11325	17218	18307	14003	12997	4456	6236	19270	8499	7402
	5087	10067	3938	3953	3939	17913	19475	5664	3918	12870	8678	9700
259:	12266	4307	12661	6842	6841	6413	17238	16962	15778	516	7796	7046
	7155	8841	7260	9891	13142	2868	8814	9110	9530	14169	17860	3971
	6657	13557	14715	9995	14954	9514	14428	5521	11000	8008	6231	6796
	6894	12969	15894	13220	469	19780	17022	2260	17210	1668	1624	17554
	9991	2888	10752	8717	1792	7940	17358	7238	580	582	583	2828
	4970	4995	18273	1838	4998	4967	8488	14536	1477	13690	10891	14229
	7730	15048	9774	18163	9481	10644	2992	6652	4943	7469	6637	19611
	6346	6577	5487	15216	8436	7531	19737	9498	7963	8585	17924	10815
	10055	9630	14139	8171	9921	12928	12473	13070	793	5190	2500	9328
	1480	13432	15272	14189	13728
260:	14746	16460	18053	7754	9978	16450	18050	18466	8866	10337	10391	8245
	1604	8568	8843	13606	19627	18283	15825	15808	15611	13871	2627	2601
	19077	1485	5898	5916	8835	2570	9910	10297	3025	18486	8908	14474
	11654	2349	10867	14488	9940	13901	7336	11511	19746	14836	1906	13400
	19527	5568	4351	18319	6689	10519	15977	15760	7605	16652	14204	14665
	2776	9225	1972	12804	727	14258	18708	1882	6435	11108	8324	18239
	3913	10328	4357	12240	6728	18983	18808	5969	9555	13551	18458	4622
	13553	10797	1818	14064	18410	6283	10854	2771	9475	10873	15912	15530
	8946	16216	7038	957	13243	13853	11908	12331	10718	11237	5623	10262
	12248	6541	17142	7053	14011	4480	2822	17981	7795	6691	3561	1473
	17141	4545	13347	1348	11256	6511	11356	8651	13955	6699	6555	16166
	6559	3185	17894	9307	6011	10635	1391	13177	702	16458	18740	16787
	4175	10950	3412	4193	951	13870	19183	18644	947	18835	6184	13111
	3667	10338	4125	14782	18479	18004	19084	12471	16645	866	1895	10049
	11122	12521	7132	14534	2660	12759	18429	17611	15615	12704	1525	9347
	7492	3451	18373	17125	5972	9777	16039	12350	4073	8962	17221	1961
	1958	11156	12899	18769	11606	3225	3930	12985	3908	7429	18285	10977
	15040	16234	17004	17293	13355	15499	2062	11463	11416	11708	6710	4422
	5817	13502	14378	14379	13504	1451	19815	12842	4405	10999	9565	11330
	5263	7189	6160	2372	3858	1248	6567	14455	1766	14550	2516	1656
	14989	16864	9312	19300	19408	13516	2556	2969	1952	2541	11207	10625
	19674	9198	10334	18065	3495	8519	12201	3921	13249	18592	857	17545
	11757	15455	2164	789	4350	3117	14133	12220	7087	11452	6125	4977
261:	6807	480	6337	12236	4678	12917	9439	9382	11513	11500	16670	16646
	16691	16660	11516	11515	11496	11498	16668	16683	16686	16664	11519	16659
	16643	14322	2737	1932	12955	12317	19334	12435	12976	5809	18730	11183
	705	12278	16937	13026	12999	1928	15878	8321	12130	11053	9846	17306
	15324	10531	11328	12167	19474	12203	2827	878	9720	4228	5762	4288
	13228	10010	14908	1017	679	14784	677	1487	10919	18296	10669	11200
	9055	18733	17838	16389	728	715	1375	1396	6901	3743	12599
262:	11852	1409	16572	9074	6600	4427	824	15705	6709	10791	14587	9823
	18325	515	12450	18527	604	6043	12455	2071	10310	18162	3176	2577
	15203	12952	14467	6645	6698	12807	6113	4709	8824	6274	8311	3411
	18810	17940	19805	17162	665	12693	1309	15412	18084	5235	8314	13722
	15858	15969	18135	2305	13611	4030	14864	5363	4470	8271	5178	12458
263:	8837	481	15637	10951	10934	19841	460	3504	15638	1014	1011	459
	1750	456	12273	4264	8627	13584	13749	19375	12485	18749	7057	408
	7233	12207	13068	5215	5515	14163	8781	1228	1226	5775	5424	8505
	18275	18314	10203	8674	2693	19005	14053	9051	8386	8477	4549	6597
	15215	10149	13783	1372	1370	13027	13294	4287	12578	1419	15077	2147
	2905	14574	17504	6516	6455	9181	2520	7135	7288	7290	2679	4618
	7286	551	9787	11002	1701	8942	8928	8930	1097	17269	12778	13750
	18947	15439	17593	1453	19108	8061	17556	5642	16885	8964	10019	2902
	1548	5257	1728	7766	17357	5647	10631	15019	1888	18777	18221	17786
	9562	9563	5156	2386	3903	18001	547	12480	13049	8197	2495	8339
	19466	12208	18977	11974	4385	19014	8302	19012	17408	8291	16077	5878
	16024	16026	16016	16018	16022	4841	9828	9829	9830	9827	14131	13874
	9849	9847	4690	2646	9842	17831	8403	9851	9865	8405	8404	6995
	9844	7012	4019	15785	10234	7448	17896	1513	13878	7287
264:	10139	8837	1318	481	15637	10951	10934	19841	460	3504	15638	1014
	1011	459	456	1750	17473	5931	11424	7490	1243	2370	12273	4264
	8627	13584	13749	14339	12485	18749	7233	12207	408	7057	19033	6702
	13068	5215	5515	14163	8781	5775	1228	5424	1226	8505	8674	18275
	18314	10203	11707	15070	2693	15215	13884	10149	13783	13027	13294	15807
	4287	9926	12578	2905	17504	6516	7290	9181	6455	7135	2679	2520
	7286	4618	551	7288	9787	11002	1701	8928	8930	8942	1097	17269
	15267	13750	18947	15439	17593	9816	11871	13684	1453	19108	8061	5642
	10443	12994	4925	6825	16676	7766	13011	12993	13010	17357	9582	4071
	15183	1888	11394	1145	18221	17786	9563	9562	18001	3903	2495	17171
	19146	18977	12208	8339	19466	10488	15681	4385	17408	19012	8291	5878
	16077	19014	8302	16018	16024	16026	16016	16022	4841	9842	9830	9828
	8403	4690	2646	9827	9829	13874	14131	17831	9847	9849	9844	9851
	8405	9865	8404	6995	7012	4019	7448	538	4587	4576	14863	2709
	10770
265:	10339	11051	4273	6693	11042	16475	7274	1052	18146	6194	6195	18166
	10865	4396	638	10021	15834	6518
266:	12371	1790	10658	8747	10474	4583
267:	19068	13736	19730	2398	4156	8189	14176	3642	16457	13680	12542
268:	16601	2370	12273	10651	18749	7233	7057	12207	408	14163	1226	1228
	5775	5424	8505	18314	10203	18275	8674	11707	14053	19005	8477	8386
	7370	6597	2693	9051	4549	13649	15215	10149	13027	9926	8169	1419
	6285	2147	15077	2905	17504	13510	7290	7286	7135	7288	2520	6455
	9181	9787	2679	4618	551	11002	1701	8930	8942	8928	1097	12778
	13750	15439	18947	17593	13684	19108	8061	16885	5642	8964	8881	18841
	15311	10703	8544	18226	18698	7766	13011	12993	17357	6778	15189	13002
	10083	1067	7944	13701	18247	8858	2375	10780	12562	6618	1888	13569
	368	16443	17735	14088	6201	18221	17786	9563	9562	5156	3903	2386
	547	12480	18001	13049	8197	2495	8339	12208	19466	18977	10488	11974
	4385	8302	19014	8291	16077	19012	16024	5878	16026	17408	16018	16022
	16016	4841	4690	2646	17831	9830	9842	9828	9829	13874	14131	9849
	7012	9847	9827	8403	9851	9865	8405	9844	8406	4019	10234	7448
	538	12803	17808	10782	7592	17774	7855	9687	15478	3508	2475	17776
	12927	17662	982	4005	17990	6216	13658
269:	14096	14101	3422	9935	9907	11404	3149	1688	10616	10023	13889	4400
	13959	6604	8205	13382	8564	13019	14460	15364	2532	8729	14099	19644
	19650	19653	11186	19315	2228	13473	8252	19754	7420	12978	18550	13931
	11080	8842	17395	1271	18960	9745	4617	1713	12914	5411	11902	15306
	3958	14094	14091	14074	7042	19574	7859	17118	9165	9458	1459	14832
	13165	14520	10512	17335	15352	14170	13097	5335	9461	13601	12995	8362
	2662	13567	14785	3435	11646	11805	18390	4017	8969	6711	4962	8794
	9291	7926	18526	14047	14393	7407	3866	5766	11775	19021	15525	1840
	13661	14072	18260
270:	16324	10027	16399	19626	19600	18167	7908	9950	15476	16909	13757	17032
	10125	16306	5372	19301	1764	9641	11812	14642	18106	18647	13221	9050
	13225	2608	858	9561	5437	14594	5439	6002	12210	12290	5485	6003
	13981	16840	17398	15782	13385	4464	894	19838	9505	11810	18088	18145
	16187	1329	14353	14351	14349	10819	8846	9429	16618	17714	16634	13050
	10112	19589	3140	14486	3444	16943	12766	19496	9070
271:	2051	2869	19263	12625	10041	11778	3745	15945	19372	16735	5498	12285
	7206	9395	16559	5318	10659	1955	8048	19028	5197	13490	4306	15568
	14829	8639
272:	16396	2024	2104	12134	16012	12790	10384	16093	7594	16424	3265	5193
	1401	2076	7270	19843	9331	4976	4079	18320	7860	6861	14631	5218
	2559	6330	4039	736	7978	11712	7058	13306	9913	17623	5261	3627
	1734	12108	18532	10878	3240	10925	8343	13484	10007	17652	6295	15677
	4058	5272
273:	12662	15327	19093	953	5512	10971	2513	18064	17932	1221	19584	4462
	3536	19638	12776	5617	18924	7810	19428	13350	11809	13768	18047	11036
	10750	3825	17782	18804	2831	18060	18535	9083	17800	9822	14551	11830
	6598	8161	6081	5049	6923	6951	12249
274:	4873	8617	18355	6309	6356	19579	11946	18342	7376	17347	13047	4927
	14649	12482	15068	14383	10603	3075	5557	16054	5542	5206	17331	19720
	11637	5366	16537	14444	11823	2345	18756	7228	11287	3136	3972	2054
	13176	4279	12726	19609	19002	3887	18393	12190	2873	4093	15745	19571
	15950	5128
275:	3391	5673	17748	12970	2049	2756	10716	780	5570	15625	17600	2057
	17079	14041	6494	10663	7787	10440	10550	12319	2852	13899	6288	6170
	14404	7408	2857	4218	2808	18604	6362	17551	11818	511	5580	16146
	1803	12576	13505	7130	9614	2287	6383	500	13389	19443	13855	8834
	1779	5989	19035	7368	11731	19702	17630	783	15741	15774	15442	17674
	2205	5474	2153	18915	13290	9728	17044	8267	18171	1334	15222	15195
	7733	5047	8922	18281	12929	8992	12922	5696	12619	2026	6208	13543
	17275	13703	15244	18936	16496	17942	12986	13118	16224	18419	10277	4864
	9332	2640	16067	11844	6736	15403	4550	3500	4202
276:	9040	9153	13748	18710	9288	19447	1096	7103	8455	3624	18607	16311
	18304	14306	9854	17310	16393	9187	17043	17574	17689	8273	13937	14386
	4911	19063	18212	18817	18819	3310	3329	18815	3312	18959	8476	1386
	2263	8742	15253	7396	3159	5367	18632	11510	4953	18618	495	19426
	10261	849	13607	1099	15505	16314	14154	11065	14523	19079	3856
277:	6298	11547	9455	3560	3298	1224	6573	17886	3688	17535	6514	11001
	16832
278:	9342	1148	8880	13995	9598	18620	16354	596	3713	12096	7035	13429
	19465	13583	8623	4778	4775	4758	6813	14722	2970	16245	18993	16658
	3106	6966	18030	8468	3622	3111	5706	1317	7832	18473	17421	13587
	13609	3484	2614	11857	11213	7994	14581	14105	8667	8513	779	14862
	6228	14830	2526	13752	2961	4199	676	14238	15588	17472	5776	1150
	15882	3232	11072	7812	12851	3715	13664	13123	9048	17974	449	2155
	3081	9548	12652	8280	15483	16721	8031	13645	18580	7580	3619	3479
	6971	18732	15594	9508	10074	14377	14469	1702	11710	13599	19866	8968
	12053	12047	4245	4259
279:	19107	3731	10544	1365	18558	3521	315	15072	9222	995	13810	10579
	12184	1169	10820	8682	17063	17115	11426	7610	8335	6680	19590	669
	12808	2095	3210	7981	8975	13797	19673	10108	12140	14891	4849	9589
	10013	11489	5848	8508	16961	14635	3335	5341	9626	18112	14644	12785
	9168	9484	8109	9837	3294	15636	3494	2017	19052	19769	16972	7811
	19903	14579	7377	4121	7557	13806	6626	9547	10641	12326	19729	7235
	1411	3644	10157	10154	2648	7794	10100	12628	15633	12168	7589	15853
	19745	14933	10255	1291	17736	2414	7188	16138	9089	9415	11728	12818
	6609	8114	10065	12609	4638	16498	2517	12709	17410	15136	17048	16713
	18469	19832	12689	1479	15584	12811	17562	13267	13892	10949	9327	472
	5713	11273	9716	15698	5082	17282	1612	7565	10794	11434	8216	10240
	6588	15316	15700	6461	7231	10826	9945	1966	767	9017	8428	9216
	1791	12429	5223	4910	19009	13274	12648	16308	4798	9611	5747	13303
	2839	10405	4049	4046	969	5871	13542	17764	7597	9272	18722	14315
	13433	12396	17102	19120	3267	2911	2934	2480	15593	1566	15118	17099
	13495	18798	16391	13630	12936	4072	17390	11981	14556	12247	14559	12268
	14560	14577	14558	12267	14576	12226	9387	332	2109	7841	18828	9615
	11137	19936	14986	3553	9388	17887	11929	4554	18866	5440	3007	845
	18184	4097	2805	16099	12066	16033	3934	3003	1594	10929	12343	16214
	17420	5606	4643	10415	14917	6601	15702	10449	19711	13552	1507	19835
	1698	9321	11696	2863	14802	17698	1498	17572	14405	13598	12043	2262
	567	1583	2618	3734	18793	19614	14449	16036	8175	4878	19636	13279
	17286	5486	17542	9087	15980	2290	11904	428	10747	9550	13537	3804
	8971	5843	3250	1514	18175	14056	11021	12535	9436	8113	10697	15175
	13365	7725	5679	15598	19442	16718	6306	9803	17134	10762	2355	13282
	9344	18414	962	10707	13732	15247	16140	14944	8665	3901	12556	13238
	3850	16213	15720	5407	7133	17222	15571	4342	952	16118	708	14879
	19403	2997	14894	498	14617	11730	18476	3794	6451	3609	16585	3917
	3066	16371	13698	6380	5627	5614	3881	18493	6416	12573	15178	11348
	7043	8200	17880	11947	15915	7621	7041	8209	18926	10681	14294	3702
	8567	18263	9236	1978	3932	19800	9976	13414	8046	11624	1429	3768
	17319	17779	7609	16512	3551	630	3171	7227	9996	16499	5172	8855
	13654	9117	3812	16292	19434	10320	1982	3126	14582	9645	1807	19478
	7519	12941	17861	14481	4909	1899	3459	1922	19333	4083	1541	12260
	11304	16432	3209	9163	2838	17263	2745	9173	17072	5826	7351	9534
	19739	19536	7374	17185	14542	17759	5670	4857	6297	13448	7546	11655
	18214	11988	3497	12119	18421	2238	9138	8088	14015	6428	10563	6419
	14637	4955	9470	1521	11999	10694	19034	314	18961	1819	15545	10660
	10122	1038	15537	16448	8891	10556	10553	13996	16085	16717	16720	16629
	3416	6712	18028	18027	18046	18045	18018	2046	2280	11438	7044	432
	10580	10575	17329	2438	2435	12466	13227	13331	3373	4354	1137	14182
	14180	17288	17285	17287	17309	18279	17281	5134	13133	13134	7591	7579
	7578	7590	7622	7593	7619	8710	11257	10155	17393	5497	7611	7613
	3368	3371	3345	3347	3346	11146	18562	15875	3327	11270	617	12175
	16942	6398	14919	6019	6427	16524	15844	4568	13742	8545	8646	17237
	10441	15117	4903	16684	7548	4237	4891	11339	18131	14205	916	15666
	812	8359	2239	6482	16408	10426	1946	16250	7313	7296	10169	13861
	16153	7466	6375	11598	1049	14326	7059	9757	6237	2331	4295	3739
	1600	4339	9125	15701	18725	10147	17364	5245	694	5869	7830	14712
	3680	8784	12459	4338	15013	3889	4760	2323	2136	16586	3470	11223
	13993	8676	4320	6154	958	7689	4997	6877	12357	17840	823	1007
	2433	5629	7080	8534	2047	12554	18572	15080	13812	14010	15955	19762
	16570	11714	12363	13476	19136	3340	8548	16230	13481	15749	6845	2819
	2803	2874	2798	2851	2774	2772	2847	2769	11161	2801	14362	3590
	1712	8501	2235	2242	2245	2243	18869	17430	17096	16063	16061	4419
	4399	7500	7499	7479	7507	7501	7478	2530	9228	852	5988	15329
	19925	15542	16445	15520	16918	16920	16451	14285	16922	15560	3473	2729
	2751	2748	2730	1449	313	6008	10997	19320	14355	13633
280:	15402	17754	15436	17629	9518	8079	14324	10849	9618	16771	13444	11169
	12000	16733	11507	10938	18192	18292	19813	13096	7912	8166	14940	10205
	3198	12339	17093	6121	18940	9368	16294	13760	16750	10258	19864	717
	17453	996	3004	19619	15600	2042	7099	14437	10711	13380	19640	16532
	2201	1802	2309	6647	9806	6879	9457	8208	12113	10109	11945	9605
	12639	15237	13105	11307	15891	3950	15781	412	17950	8118	5602	1129
	18116	15989	10069	5174	1902	2069	8549	11084	3460	2119	3544	5463
	19760	6025	2277	7245	14680	12142	11315	10103	11686	7410	5674	18381
	3114	6700	7140	6390	1801	5927	9743	14343	16776	14417	6038	14869
	8795	12626	14704	15315	19906	16747	15411	2945	6780	4297	8158	3758
	5621	3569	4034	12467	19872	13169	1081	8095	1736	18688	9902	11293
	1423	19566	17541	1444	2075	19482	17202	17020	15746	15846	16117	2321
	9835	4123	7173	5998	4409	19765	10646	9885	10056	18669	12506	11744
	14243	2630	5877	7111	5671	1529	1675	7444	6146	3023	3898	18187
	1165	11993	15839	10330	6061	5491	4227	3781	9058	17595	11079	11202
	7362	10144	11840	8333	13318	3170	6878	6916	16201	15777	19884	5750
	12644	18885	13200	8731	18368	8181	14701	7566	13576	7966	16096	3440
	4942	4573	2833	10605	7925	14032	3404	4390	11831	17429	15424	19414
	16495	16622	11298	10131	6694	18316	8214	4497	14461	11010	15850	4991
	15394	15401	5554	7553	7642	6491	5072	16712	18970	17407	1013	5799
	15429	13670	4021	17706	15034	10501	15109	5127	9817	12262	12837	1717
	18387	17051	1748	3020	8327	5847	16045	1172	5230	13057	10892	8743
	3975	4171	5329	579	17409	16177	5306	5472	11154	9931	4136	5374
	2437	15963	2773	17124	17810	18806	13785	2757	10243	16360	5612	16322
	12186	2514	18051	19245	15150	17624	1481	7727	15029	17521	3890	2359
	5655	15000	19595	10596	9889	9655	9987	11358	19111	11956	4272	6625
	9408	15892	4615	17792	13508	980	2199	5653	15516	15373	15610	13373
	9133	4362	10774	17361	10744	477	554	11553	14415	17110	12358	14292
	13332	11804	11811	9941	13978	11248	18909	19512	16233	3315	13090	18834
	11850	7862	19388	5804	19237	19460	4704	13932	6772	6661	6659	2180
	12650	18850	12384	12309	430	13903	13110	18218	9954	6627	8550	4388
	18332	16577	10907	816	14925	18100	1185	19304	652	9511	10006	6151
	12106	6150	12493	7145	15562	2025	18478	7025	7026	4485	14496	7121
	15494	13310	16051	8638	5236	11905	7716	16724	17428	13977	12065	14350
	8192	14225	12919	19150	3456	17883	4875	7007	6429	17497	12548	9527
	12164	398	13983	6190	3241	2352	5757	2425	19500	3188	13614	16480
	1042	2584	18280	17452	1855	15112	8640	9646	8269	17863	2113	19671
	4472	8213	10111	7758	11259	15393	17215	10615	14115	18114	19469	2426
	8337	9601	19412	1557	13838	5949	16547	12016	6009	17996	16020	13608
	18118	4335	2510	2358	6156	10954	3031	10072	16042	9333	971	1426
	6023	16744	18147	18700	17680	17679	4200	2825	14313	17189	9300	3978
	3376	2704	12344	17083	7635	8941	10128	19694	13357	12141	8860	8119
	8265	15908	3690	8287	5054	16229	19707	8187	12767	8492	1917	743
	3160	17877	8353	12737	8570	6610	16241	17568	17643	18099	7570	6986
	13046	14790	11141	18103	19929	4945	4863	7823	1957	13295	8615	7666
	19503	18182	17400	18683	13167	474	4854	8015	15919	17049	6539	8967
	18743	18229	1022	7970	16839	19367	17290	18980	13370	3941	6507	16184
	8973	2098	2078	3254	17794	10311	17483	2883	18643	13517	3729	1887
	1368	14447	2804	10407	12834	17444	18805	15242	15905	18413	11332	15147
	5790	12206	12866	11142	2298	19828	19319	18179	10150	5563	17462	11283
	10674	17994	7653	11921	8670	18362	11531	10803	14348	18130	4425	7792
	3742	4069	13020	8929	12909	5891	3491	11742	10960	1707	7447	11965
	14102	16993	19751	4688	6075	5409	928	14103	17862	5849	11486	3424
	13098	6417	1263	14274	3937	4584	19652	3860	4922	7959	16775
281:	8694	12679	18529	10066	2678	7179	6485	2766	9063	12300	13395	10238
	7333	12286	11528	7686	10627	19423	16297	3477	14307	14332	4683	6386
	14764	18946	11842	6708	4206	7511	14161	4948	18236	16555	19706	16935
	9276	1705	8251	17729	14148	10222	1114	14562	7443	7171	12646	525
	3814	3651	11471	13348	17253	3355	5958	18560	9986	5060	4869	4769
	4656	9642	648	1410	2931	18886	8791	11423	16482	11075	17009	10151
	4552	4158	14670	5141	19526	19457	6070	18446	10680	4669	10087	10957
	4753	6048	16338	807	6752	7620	2892	903	10046	10520	10397	14613
	1203	19013	13180	13847	9021	8726	11936	9469	19630	17914	1539	16852
	2684	10230	15144	7414	15169	16341	18719	4705	14987	1054	1301	8183
	10260	12916	14291	9101	5800	9224	19194	2786	14896	17070	13453	10805
	8590	1236	3657	17563	5588	19064	6364	15198	2518	14895	12534	11548
	11147	5143	4729	18858	19784	5715	6997	19753	10859	13393	5535	514
	6792	15326	7576	4957	14042	1374	8856	9698	10313	12942	14998	18662
	4919	12571	16319	920	17169	6335	5044	6370	18927	5610	2268	12436
	6087	3030	2039	16323	8447	18750	16277	2412	18682	18358	12549	18406
	1390	8657	15832	8609	6974	6281	8591	1357	5973	16208	12678	10423
	18094	17711	4111	10235	10848	1743	3109	9476	6992	16202	18853	17948
	2213	12570	4347	4954	1510	15130	3018	12888	12264	16727	16884	19498
	10768	14695	16944	12594	14452	18055	12881	19189	10784	2720	13143	5352
	7662	15194	18003	5874	16708	14822	14749	15941	2031	15715	10075	12830
	6407	9819	6114	8177	6890	5432	15138	19857	18139	1174	8978	8120
	9900	6781	18900
282:	9488	13616	15590	2297	2284	4876	8155	4503	12483	1657	3791	899
	884	18026	18515	18521	18025	17955	559	18537	557	16157	10429	13191
	7467	11321	7432	14150	2723	12764	12504	16859	19196	19198	19204	18436
	19288	19227	19280	19200	19248	19885	18438	19282	18463	19259	19224	19904
	19285	18461	19231	19909	19908	19292	19887	19310	19924	19920	19912	19255
	19201	19905	18443	19860	19222	18441	18425	19252	19253	19233	16129	15836
	3701	13372	6579	3072	10251	10921	11241	1852	14936	13471	2795	13570
	13592	5773	13546	3179	4658	5425	19564	6136	5441	19623	527	19056
	16702	16685	2755	4581	1965	4319	3694	3892	6668	6060	18290	1483
	12475	6925	2154	2749	11421	12831	15432	10442	12991	12491	11934	17139
	10324	10227	2579	15389	7706	18794	1036	10124	11030	10073	15797	12617
	3046	10666	11189	6165	6563	8600	2237	11718	1494	17677	1968	11225
	3759	19617	1963	1230	9162	14979	2576	10989	16101	1141	17522	11388
	19518	14314	9391	12490	9259	9858	9861	9878	3027	8297	6634	2580
	564	536	513	13257	12751	2800	4947	6596	6727	12672	13807	4898
	6067	7002	9776	9305	5205	17859	10706	9919	5631	3026	14137	17947
	13952	10969	17270	11134	11135	1464	1994	7337	4302	19858	14835	3132
	18541	18685	15115	13477	7242	5034	4232	4229	7849	3936	9400	7074
	1711	13593	1267	7020	6203	5752	11017	19211	16500	5464	16481	16479
	11262	9474	2657	9912	17012	17455	16989	2421	11405	4134	4135	13791
	3760	4746	7250	11301	16165	19238	6369	8863	6260	10640	806	1373
	1942	9798	10528	1672	6827	6035	18378	2844	12503	12481	18439	9335
	6502	16929	6137	3738	14085	11558	7893	670	2543	714	7694	12393
	8619	7916	5416	4052	6540	18074	16465	12699	15044	7738	6074	5428
	10870	18932	19505	13905	15650	18814	14236	16809	16695	16811	6358	6377
	9099	13233	15830	15855	16971	9952	13720	19312	2882	17849	13346	11894
	13927	10068	15728	15724	1669	10842	5211	5573	11524	15993	10964	11561
	8701	8076	2340	9030	12177	8173	15874	13078	13080	15578	16552	11954
	17694	13066	11930	9502	8839	10015	14690	15514	14413	543	5118	19602
	14943	17995	1533	15898	17708	13695	8703	7375	12243	12246	10408	1134
	7047	7069	1384	664	14725	13989	14727	12311	550	588	2405	3105
	1325	3085	4328	12896	10416	5716	5734	5080	10206	14083	5736	12511
	7935	9350	11129	2367	15487	18863	6310	16136	16616	17976	3605	11762
	6296	7735	3664	15502	16857	13747	16858	1704	8970	8242	3966	18776
	2037	13001	18364	8423	12792	14596	1694	2481	2503	17868	531	18007
	7674	7678	2470	5590	15264	15917	13152	11243	11441	18206	19145	19302
	5018	2605	15951	15519	11689	3205	8293	4752	4648	2589	3319	3377
	6240	2569	2542	4651	4796	4774	4647	4644	4101	4693	4777	4022
	4018	4020	4714	3995	4133	4060	4668	4197	1988	16865	1441	16612
	758	17827	17825	6420	6905	12143	5753	17242	18540	553	5233	15097
	3540	2308	14280	13379	15914	9672	9677	653	5107	10689	462	16974
	6046	7495	7028	14188	17597	18006	10434	5334	13292	9733	15419	1170
	1187	16770	17137	16828	16680	5597	11937	16404	17267	5157	13849	8607
	18671	4234	14051	1528	11389	17864	17893	17962	561	12269	12414	14893
	13196	10758	18547	563	558	18543	532	5357	13969	17925	3015	560
	3362	5820	7763	960	17879	10047	537	3873	8224	535	555	556
	585	17895	562	2019	11745	11746	8692	18545	533	19830	13796	13716
	3761	15630	15770	10070	7827	6148	2018	7825	7828	8464	8157	7805
	3409	3407	3366	3430	3387	3429	7806	7768	7804	7797	7772	7799
	7771	7776	7822	7820	5838	18949	16551	2875	14511	15973	10383	9041
	11443	17876	17871	1216	3604	18867	11364	7984	7564	5619	16508	9249
	19817	11408	4489	7775	16600	11204	11343	6760	16109	12509	11456	1471
	12963	1159	14149	17898	4525	8069	5516	3522	17718	17147	2073	10352
	9958	12355	4590	8758	17922	17979	15581	2550	4547	11843	1492	9863
	9732	12824	4458	4446	2432	8850	10795	2573	12282	1601	2523	1313
	4569	6686	7903	18104	7660	11455	7569	10308	14385	17379	16758
283:	15531	6256	3677	12101	13037	10190	10248	11043	822	16817	7460	8374
	602	15713	1872	6858	11835	10862	12518	10351	1380	17170	18399	10269
	9599	10039	5643	1398	5309	2941	13830	626	17116	19140	11467	18598
	13371	8055	11493	2929	12146	10801	14962	19804	16176	17439	2811	496
	2695	19371	12254	9322	16426	17763	3303	7474	8440	6587	7356	10022
	7182	7542	19708	4269	14632	12375	12229	13513	5894	17174	11372	14162
	15843	13669	2318	4478	4374	2133	1853	5420	9504	9838	8996	1878
	5702	13717	1634	18754	3402	15053	14759	16550	3082	4918	14755	14416
	9281	8312	16172	11469	9564	18095	19075	13665	14192	9419	14729	2368
	10551	14620	6612	19450	454	8874	19239	7854	8878	17842	15819	18002
	13934	12440	12425	7219	13659	3489	18080	14572	6789	15128	8759	17772
	11836	13231	12637	8764	17762	17882	18242	8340	4916	1839	12027	518
	6044	6520	14817	9982	15655	16991	6478	8575	17011	7819	19454	11008
	3852	16238	12816	18889	19856	3049	6004	4420	14200	19451	3399	3287
	757	4411	3141	3803	3354	2485	600	11184	11333	15248	4728	19407
	13987	16072	13120	493	7888	811	14946	2583	8687	9939	14742	4000
	19160	16329	17352	7798	9075	11087	16289	13321	3576	5304	6605	16610
	12700	5370	15030	7544	2014	8691	16557	17396	12352	3485	16266	13480
	19873	8847	11387	13745	2392	8223	5726	12517	14311	13416	14639	10763
	14203	18402	3129	12616	9840	18774	18715	15765	15812	8125	18204	4445
	799	8531	4265	17783	16560	14953	11093	3886	5539	6863	15521	13084
	19629	18230	15314	6456	5130	7907	9874	4068	9185	2982	8263	8178
	11555	12456	2286	17432	6568	1183	1923	13056	400	18905	11046	17586
	15182	10135	12752	9213	14279	7205	650	6593	6212	18450	15704	9964
	7123	5422	14997	16892	14156	16838	19853	13287	1951	6717	14456	16168
	7220	6525	16069	12673	11280	9572	7263	16613	1000	3100	1497	17231
	9541	2750	6439	3980	10290	15824	17100	4572	9670	8215	6984	12801
	10455	3696	17104	11037	10557	7817	7141	11642	18768	18492	12742	7415
	8653	14608	13015	13405	13121	8857	17133	16228	14222	5302	7237	7488
	5999	3641	566	2958	11175	2130	2764	16189	18380	6548	9673	14038
	14035	7139	15190	5426	19562	18437	17501	644	2919	7071	7261	14062
	13482	15191	11743	19472	19437	6920	13727	15618	15631	19891	11927	10932
	8010	16223	14499	6723	19485	6581	14854	14856	7405	12499	14495	885
	6550	15800	14507	17436	16225	11331	3216	8486	4438	7918	5382	661
	9656	5184	1208	3516	15538	15408	11613	11439	5451	15131	18608	4901
	4791	17602	8576	6930	9997	16304	16968
284:	14546	4993	1029	7514	2917	10152	11335	9594	14726	17937	6935	14151
	10273	5997	7000	10002	6010	16908	7529	2137	5389	15290	8071	6027
	4413	15840	17540	6072	10477	13780	16667	4735	851	17780	8426	4344
	3194	6508	4023	12288	16472	15236	18423	2712	6253	7180	8745	16575
	12645	11798	15025	14872	12821	12055	5069	8763	5400	5829	7669	2817
	5344	1590	8606	18818	16682	18975	16411	12120	1632	2145	10107	3618
	17320	7159	19374	8892	12001	19521	2499	7464	11752	2395	19468	2094
	17945	13685	3703	7073	8473	15278	11750	6376	722	1788	12276	2177
	3659	2253	13161	10545	826	3249	6401	10373	5991	4410	9993	9994
	4785	9814	10962	18365	854	5075	4178	13328	18181	19525	14890	1603
	6161	1186	310	587	2986	3592	12649	3080	15617	11601	13501	13181
	6206	12788	7897	2732	11732	7914	4586	18225	17758	4002	14369	2043
	9570	3991	19823	19209	11521	18784	14970	6131	8658	15987	11465	8315
	19681	17975	1559	5862	3880	11271	2108	14500	11285	16316	19436	10432
	7652	11110	19523	2843	17746	1118	7774	19578	18424	4835	14580	15727
	5582	2332	2821	17034	16853	10210	12320	9478	7636	16940	5379	13944
	8103	651	3131	7232	11772	19236	15809	4006	529	19806	7702	4689
	17509	5782	10158	6488	7040	13260	10093	686	10592	15289	19347	9038
	13009	13925	7052	14143	8739	14688	12107	15485	11400	6474	3104	3107
	14423	1690	5872	1941	9668	3685	11900	10682	15943	14081	8352	10943
	8232	18499	14024	18737	9701	5604	13245	3263	5326	14254	14253	14255
	14251	19565	4990	2198	4887	16593	4192	623	16711	18391	10980	7532
	10316	16098	911	19278	8228	17323	16757	2097	18771	1562	8768	11770
	13930	13210	9302	4394	18666	1679	19230	14127	8256	3565	3486	8305
	17338	12200	14095	16106	16554	5101	9627	11323	13709	19928	15448	7178
	6851	9856	17693	5450	14758	6440	4840	16948	10481	18098	12809	9820
	8612	5560	2328	16487	4316	9793	9587	6024	4610	3518	14472	8379
	17788	16283	940	18073	12073	12239	12641	1517	4520	4253	19655	19157
	7457	14257	13738	9744	6631	11117	7100	10958	1663	19658	830	850
	981	19109	1871	7127	18552	12232	3002	16895	13330	7142	5045	8449
	15114	15111	19687	19859	2450	5262	5468	1622	15404	10115	3091	1432
	7857	9421	10367	16178	16767	18032	14517	10922	4845	5596	17422	17656
	14974	16264	17181	461	10535	13155	18603	4629	7200	12897	12379	19551
	3158	16835	2030	2535	10521	8602	5328	6340	5196	12908	4510	6303
	5493	710	15944	18673	17709	16365	17701	3274	14621	2173	3229	12063
	8399	9226	19709	3984	17274	5355	11541	16698	15860	19429	17598	1573
	5354	3074	10753	6512	4856	14767	3042	14876	3811	856	14273	14271
	2338	13640	5456	5459	8719	10281	6810	7883	15726	15508	17683	3047
	16285	12882	13574	3723	18442	11447	14287	18277	8323	8320	19125	8734
	18846	8429	8735	6351	18016	7745	18581	16456	13515	4296	16800	3915
	7965	11716	12382	5285	552	18741	19308	12228	713	1275	3193	601
	4292	18067	7027	15113
285:	14398	1558	18339	13431	13428	4182	16260	10619	6127	12005	7704	5064
	13072	4395	9032	19907	9588	478	12782	9917	13100	5202	10562	2044
	4452	10775	3543	14840	14071	14020	10406	14218	1797	16955	3879	10312
	12432	14824	7082	14554	15744	1508	15866	16232	6797	7077	5816	3596
	3597	3311	15022	881	2638	906	212	12037	12039	3458	9042	8812
	3217	9466	2270	18102	610	2431	16650	14418	2251	1244	4235	6826
	16651	14435	13004	7852	18349	9553	9262	4340	9235	15603	1413	17223
	8296	10538	16071	16048	2120	19847	11239	10420	18968	12345	2995	2159
	2549	3987	7551	19585	5685	4861	16862	8677	13466	11435	11230	6015
	6649	8097	16025	9440	1353	16249	16151	9398	15930	11226	2870	12373
	15043	11765	10145	11071	9537	9095	5496	2350	11756	7967	13362	2624
	7924	6715	15074	17068	9275	10177	11007	14857	8951	12182	11643	13590
	1740	7684	910	929	9093	10018	13344	16978	6305	11378	17544	11969
	1393	15937	11803	16631	13126	14108	18524	9644	8425	8421	8420	10755
	16696	15994	10757	1143	3549	5868	5126	7506	19032	12669	12547	4707
	4710	4708	4732	4736	4733	8772	4730	4711	3385	1831	2528	2304
	13223	15197	15098	11673	4386	11884	2336	9633	9118	14693	2399	12725
	14548	11011	18359	14692	5652	5434	9755	9513	2681	4364	19777	13525
	12903	6859	4326	6088	11611	3068	3578	13940	8582	14440	19618	9826
	13275	18012	9371	10057	5985	18222	15021	11502	14888	14001	1279	17431
	18172	7826	14232	8317	10294	7425	15565	5531	17742	5564	18144	14389
	13857	7113	16444	7400	10835	13461	4722	2115	13951	19022	5256	18254
	7276	16719	1045	2920	3279	19766	684	19811	1237	15371	10076	8004
	18712	2781	4940	11219	2673	19836	18985	14312	15772	3933	6426	7906
	15599	11860	1907	18897	14263	16522	4672	13737	574	16112	15415	11543
	3433	7746	4115	9525	5392	9445	2252	14696	17114	1400	9349	4881
	14484	18930	2533	10428	7616	18415	10497	12585	10236	4874	19793	4633
	12544	1995	17255	6130	10439	8504	7487	15261	6768	12863	3628	15985
	17752	5019	2036	2840	18127	12020	9264	14875	7498	1448	1059	9636
	2644	15865	6207	9893	3161	10031	16927	2065	7894	2171	4174	1254
	18701	10035	19271	1389	12810	4747	12671	19586	10698	5651	9018	1931
	4286	7712	3519	12527	13943	6181	5039	13948	16797	16826	18966	5145
	17610	6304	8895	7744	9280	9284	10647	16558
286:	9313	1653	17351	14049	14657	1383	1015	18068	4392	10779	16383	9920
	487	9934	2830	3247	2351	18430	10228	17404	17658	5336	5337	17655
	1350	17686	17699	17685	5353	17632	5339	17682	1645	1642	1640	1620
	1616	8916	8212	9895	2832	2783	4293	6486	10048	4251	5819	5822
	5393	7543	8453	7971	5457	12283	11526	4620	4594	4593	12447	12446
	12443	12421	12418	12419	4579	6639	4625	4624	4598	8851	4628	4580
	4631	14489	13819	14308	18593	5807	15337	2081	12213	5581	14765	7934
	18948	16079	6446	10336	6213	8673	10129	12010	19004	19003	13184	10392
	11721	7649	9054	15693	14867	14868	7482	1784	9674	1270	19176	1199
	8500	19667	4212	4448	3986	12515	15430	7679	18854	16043	4242	10959
	13366	2878	11272	5100	3297	7169	19051	3475	13999	17136	17946	7102
	9260	9177	5942	18576	8622	9353	18500	14477	16377	4784	16796	16425
	17653	13145	16135	7307	4655	9544	12992	17516	2232	1408	2230	1599
	11103	9661	8918	17531	7259	10941	16332	19728	2216	11076	2670	18069
	18160	18744	1206	2891	5478	5142	4382	2683	19169	7606	11398	12629
	18745	3786	17936	5887	3425	5214	8401	14931	18934	15251	6533	4671
	6531	17015	1412	5092	8805	5545	6049	12372	578	17463	10263	1161
	2814	5758	10686	7449	1330	10508	15984	8027	1288	5176	19668	1710
	19355	3454	13918	14445	2162	14905	2727	17196	7122	9749	10816	13053
	15837	11705	1700	2315	17710	12014	19134	14514	14744	2761	1547	12385
	7818	16211	18823	9872	2759	2806	12795	9882	6415	11514	9287	8821
	10229	14451	7419	9477	11295	15793	15660	11783	701	2955	17659	2504
	17348	16342	15186	6406	8247	5380	13891	1315	1256	19915	1724	2012
	14065	10299	8282	616	13375	9102	16056	11991	9014	9223	19193	5659
	12275	17960	6092	13451	8592	8527	7016	15734	1394	3654	2224	9086
	6674	15201	5282	3178	18367	19531	17843	8903	4152	2519	4236	14485
	8563	18395	2904	12192	17438	7558	8708	17132	14463	2926	3606	14880
	4126	13192	11148	18831	19343	3854	3197	1662	15209	1164	6993	19768
	5846	5825	13392	3964	18837	3676	6815	1563	14197	4983	16851	1080
	10309	13336	6679	6681	19226	1139	18217	6677	11476	10458	11376	11534
	15281	15284	4323	17519	4170	18686	3737	12407	9314	18763	18235	2522
	424	12163	6909	13949	13705	2221	17613	6620	10223	18017	9192	4434
	7858	4011	19099	15849	2703	7837	16579	10683	5630	13463	4008	5853
	15102	13390	15974	3534	9370	7120	13281	10677	6277	7442	19758	3296
	12002	5857	6669	12717	15152	973	15151	5971	2689	2692	14737	15184
	19235	11966	9246	1051	16723	7949	2967	13928	1691	17376	8438	6021
	5801	5705	16980	13383	18063	14100	12568	5238	12085	17418	17386	7929
	19078	16133	15135	4577	8414	19149	3621	10044	6007	16987	3208	3567
	14540	9431	6837	9742	14981	18407	10889	5572	15933	2966	14048	10199
	19043	8146	11747	2107	1220	19179	13356	3167	14446	7973	13997	10221
	14201	10975	11488	8663	6381	2578	2575	14911	6552	16339	768	1468
	18066	12089	17874	13394	9319	919	12747	7881	17584	17587	6226	19910
	7627	18249	9111	10610	1083	6509	17564	5851	15935	14750	1415	7919
	8561	13034	1757	7298	10482	17985	2674	18011	18159	6510	8905	4745
	13968	5850	6365	18717	6735	7063	6097	7741	6692	16850	9791	1250
	1976
287:	14033	3069	10887	17423	15804	10725	1039	8823	13656	1864	18512	3164
	11788	9149	8808	5438	15880	3992	800	12872	18261	8766	14438	3187
	10085	4070	901	13535	2677	8285	10764	19705	18563	18309	4608	19842
	15814	13706	2652	1108	2600	11436	8221	18056	9465	14769	5688	5397
	3900	1598	6393	5004	17760	15162	12125	12647	7151	11629	14327	14070
	16315	15318	15939	1024	17173	7378	11794	17214	14034	2307	3146	11136
	745	14629	1219	8349	9131	14106	2574	6164	13786	12212	288	9930
	5003	15379	17113	11587	10350	16113	1863	10534	16794	10418	3427	14528
	9660	19113	14120	18931	693	10649	15876	620	11764	4473	14614	15160
	2527	7562	15931	15942	9412	10452	1278	9417	14663	6750	3059	18739
	5729	11588	8690	1571	1240	12230	10197	3943	9396	19704	5587	3849
	6395	2512	1364	4248	9916	17140	19660	9460	2383	2545	10421	10451
	7528	17188	5982	10710	4743	17869	13867	15895	18341	13127	9124	11340
	15482	4846	15991	3001	8402	17663	13283	11123	13596	1151	10214	18536
	11869	2959	17628	2207	1422	5187	12698	16068	18921	3969	18923	15856
	13136	15313	11878	7997	15604	14266	18654	16893	14982	19298	8644	8268
	18840	18022	3726	3772	13530	12761	10851	9602	19087	12685	5286	4627
	16074	10376	1178	17450	2560	825	10966	6542	5902	17622	13520	16539
	494	18321	9756	4078	13338	11983	15481	17256	1850	10700	6895	19331
	5603	16542	8629	14464	8541	11955	15582	1615	9393	11799	9705	10217
	3894	5124	17122	10037	19916	3503	17417	13729	5423	16076	1843	12859
	17649	1593	15088	11536	19105	6404	17208	9567	4208	3180	3864	9156
	12512	15134	8390	16883	10879	8150	9719	12593	14926	14282	17273	6293
	6538	13207	13438	1179	13854	1534	12605	12235	11858	6290	13936	15363
	13652	15361	19700	3899	1614	9390	19686	12223	19528	1763	6129	2975
	18334	4649	2415	18042	5063	4001	2807	5550	868	2976	14624	3321
	10604	2149	11085	14039	5615	8502	14424	9369	14136	4541	3019	11951
	18036	14808	13308	6822	19066	15323	12540	9754	11217	12299	6562	19219
	2211	1980	409	1970	8961	14118	1424	14183	13802	3223	12957	11050
	12155	17192	14643	15307	6911	12324	7373	11544	13247	17437	12217	11827
	14360	17578	12753	6662	19268	1665	7212	10672	7843	1289	5216	4585
	19139	7937	16633	5644	16320	11433	9472	12965	12360	3202	11429	11427
	19689	1587	773	8864	14046	5266	9201	17227	10510	7291	10904	10655
	4965	17040	2183	16534	15799	9967	13632	6850	19691	8788	16409	4673
	17193	14098	11916	2451	4834	18460	18183	14691	19318	1489	12987	16441
	17707	16821	18525	6452	5795	10306	808	5051	9434	4528	13359	1168
	13904	3700	15002	12152	6475	2944	15440	18551	1414	4231	14124	8358
	6492	16565	17743	5954	16843	954	5541	5859	18096	12827	15357	11338
	14884	7974	10246	5834	3697	450	16197	8091	11058	8780	10822	12036
	3632	1407	632	5735	2700	8621	4284	18605	5544	3482	7892	18630
	6701	13975	18530	11623	10209	10341	12178	19162	4832	13434	19639	7936
	1306	11386	4376	19409	7887	13961	19229	11753	10931	1751	18762	698
	7891	10920	8887	5093	14119	434	10475	8949	9491	15868	17642	9386
	8713	17515	11664	12640	4630	9414	7285	9296	14419	9418	14870	5136
	2943	8767	9080	9232	9233	9981	8666	11032	6927	1544	17476	16917
	19548	12188	2244	15586	11795	14909	4751	13962	9106	429	18945	4616
	5199	10178	18224	7863
288:	14033	3069	10887	17423	15804	10725	1039	8823	287	9557	13656	1864
	18512	3164	11788	9149	8808	5438	15880	3992	800	12872	18261	8766
	14438	3187	10085	4070	901	13535	2677	8285	10764	19705	18563	18309
	4608	19842	15814	13706	2652	1108	2600	11436	8221	18056	9465	14769
	5688	5397	3900	1598	6393	5004	17760	15162	12125	12647	7151	11629
	14327	14070	16315	15318	15939	1024	17173	7378	11794	17214	14034	2307
	3146	11136	745	14629	1219	8349	9131	14106	2574	6164	13786	12212
	5003	15379	17113	11587	10350	16113	1863	10534	16794	10418	3427	14528
	9660	19113	14120	18931	693	10649	15876	620	11764	4473	14614	15160
	2527	7562	9412	15931	15942	10452	1278	9417	14663	6750	3059	18739
	5729	11588	8690	1571	1240	12230	10197	3943	9396	19704	5587	3849
	6395	2512	1364	4248	9916	17140	19660	5811	9460	2383	2545	10421
	10451	7528	17188	5982	10710	4743	17869	13867	15895	18341	13127	9124
	11340	15482	4846	15991	3001	8402	17663	13283	11123	13596	1151	10214
	18536	11869	2959	17628	2207	1422	5187	12698	16068	18923	3969	18921
	15856	13136	15313	11878	7997	15604	14266	18654	16893	14982	19298	8644
	8268	18840	18022	3726	3772	13530	12761	10851	9602	19087	12685	5286
	4627	16074	10376	1178	17450	2560	825	10966	6542	5902	17622	13520
	16539	494	18321	9756	4078	13338	11983	15481	17256	1850	10700	6895
	19331	5603	16542	8629	14464	8541	11955	15582	1615	9393	11799	9705
	10217	3894	5124	17122	10037	19916	3503	17417	13729	5423	16076	1843
	12859	17649	1593	15088	11536	19105	6404	17208	9567	4208	3180	3864
	9156	12512	15134	8390	16883	10879	8150	9719	12593	14926	14282	17273
	6293	6538	13207	13438	1179	13854	1534	12605	12235	11858	6290	13936
	15363	15361	13652	19700	3899	1614	9390	19686	12223	19528	1763	2975
	6129	14424	9369	868	4001	2807	5550	18042	4649	2415	5063	14136
	18334	4541	2976	3321	2149	14624	10604	14865	4260	2050	11085	14039
	5615	11951	13308	18036	12540	14808	6822	19066	15323	9754	11217	6562
	19219	2211	1980	409	1970	1424	14183	3223	14118	17192	13802	12957
	12155	11050	12753	8961	6911	12324	7373	14643	15307	11544	6662	13247
	17578	14360	17437	1289	5216	7937	4585	19139	16633	5644	16320	11433
	9472	12965	12360	3202	11429	11427	19689	1587	773	8864	14046	5266
	9201	10510	17227	16534	7291	9967	10904	10655	19691	8788	16409	4673
	14098	2451	17193	11916	14691	4834	18460	12987	18183	2944	1489	19318
	17707	10306	5795	6452	13359	16821	18525	16441	808	5051	4528	9434
	1168	13904	954	5541	5859	17743	14884	5954	16843	18096	12827	15357
	6492	16565	11338	19639	5834	10246	3697	7974	4376	19409	11058	16197
	8091	1751	18762	450	1407	3632	8780	2700	8621	12036	18605	5735
	632	10822	5544	6701	7892	10341	13975	4284	3482	5093	14119	434
	10475	8949	9491	15868	17642	9386	8713	17515	11664	12640	4630	9414
	7285	9296	14419	9418	14870	5136	2943	8767	9080	9232	9233	9981
	8666	11032	6927	1544	16917	19548	12188	2244	15586	11795	14909	4751
	13962	9106	429	18945	4616	5199	10178	18224	7863
289:	5523	4313	17448	17841	12777	10355	19605	19897	13150	13788	8075	5624
	13087	8533	15156	16282	4418	5362	17314	16470	9691	6830	3539	8947
	11303	15847	12513	12958	12763	11550	1760	7062	5192	9953	3474	3481
	8116	13424	12304	5690	13692	18678	7503	12740	12452	4939	13248	3994
	5264	3961	4726	4657	19545	17062	16253	1040	7214	5231	11985	5953
	3077	10399	11073	3556	17461	15325	9938	4761	10626	12944	12889	11220
	6105	15005	17775	7340	16006	1428	19624	11919	12601	642	3843	3912
	4096	12974	13254	2664	16161	8861	13261	13575	9768	14949	19126	4880
	5824	9299	14874	5687	2782	17505	17726	6275	3175	13677	4398	4025
	5376	4677	18843	7388	15255	14128	2596	1938	14569	13062	4742	13091
	16870	9122	11112	12762	3052	11166	2182	15922	13893	6314	10549	17230
	10781	6932	11582	16011	6946	9000	6251	9407	11924	9877	6838	15556
	15794	11808	19431	2295	1090	12514	7454	18403	2529	18862	19419	12173
	3773	11501	1975	14174	16124	8277	11572	2186	4994	16607	12215	7089
	1851	16812	1689	3238	11697	1993	14319	8424	1113	3997	2880	18417
	15224	9191	8757	16205	13252	3135	18125	1294	2016	407	744	18246
	19479	15883	18721	4036	14388	15680	19029	5232	12705	8217	16267	8222
	11264	961	12449	16916	13761	3615	7172	14831	14301	16476	14882	7390
	14607	14441	5660	11599	11359	4092	17343	13568	581	5071	859	5934
	10571	12380	8016	725	9697	6714	8705	17074	1644	11944	12826	11957
	4502	5499	5549	14898	1200	7101	18165	6291	6133	13712	11143	13419
	3259	14157	11554	16748	6359	2357	941	11901	18451	1984	931	4926
	6943	18727	6551	17793	4278	4905	3893	11593	10509	15596	4274	3302
	3510	8231	18704	13258	4695	1347	8641	4776	16869	9696	16749	12938
	4466	5831	2176	13023	7184	5489	8886	12920	6529	871	15574	8579
	17493	7527	18633	2515	3708	13536	7991	3379	16897
290:	13013	2572	15390	1939	14078	15522	9968	9379	18412	18308	9944	6870
	12684	3058	2845	13741	4261	19592	4701	17626	13030	8728	11077	4091
	4089	17112	14261	13039	2448	19588	19632	10171	3875	16215	6248	6904
	17027	14757	14672	18336	6783	16361	13415	7175	19494	2544	3982	12302
	12865	16091	6469	16191	15776	6721	2879	4779	2699	14780	16535	10778
	10809	10295	3281	8912	10017	11633	16868	2259	19421	16353	2746	14045
	11766	14772	5191	8574	17749	1617	17592	15384
291:	17667	17054	19440	15213	7334	2141	3727	9845	11081	9526	12337	15918
	15597	3255	10276	970	16639	19309	2222	15291	19583	14945	7813	9966
	1286	1910	10491	19293	16030	19104	463	11758	9810	14676	9957	4241
	14669
292:	9221	9220	6193	3253	14842	13721	4160	4741	7322	10303	16057	13497
	13494	13514	3142	3155	3203	3201	3204	3231	3227	3230	3228	5987
	3206	17127	17830	17527	19410	19413	12404	13866	13360	13864	13860	1092
	18317	16638	3344	11672	1048	5311	11768	18957	9486	5520	4119	3271
	14470	11906	13464	5650	8589	1858	2994	2964	2947	2923	14662	12783
	5730	10701	3718	10132	8593	12855	6873	15376	15374	19023	14646	14645
	9650	10687	844	17322	17326	6887	4652	3798	14561	15275	12003	17738
	3837	3822	3819	3846	15647	8778	10880	17001	8637	3841	8776	18718
	14843	3863	3817	13443	13421	13442	13498	3923	13787	3280	3276	18190
	4216	12478	8586	7030	8614
293:	13730	7076	16121	2661	2089	15753	13834	1549	18755	5880	14792	946
	10952	15392	19376	15572	11083	1945	12301
294:	19341	4724	19896	3258	5111	599	6431	13589	6942	6192	6252	13509
	19882	4075	1265	10657	10589	5296	15003	18223	18248	2327	1261	9965
	4027	15223	16926	14122	18287	13182	19642	13929	3304	730	11185	4607
	2628	9008	18151	2691	11457	19048	6876	6857	15174	15158	17254	6991
	11670	4110	4109	12772	10184	4872	12819	12694	6264	7048	9607	9648
	12614	8990	7650	19393	5863	3904	10368	7157	16438	13128	17560	7535
	1575	10105	10134	568	13578	4304	9818	7538	9257	18048	15120	18444
	17647	18149	3239	2052	591	16792	10643	9825	9839	18609	3056	3720
	12370	9361	1519	17206	2733	2404	2274	10638	1810	1681	794	5509
	8149	6109	16956	4055	7526	9906	3806	4482	3406	8669	11941	8434
	17353	1718	16055	16094	11109	13291	14317	16938	8688	12395	11729	19065
	14012	4384	7554	19069	18471	16995	10788	16414	3527	4972	4041	7910
	6178	2598	12998	9686	12367	8259	9712	9092	15589	17494	13102	3555
	4223	2428	5186	13868	17026	19357	3670	16415	4465	5164	14396	8679
	11667	4509	11014	4526	10211	3983	11652	13984	11918	3989	1532	19637
	1516	8361	9956	2740	19492	19752	7125	504	18859	1252	13539	6656
	17606	8695	18363	828	4343	4885	16387	12180	16021	6095	15293	7951
	15342	16946	16808	5255	15684	11760	19481	5992	13140	3529	12315	13875
	7700	1035	5477	5476	17832	8331	8176	7166	12559	4721	7461	1735
	1862	4162	19901	18566	16648	17875	1044	17901	16027	3656	5177	4493
	8389	7751	16700	19251	7033	8539	8515	6547	17753	6527	7218	4305
	18907	2441	16894	8368	15641	7365	8148	6445	5873	7736	9634	9685
	9688	5086	9147	19697	6980	13974	14084	14057	14234	16981	7596	2200
	9879	6560	7032	4346	9104	12902	9061	16803	18628	5254	6524	19495
	6553	1235	13979	4589	5122	8755	5028	18168	8279	14683	17291	3546
	19846	17369	19927	12627	14146	16105	17234	15988	10866	12145	8926	6958
	8927	4271	16119	10894	10871	14052	4521	4546	19411	17524	542	18878
	6595	2294	11153	9855	9127	6844	8065	9659	15206	10237	10286	5228
	7708	16193	16173	1816	2487	7234	18474	15871	18498	18386	9860	16437
	13117	11881	10754	18738	9129	5234	17737	539	13246	11876	5966	6301
	18615	2022	1249	15906	6134	1486	1737	17536	16641	16126	2117	18070
	1727	19256	772	771	16084	13449	415	1588	4731	11912	13036	12904
	10167	10170	10181	11796	7931	4436	19552	15340	9069	6065	9576	14476
	4033	6144	6355	9580	11373	3845	10555	6026	1538	14252	11149	12524
	19249	7977	8112	14058	7344	7310	12622	12584	4642	6874	16745	440
	1628	829	15558	18297	1682	2486	4188	5990	15852	3101	17511	647
	6316	8897	18910	1311	5918	17076	9692	4085	13054	13063	16969	6300
295:	10020	1439	9915	16163	18062	10972	18887	18356	4431	1884	10727	13800
	12798	2858	7343	14738	2791	14434	9539	17180	8662	5767	8275	2125
	6101	2121	7866	9540	2780	5578	19289	18868	12356	8526	2380	13296
	3693	16875	2379	13074	19810	949	14344	1531	16649	14889	14044	4090
	3535	14795	14076	11583	15037	3570	4564	3572	16352	16416	1362	5033
	6120	979	439	15995	19886	17585	14283	1830	6945	9194	11025	1647
	6731	8953	4154	14591	490	17151	13499	11392	12621	8859	12715	16405
	19405	18614	9282	12579	6899	1876	7114	15543	19677	5323	11508	12990
	19006	18475	3862	7010	19748	1860	15260	4534
296:	4540	6354	1755	1754	17371	3891	9752	4896	3390	13183	12597	8675
	19057	6229	4548	17590	5708	4363	6198	17103	9094	2389	11801	14837
	6762
297:	4370	7533	2597	5919	4081	1338	1069	10498	19778	13926	7098	7281
	7968	12746	5461	13420	11565	11748	7477	1523	7224	17897	18082	14625
	17717	1367	15055	3466	18152	3922	5925	14810	16421	9929	17561	14752
	18760	19598	12786	11882	9247	5551	4562	3594	11415	17495	11692	12861
	16921	838	16714	10602	15187	4256	6989	12555	10731	13076	7618	12876
	10208	869	12690	7839	12981	17982	15557	6454	5181	4327	12171	8233
	18590	17657
298:	16642	4906	6546	8807	15527	1974	3476	14666	13472	17605	12833	9706
	9679	4373	19761	15703	11680	18191	19790	19596	6939	14155	3044	7880
	10088	3490
299:	13175	3648	1095	19091	6471	18453	11391	11863	12390	14399	8543	2686
	18964	17580	17565
300:	15769	8388	13307	16886	9617	9239	11198	2854	892	2335	11948	5159
	11294	8727	8073	11276	15356	19272	11054
301:	10572	5247	19764	5097	11353	18884	8479	9341	11247	19532	7752	2918
	6108	14397	6315
302:	17017	11635	5436	17789	15563	8900	12793	3944	11383	6001	6054	7021
	17088	19664	6262	2642	18990	4151	14818	7272	8345	5258	16301	1266
	4095	18986	14878	10156
303:	9378	2246	16919	2665	10325	6537	6515	8525	17336	2540
304:	3039	5198	11864	16540	18772	6379	16878	17787	1436	12983	13229	16103
	16127	8943	12794	16102	16100	4759	19321	8190	2913	15179	3181	5096
	12048	6045	692	3816	5920	473	489	14828	10454	1781	15219	3647
	12472	10767	4582	2698	3361	17036	3796	8573	19690	16677	8338	13554
	16002	17464	6225	3244	7491	16139	13938	11138	14770	18781	12979	6776
	12028	7778	6586
305:	6411	7134	8906	16488	13440	2289	8244	2283	7468	6684	10347	4285
	18467	13646	6223	12937	4443	18455	1856	14107	6321	2537	3970	3246
	12156	16596	16256	12890	15409	19151	4718	5175	11879	18126	18711	9204
	13563	9579	16836
306:	12951	10398	11346	19186	15452	7802	7096	13491	13681	11482	14807	3733
	17000	671	17496	8110	12773	17405	2034	7911	17108	17121	17123	10527
	13104	9923	15163	15161	15672	1133	7107	15159	16049	2789	5414	19524
	9684
307:	17075	12342	704	19539	6820	19480	17050	17008	19402	15902	18440	5435
	2820	13268	13272	6889	3099	441	18043	8140	7516	17639	18575	12426
	10811	14604	7842	4222	5559	17265	12910	16743	603	5928	7036
308:	9770	17349	9595	3084	14563	12042	12040	1361	7692	7690	7693	11111
	13156	9807	11464	16693	16672	16674	16694	16675	11027	16798	4507	7680
	7462	15210	11446	7522	12111	10983	13675	15116	10650	17747	18142	6554
	18344	14846	3610	1916	1918	1920	10514	10515	5814	16298	16963	8419
	5633	10896	11971	11972	11360	9381	15416	8935	9451	16130	7252	12038
	12592	11897	11899	9157	12294	15591	3634	3640	6918	1028	1025	1010
	7954	6924	6937	3533	5048	13534	13474	10893	902	10939	9585	2197
	2948	10393	11720	14453	7240	11026	11092	11088	13756	13185	13798	13762
	13187	13799	16822	9450	14147	11126	10984	11917	14454	6394	8064	8050
	1033	4682	5085	13556	3724	3725	3721	3722	3710	18855	16128	4243
	13813	13676	13159	17397	9345	13367	5940	18157	464	11157	3264	3704
	3705	16760	646	11952	7153	3598	8442	9161	5867	7339	1404	15165
	12587	12670	19102	11078	3824	18746	9973	5144	1144	7739	4130	12607
	1009	1004	1006	11992	1285	16777	7909	4484	8987	406	3215	13397
	1047	5188	16783	5519	18583	11625	11004	11056	11059	6050	3963	13604
	8117	14971	18716	17730	12013	7877	17204	5195	19070	4915	13843	19554
	4417	15788	11190	1693	2846	16330	11950	7243	10375	3691	3684	3687
	17915	16005	1671	19540	10499	10225	15761	1933	15767	2505	8246	16343
	19493	18295	12056	10278	16556	16134	13206	13209	16090	16088	16110	9103
	10581	9834	3635	13203	4829	5828	1395	2655	597	19923	5896	16913
	5772	1466	3658	3683	821	19840	795	820	15202	10661	15913	19756
	19772	405	19504	5314	3672	6313	9762	9767	14249	2904	2899	2900
	8635	18813	7724	8042	16951	14555	9160	2926	14241	2897	2907	6915
	6922	6897	9718	18623	16845	656	654	659	16108	18227	12270	12018
	10063	10061	13077	8431	11119	11118	16150	16966	13427	3374	18426	7386
	11478	3612	3613	3636	6900	6903	16373	19217	6757	19030	10360	13103
	6818	14125	6759	13921	13923	1196	13924	4819	16656	1658	8471	1659
	11962	14022	10872	13157	16640	7948	18681	18699	9529	8931	12442	1512
	3143	16501	19154	6032	6908	18549	9542	12945	3916	18656	12708	15592
	14240	9740	9737	14661	1110	13204	886	14017	9925	2939	15031	19131
	1242	483	2701	9085	19779	2445	3183	8143	14002	9189	19747	2742
	4298	18369	5914	14761	2103	10374	8783	8779	8777	17066	2087	8596
	4717	10877	14774	8813	8334	16562	10853	8024	16960	13916	13919	15016
	15751	12115	10855	2038	746	14851	19743	15226	16549	2267	6875	7927
	1005	9649	9959	7485	19025	14411	14374	9783	13960	2711	9164	3716
	2547	17424	18941	14075	13323	5543	14815	2165	1756	13839	16367	15644
	11187	13216	6388	12024	17755	13911	9905	17809	19499	10196	18637	7216
	7392	11656	13835	19161	16251	11503	6720	2060	15709	5305	7634	17852
	18207	16818	3165	3167	12327	9373	14446	11406	9769	2446	11776	15103
	15104	9805	5938	19634	3749	1241	11031	13445	16440	11033	3821	17474
	17796	16654	11605	11600	17919	4230	11602	17229	15737	7785	3844	3847
	519	688	4144	11023	1132	10390	15833	11665	4077	12918	16563	15661
	7525	9509	17053	887	778	9741	9759	13398	9612	8193	17328	1726
	10300	10279	9459	4992	8844	9571	11468	16657	10900	10059	4404	16431
	11209	1678	3452	10909	10062	4426	1897	4429	8284	16799	18573	16149
	16964	6742	6744	5361	16614	5359	12651	15347	15877	15350	15344	4440
	3577	3488	4442	10910	10911	14004	10912	10807	15901	13213	4453	4455
	4477	4149	10914	4145	4506	4504	4500	4481	577	17702	16492	16427
	16446	11291	16449	16422	11428	16623	11430	19757	14384	11120	19257	11484
	6191	11608	846	12045	12044	14753	16772	3629	13129	8555	3650	3653
	10024	12068	12069	1079	9792	9323	9336	9318	12730	11003	10356	10985
	6919	18233	18757	9875	9790	9802	11466	5912	9363	883	11049	11994
	14457	3872	11895	9736	10974	19097	11659	19098	11658	9788
309:	16801	13132	15648	16447	8652	17833	5712	8074	10690	3442	8077	16017
	4336	4334	18570	13315	10785	16462	16900	17538	1921	19152	17383	12486
	10936	16527	2668	17086	13286	16325	15106	13376	6034	10789	18519	10507
	14473	19441	13022	10831	1501	10537	763	17615	18865	8762	11975	17249
	1355	1352	1731	3928	897	5821	9053	17271	7633	3483	11371	720
	6786	10425	4104	19770	14043	16580	16413	15086	13278	2632	19416	11567
	16915	397	7115	6155	10323	4935	10287	7095	9624	14788	15196	8456
	15783	6496	1418	747	8160	15292	7226	422	8460	777	12388	7677
	4483	18613	12189	5109	11791	10558	6465	10804	9227	17539	7309	3145
	8845	16327	2894	1639	629	7456	18787	8517	4205	13792	7154	8540
	19386	12121	8153	18037	14847	2324	11604	5469	9801	18468	17366	14140
	12739	8760	7404	15266	14677	8556	8459	8628	6999	13739	10675	6389
	19049	14756	908	978	13391	15622	2116	3418	6687	7203	11089	7349
	10890	12729	12757	12691	8258	15154	5394	2810	6594	13059	13162	13166
	13276	13782	13550	9631	11740	13230	18487	12114	13130	765	19329	16985
	16984	5165	16244	3083	4150	10606	8313	3214	11734	4026	14277	12261
	15383	15691	14747	10250	7726	5530	9870	13909	4204	5398	7793	3405
	7816	1214	655	5288	9771	18267	18824	18445	5386	6029	17183	14760
	17340	14748	14228	707	15468	7547	7657	4329	6352	16661	19814	2314
	3041	8145	7582	19453	4067	11236	9815	11632	10505	5399	5401	5701
	3262	7403	12127	7921	3166	6436	1351	6654	13970	6833	12197	8822
	8554	1854	17475	5955	3220	10389	18522	18779	11151	13163	16644	6673
	19122	8310	11845	11773	14281	8380	9076	16982	13329	19679	12412	10783
	16471	12667	4679	11260	17650	666	16258	15997	18585	8921	10213	7197
	5699	9843	6432	2595	1776	740	11551	15041	10033	14055	10738	16517
	16515	13985	6821	4687	3337	14627	7093	14433	13024	16428	7275	3380
	4043	18350	608	14968	2172	16860	13317	8029	18848	11693	11576	15045
	11780	19931	1062	11038	10639	11224	11221	2227	3034	6350	5148	7749
	12030	8913	7568	3467	5635	3053	4469	12468	7698	10930	8896	10567
	5095	10895	11854	16996	7668	15453	7431	7583	8611	5945	3403	2901
	7239	8207	19926	4267	5634	2493	15967	18640	17304	14193	13803	13885
	12365	19133	15544	17454	12090	5312	11677	14113	8439	6438	1093	1935
	8879	6523	2410	4492	1778	17725	1981	17098	9203	11167	16434	12877
	12668	8547	1722	11460	9512	9552	5897	11717	12745	12744	4809	4793
	17479	11627	8724	14508	4543	10264	4971	8893	12051	5043	4355	1461
	19129	13714	13778	4337	1055	12988	17873	9876	17972	770	14159	4321
	9397	9625	4982	12453	12399	15388	13950	19243	9355	1752	1060	16605
	8940	17507	5292	4698	6678	1898	8775	4290	2447	16769	3911	11807
	5120	6985	4114	3219	17056	14361	17019	4749	8281	1577	17419	16965
	17327	5163	19684	6013	18109	15816	18702	17013	15234	16355	14223	9490
	1545	18506	18978	18616	9311	10823	8457	8366	18318	6280	1223	3810
	19685	10902	7638	4578	7075	3196	14114	9172	3199	14132	12017	11925
	1300	1299	18838	3719	12015	11644	5046	3195	19721	18624	15377	19934
	6278	1999	1378	12351	5552	13478	7204	16346	1190	12660	10133	10832
	11577	11662	11896	13158	19477	7317	4352	1153	5239	13031	8108	17846
	9200	7760	2474	1105	8336	10576	16823	13973	16952	16876	12397	479
	1881	1602	6077	2802	12398	4605	2551	8786	2140	2855	2834	9841
	17023	18871	17373	17344	16728	10808	18676	16746	8129	2385	8180	3562
	5933	4787	2996	10676	15651	12169	19544	7801	7900	2278	855	7521
	18383	2546	1234	12032	19919	3652	721	8963	1814	10032	8605	10275
	18902	12424	12526
310:	4993	14546	1029	7514	2917	10152	11335	9594	14726	1405	17937	6935
	14151	10273	685	5997	7222	14658	11749	10121	7000	10002	6010	16908
	7529	2137	5389	15290	8071	6027	4413	15840	17540	6072	5406	10477
	13780	15060	14826	18210	10116	10120	10142	16667	851	4735	17780	8426
	10161	10163	18631	4975	4344	3194	6508	3557	11735	16259	4023	12288
	16472	15236	18423	2712	6253	7180	8745	16575	12645	11798	15025	14872
	12821	12055	5069	8763	5400	5829	7705	7669	2817	5344	1590	8606
	18818	5065	13075	16682	18975	16411	12120	1632	2145	10107	6360	3618
	17320	18641	7159	19374	8892	1683	10110	12001	19521	2499	7464	11752
	11792	2395	3322	19468	1246	2094	17945	13685	3703	7073	8473	15278
	11750	6376	722	8952	1788	12276	2177	8655	3659	2253	15358	9315
	2534	8070	15012	13161	10545	826	3249	6401	10373	13341	5991	4410
	9993	9994	4785	3349	11306	9814	13902	10962	18365	854	5075	13311
	4178	13328	18181	19894	19525	14890	1603	6161	14478	9243	587	2986
	3592	12649	3080	15617	11601	13501	13181	6206	12788	7897	11732	2732
	7914	3063	8862	19937	4586	2634	18225	17758	1058	4002	4561	14369
	2043	9570	3991	19823	19209	11521	18784	14970	6473	6131	8658	7280
	15987	11465	8315	15256	19681	16402	17975	1559	5862	3880	11271	16235
	13872	2108	14500	11285	16316	19436	10432	7652	11110	16345	19523	2843
	17746	1118	7774	19578	18424	4835	14580	15727	5582	2332	15462	2821
	17034	16853	10210	605	14264	12320	9478	13425	7636	16940	15239	5379
	13944	8103	651	15792	12912	15640	3131	7232	11772	19236	15809	4006
	529	19806	7702	4689	17509	3421	19306	5782	5183	10158	6488	7040
	13260	10093	13627	8732	686	10592	15289	19347	9038	13009	13925	7052
	14143	12780	12107	8739	14688	11400	7874	6474	3104	3107	14423	1690
	5872	1941	9668	3685	11900	10682	15943	14081	19665	8352	10943	8232
	4088	18499	14024	14211	18737	9701	5604	13245	3263	5326	14254	14253
	14255	14251	19565	4990	2198	4887	7549	16593	10140	4192	623	1967
	10165	10137	16711	3289	18400	18391	18244	10980	7532	10316	16098	911
	19278	11727	8228	17323	16757	8819	2097	7207	18771	1562	7815	9992
	16477	8768	11770	13930	10114	10138	10097	10099	13210	9302	4394	5658
	18666	1679	11104	1773	19230	14524	6799	14127	8979	14095	16554	5101
	8256	3565	16106	3486	17338	8305	12200	9627	7178	11323	13709	19928
	6851	15448	5450	14758	6440	4840	16948	9856	17693	10481	18098	12809
	9820	8612	5560	2328	16487	4312	4316	9793	17788	16283	4610	8379
	6024	3518	14472	940	18073	4253	1517	12073	19157	19655	12239	12641
	4520	7457	14257	13738	6631	9744	7100	10958	1663	19658	19657	850
	830	981	19109	1871	7127	18552	12232	3002	16895	13330	7142	5045
	8449	15114	15111	19687	19859	2450	5262	5468	1622	15404	3091	1432
	7857	10115	9421	16767	16178	10367	18032	14517	10922	5596	4845	17422
	17656	15742	14974	16264	17181	461	10535	7200	13155	18603	12379	4629
	12897	19551	3158	16835	2030	2535	10521	5328	8602	6303	12908	6340
	16365	17709	15944	18673	710	17701	14621	2173	12063	3274	3229	8399
	3984	9226	19429	19709	17274	5355	16698	11541	15860	3074	17598	1573
	5354	6512	10753	4856	3042	14767	3811	14876	856	14273	14271	2338
	13640	5456	5459	8719	10281	6810	7883	15726	15508	17683	3047	16285
	12882	13574	3723	18442	11447	18277	8323	8320	19125	8734	18846	8429
	8735	6351	18016	7745	18581	16456	13515	4296	16800	3915	7965	11716
	9558	12382	5285	893	552	10143	18741	19308	284	3200	12228	1654
	713	1275	3193	1053	601	4292	7027	15113	11082	1186
311:	13531	15014	19184	4117	8168	10792	10850	19284	5526	10160	888	19833
	19024	13115	9575	3212	14092	11196	16286	14686	5558	17090	17081	16907
	15798	5743	12604	593	8040	15926	19225	11909	5613	8553	2850	19417
	15940	5913	17199	13547	15258	14739	402	14718	10577	2249	2275	7788
	11681	6460	1636	8604	12864	2293	8498	7265	8520	7952	12354	16688
	12128	1747	6881	10130	17339	3222	18097	865	18477	16994	5237	19366
	395	5648	7676	8241	14849	16511	1950	3133	15141	12703	6805	4165
	17466	13619	14951	10472	5901	7341	11090	7199	6418	19254	13897	13967
	6098	8683	6978	11297	10739	18490	11417	13326	15170	8985	4283	10620
	10050	2430	5975	15405	7764	12075	11938	5271	18351	2785	12328	14687
	15954	10173	10759	17131	7683	6392	9694	19508	11227	14431	10991	10955
	5333	17596	14716	12198	17690	13772	11828	9888	1787	3935	15629	403
	11568	12658	9592	16152	16381	18209	19448	16752	14848	2937	5447	7889
	8469	9447	13876	12634	18731	15627	12984	10207	3116	8826	6318	8934
	3762	3827	1708	7049	9274	12720	9444	9420	7354	14664	6570	4045
	1225	17512	4782	7308	1264	7406	3352	15122	18219	1822	2876	14134
	2291	3261	16739	2348	11942	11875	9500	6270	9871	6173	5722	16368
	13322	16507	13154	9751	11960	1438	3048	4276	19604	2680	11597	11557
	8211	1536	11015	10386	6448	17614	15609	11101	10314	7156	6322	4619
	11816	14775	1023	3639	18377	18621	18433	10468	17302	1949	17435	9669
	4544	1990	7409	11278	9214	16595	11711	18807	4491	14566	15498	15400
	17691	19615	14984	2401	18622	13403	8647	3919	733	12287	9690	2714
	4040	7221	18345	19682	16469	10317	16625	3496	2388	12510	10301	19547
	15032	8347	1998	2048	14532	10402	4795	13067	6630	12135	4047	7346
	15387	1896	11660	3389	19879	2061	6988	19738	13900	4474	2621	17457
	11124	8595	8920	19461	11829	16912	9638	10219	14338	4225	12172	9374
	10078	11281	3024	15023	11963	14871	13655	10963	11216	16949	17195	6722
	17870	4862	12097	5511	1927	6433	11365	12620	12118	8817	4187	18833
	16333	4734	2143	19881	4183	19538	447	7211	18329	5917	9943	14372
	8806	16953
312:	9795	6261	5149	5170	5168	12820	7573	12233	9643	9693	2068	11819
	18352	16729	2793	17847	7225	12946	8557	1832	444	10898	1956	4691
	7777	13647	19902	13284	13061	12536	5153	5151	16950	2990	2989	2988
	3487	2993	9433	3848	1198	1195	12880	1340	18680	17799	7617	13361
	9528	3585	6230	9821	3940	6243	18903	7878	9794	11915	6414	8130
	3463	15900	17558	2602
313:	19107	3731	10544	1365	315	15072	9222	995	13810	10579	12184	1169
	10820	8682	17063	17115	11426	7610	8335	6680	19590	669	12808	2095
	3210	7981	8975	13797	19673	10108	12140	14891	4849	9589	10013	11489
	5848	8508	16961	14635	3335	5341	9626	18112	14644	12785	9168	9484
	8109	9837	3294	15636	3494	2017	19052	19769	16972	7811	19903	14579
	7377	4121	7557	13806	6626	9547	10641	12326	19729	7235	1411	3644
	10154	10157	2648	7794	10100	12628	15633	12168	7589	15853	4368	19745
	14933	10255	1291	17736	2414	7188	16138	9089	9415	11728	12818	6609
	8114	10065	12609	4638	16498	2517	12709	17410	17048	15136	16713	18469
	19832	12689	1479	15584	12811	17562	13267	13892	10949	9327	472	5713
	11273	9716	15698	5082	17282	1612	7565	10794	11434	8216	10240	6588
	15316	15700	6461	7231	10826	9945	1966	767	9017	8428	9216	1791
	12429	5223	4910	19009	13274	12648	16308	10486	4798	9611	5747	13303
	1346	2839	10642	10405	4049	4046	969	5871	13542	17764	7597	9272
	18722	14315	13433	12396	17102	19120	3267	2911	2934	2480	15593	1566
	15118	17099	13495	18798	16391	13630	12936	4072	17390	11981	12247	14556
	14559	12268	14558	14560	14577	12267	14576	12226	9387	332	2109	7841
	18828	9615	9388	17887	3007	11137	19936	14986	3553	4554	11929	16099
	5440	845	18184	2805	4097	18866	12066	14917	16033	3934	3003	1594
	10929	12343	16214	17420	5606	4643	10415	6601	15702	10449	19711	13552
	1507	19835	1698	9321	11696	2863	14802	17698	1498	17572	14405	13598
	12043	2262	567	1583	2618	3734	18793	19614	14449	16036	8175	19636
	13279	17286	5486	17542	9087	15980	2290	4878	11904	428	10747	9550
	13537	3804	8971	5843	3250	1514	18175	14056	11021	12535	9436	8113
	10697	15175	7725	13365	5679	15598	19442	16718	6306	9803	17134	10762
	2355	13282	9344	18414	962	10707	13732	15247	16140	14944	8665	3901
	12556	13238	3850	16213	15720	5407	7133	17222	15571	4342	952	16118
	708	14879	2997	19403	14894	498	14617	11730	18476	3794	6451	3609
	16585	3917	3066	11348	16371	13698	6380	5627	5614	3881	18493	6416
	12573	15178	7043	8200	17880	11947	15915	7621	7041	8209	18926	10681
	14294	3702	8567	18263	9236	1978	3932	19800	9976	13414	8046	11624
	1429	3768	17319	17779	7609	16512	630	3171	7227	3551	9996	16499
	8855	13654	9117	3812	16292	19434	10320	5172	1982	3126	14582	9645
	1807	19478	7519	12941	17861	14481	4909	1899	3459	1922	19333	4083
	1541	12260	11304	16432	3209	9163	2838	17263	2745	9173	17072	5826
	7351	9534	19739	19536	7374	17185	14542	17759	5670	4857	6297	13448
	7546	11655	18214	11988	3497	12119	18421	2238	9138	8088	14015	6428
	10563	6419	14637	4955	9470	1521	11999	10694	314	18961	1819	15545
	10660	10122	4646	13358	18428	15805	12921	13439	13112	10688	18791	3218
	10153	5875	15881	14320	2938	13237	12193	16805	2072	13687	15276	12110
	9603	12516	3748	11719	18595	2719	7437	2981	5840	16220	2127	19699
	11867	10298	3040	4738	17635	17582	9714	2444	8326	10594	16463	11355
	16573	18034	10940	2168	14219	13085	7255	8220	15653	19511	18649	7840
	8684	2272	9676	5395	14310	13304	17528	4645	2741	19834	12291	3607
	4917	15188	1038	15537	16448	8891	10556	10553	13996	16085	16717	16720
	16629	3416	6712	18027	18028	18046	18045	18018	2046	2280	11438	7044
	432	17329	10580	2438	10575	2435	12466	13227	13331	3373	4354	1137
	14182	14180	17288	17285	17287	17309	18279	17281	5134	13133	13134	7591
	7579	7578	7590	7622	7593	7619	8710	11257	10155	17393	5497	7611
	7613	3368	3371	3345	3347	11146	3346	15875	3327	11270	617	12175
	15844	16942	6398	16524	14919	6019	6427	4568	13742	8545	8646	17237
	10441	15117	18131	812	16684	7548	4903	4891	11339	14205	916	13861
	4237	5869	14326	7059	9757	15666	8359	2239	6482	16408	10426	1600
	4339	9125	15701	10147	6375	17364	694	11598	14712	1946	16250	7313
	3680	8784	12459	4338	15013	3889	4760	2323	2136	16586	3470	11223
	13993	8676	4320	6154	958	7689	4997	6877	12357	17840	14977	10169
	1152	12474	11366	1049	7296	3453	823	1007	2433	5629	7080	8534
	2047	12554	18572	15080	13812	14010	15955	19762	16570	11714	12363	13476
	11640	13481	6845	19136	3340	15749	8548	16230	2819	2803	2874	2851
	2798	2774	2772	2847	2769	2801	11161	14362	3590	279	19320	14355
	13633	12071	4122	10856	1712	8501	2235	2242	2243	2245	18869	17430
	17096	16063	16061	4419	4399	7499	7500	7479	7507	7501	7478	2530
	9228	852	5988	15329	19925	15542	16445	15520	16918	16920	16451	14285
	15560	16922	3473	2729	2751	2748	2730	1449	6008	10997
314:	19107	3731	10544	1365	3521	18558	315	15072	9222	995	13810	10579
	12184	1169	10820	8682	17063	17115	11426	7610	8335	6680	19590	669
	12808	2095	3210	7981	8975	13797	19673	10108	12140	14891	4849	9589
	10013	11489	5848	8508	16961	14635	3335	5341	9626	18112	14644	12785
	9168	9484	8109	9837	3294	15636	3494	2017	19052	19769	16972	7811
	19903	14579	7377	4121	7557	13806	6626	9547	7168	10641	12326	19729
	7235	1411	3644	10157	10154	2648	7794	10100	12628	15633	12168	7589
	15853	4368	19745	14933	10255	1291	17736	2414	7188	16138	9089	9415
	11728	12818	6609	8114	10065	12609	4638	16498	2517	12709	17410	17048
	15136	16713	18469	19832	12689	1479	15584	12811	17562	13267	13892	10949
	9327	472	5713	11273	9716	11114	15698	5082	17282	1612	7565	10794
	11434	8216	10240	6588	15316	15700	6461	7231	10826	17284	9945	1966
	767	9017	8428	9216	1791	12429	5223	4910	19009	13274	12648	16308
	4798	9611	5747	13303	2839	10405	4049	4046	969	5871	13542	17764
	7597	9272	18722	14315	13433	12396	17102	19120	3267	2911	2934	2480
	15593	16755	1566	15118	17099	13495	18798	16391	13630	12936	4072	17390
	11981	14556	12247	14559	12268	14560	14577	14558	12267	14576	12226	9387
	332	2109	7841	18828	9615	17887	9388	3007	16099	12066	845	18184
	4097	18866	11137	19936	14986	3553	4554	11929	5440	2805	16033	3934
	3003	1594	10929	12343	16214	17420	5606	4643	10415	14917	6601	15702
	10449	19711	13552	1507	19835	1698	9321	11696	2863	14802	17698	1498
	17572	14405	13598	12043	2262	567	1583	2618	3734	18793	19614	14449
	16036	8175	19636	13279	17286	5486	17542	9087	15980	2290	4878	11904
	428	10747	9550	13537	3804	8971	5843	3250	1514	18175	14056	11021
	12535	9436	8113	10697	15175	7725	13365	5679	15598	19442	16718	6306
	9803	17134	10762	2355	13282	9344	18414	962	10707	3609	13732	15247
	16140	14944	8665	3901	12556	13238	3850	16213	15720	5407	7133	17222
	15571	4342	952	16118	708	14879	19403	2997	14894	498	14617	11730
	18476	3794	6451	16585	3917	3066	16371	13698	6380	5627	5614	3881
	18493	6416	12573	15178	11348	7043	8200	17880	11947	15915	7621	7041
	8209	18926	10681	14294	3702	8567	18263	9236	1978	3932	19800	9976
	13414	8046	11624	1429	3768	17319	17779	7609	16512	630	3171	7227
	3551	9996	16499	8855	13654	9117	3812	16292	19434	10320	5172	1982
	3126	14582	9645	1807	19478	7519	12941	17861	14481	4909	1899	3459
	1922	19333	4083	1541	12260	11304	16432	3209	9163	2838	17263	2745
	9173	17072	5826	7351	9534	19739	19536	7374	17185	14542	17759	5670
	4857	6297	13448	7546	11655	18214	11988	3497	12119	18421	2238	9138
	8088	14015	6428	10563	6419	14637	4955	9470	6734	1521	11999	10694
	1819	15545	10660	10122	4646	13358	12921	18428	15805	10688	13439	13112
	10153	5875	3218	18791	2938	12193	15881	16805	14320	13237	2072	13687
	15276	12110	9603	12516	3748	11719	18595	2719	7437	2981	5840	16220
	19699	10298	2127	3040	11867	4738	17635	17582	9714	2444	10594	8326
	16463	11355	16573	10940	2168	14219	13085	7255	18034	15653	19511	18649
	7840	8220	2272	9676	5395	14310	8684	4645	2741	19834	12291	4917
	15188	3607	13304	1038	15537	16448	8891	10556	10553	13996	16085	16717
	16720	16629	3416	6712	18028	18027	18046	18045	18018	2046	2280	11438
	7044	432	17329	10580	2438	10575	2435	12466	13227	13331	3373	4354
	1137	14182	14180	17288	17285	17287	17309	18279	17281	5134	13133	13134
	7591	7579	7578	7590	7622	7593	7619	8710	11257	10155	17393	5497
	7611	7613	3368	3371	3345	3347	11146	3346	3327	11270	15875	617
	12175	15844	16942	6398	16524	14919	6019	6427	4568	13742	8545	17237
	8646	812	10441	15117	16684	7548	11339	18131	4903	4891	14205	916
	13861	14326	4760	4997	7059	9757	15666	8359	2239	6482	16408	10426
	1600	4339	9125	15701	10147	6375	17364	694	5869	11598	14712	1946
	16250	7313	4800	3680	8784	12459	4338	15013	3889	2323	2136	16586
	3470	11223	13993	8676	4320	6154	958	7689	6877	12357	17840	14977
	10169	1152	12474	11366	1049	7296	3453	16153	10586	1007	2433	5629
	7080	8534	2047	12554	18572	15080	13812	14010	15955	823	19762	16570
	11714	12363	13476	13481	6845	19136	3340	15749	8548	16230	2803	2874
	2819	2798	2851	2774	2772	2847	2769	2801	11161	14362	3590	279
	19320	14355	13633	12071	1712	8501	2235	2242	2245	2243	18869	17430
	17096	16063	16061	4419	4399	7500	7499	7479	7507	7501	7478	2530
	9228	852	5988	15329	19925	15542	15520	16445	16918	14285	16922	15560
	3473	2729	2751	2748	2730	1449	313	6008	10997	18961
315:	2185	11361	5661	8686	17264	8328	8330	7550	10232	18956	1696	5922
	16632	14340	10579	4523	1169	11996	8012	4522	16791	8335	10038	5066
	4969	4634	17213	14401	7284	2099	15619	17243	6718	8030	14321	1903
	15554	15028	12204	3060	19178	8109	11653	9316	17858	18132	18589	9667
	17297	5418	17508	15450	6147	5012	18588	18020	862	4920	15050	16525
	3064	2884	5607	12297	11396	18052	2912	17060	1697	17604	18870	8264
	17736	1732	5968	5593	15864	18761	4044	5088	17149	10593	5242	4527
	13682	8054	16503	1962	19380	1623	10664	4597	15319	3478	16945	18584
	3108	12724	15546	15764	15754	18354	18416	4890	19678	19130	15532	5213
	11374	9753	14702	15217	17235	15921	5855	18752	1904	15721	18203	18156
	18864	8700	7560	16924	636	6811	16379	19046	11063	10405	4049	4046
	969	14929	16207	7597	9272	15425	17368	11052	14975	6574	9560	10349
	3830	8250	2911	2934	2015	18785	8645	8196	7713	11586	14490	18301
	18792	15378	7699	18803	19816	16104	14468	17390	11981	14556	12247	14559
	12268	14560	14577	14558	12267	14576	15277	12222	16710	12224	12226	9387
	332	17612	2109	7841	18828	11556	1969	9578	7424	5369	245	18873
	16602	11137	19936	14986	3553	4554	11929	5440	2805	12674	16033	3934
	3003	1594	10929	12343	16214	17420	5606	4643	10415	14917	6601	15702
	10449	19711	13552	2863	1507	19835	1698	9321	11696	14802	17698	1498
	17572	14405	13598	12043	2262	567	1583	2618	3734	18793	19614	14449
	16036	8175	19636	13279	17286	5486	17542	9087	15980	2290	4878	11904
	428	10747	9550	13537	3804	8971	5843	3250	1514	18175	14056	11021
	12535	9436	8113	10697	15175	7725	13365	5679	15598	19442	16718	6306
	9803	17134	10762	2355	13282	9344	18414	962	10707	13732	15247	16140
	14944	8665	3901	12556	13238	3850	16213	15720	5407	7133	17222	15571
	4342	952	16118	708	14879	19403	2997	14894	13269	498	14617	11730
	18476	3794	6451	4601	7256	19396	2011	16585	3917	3066	16371	13698
	6380	5627	5614	3881	18493	6416	12573	15178	8209	11348	7043	8200
	17880	11947	15915	7621	7041	18926	10681	14294	3702	8567	18263	9236
	1978	3932	19800	9976	13414	8046	11624	1429	3768	17319	17779	7609
	16512	630	3171	7227	3551	9996	16499	8855	13654	9117	3812	16292
	19434	10320	5172	1982	3126	14582	9645	1807	19478	7519	12941	17861
	14481	4909	1899	3459	1922	19333	4083	1541	11695	12260	11304	16432
	6090	18117	19599	18305	3209	9163	2838	17263	2745	9173	17072	5826
	7351	9534	19739	19536	7374	17185	14542	17759	5670	4857	6297	13448
	7546	11655	18214	11988	3497	12119	18421	2238	15982	9138	8088	14015
	6428	10563	6419	14637	4955	15467	9470	1521	11999	10694	314	1765
	1819	4359	18984	14947	13368	6080	8594	12041	17552	5525	3665	12817
	1783	2890	18428	15805	9454	10688	13439	13112	10153	5875	3218	18791
	2938	12193	15881	16805	18770	14320	13237	2072	10668	13687	15276	12110
	9603	12516	3748	11719	18595	2719	7437	2981	5840	16220	19699	10298
	2127	3040	11867	4738	17635	17582	9714	2444	10594	8326	16463	16175
	11355	16573	10940	2168	14219	13085	7255	18034	15653	19511	18649	7840
	8220	2272	9676	5395	14310	8684	4645	2741	19834	12291	1038	15537
	16448	8891	10556	10553	13996	16085	8752	6235	10378	16031	9773	9748
	8634	1983	5010	16717	16720	16629	3416	6712	18028	18027	18046	18045
	2247	17307	5751	13958	2046	2280	11815	11438	7044	432	17329	10580
	2438	10575	2435	12466	13227	13331	3373	4354	1137	14182	14180	1685
	6522	17288	17285	17287	17309	18279	17281	5134	13133	13134	7591	7579
	7578	7590	7622	7593	7619	8710	11257	10136	10155	17393	5497	7611
	7613	3368	3371	3345	3347	11146	3346	3342	14996	9789	7217	2775
	6179	6205	617	5167	7673	18690	12874	2313	15844	16942	6398	16524
	14919	6019	6427	13742	5524	12408	15818	13998	17952	15058	2676	443
	16567	8545	9337	11440	6373	12996	16653	18303	8646	8910	18008	14978
	10441	2409	16827	2374	16684	7548	18131	4903	4891	11339	14205	916
	13861	14326	7296	2136	7059	9757	15666	8359	2239	6482	1101	8053
	8470	8156	10487	1094	11152	5629	7080	6211	8534	2047	12554	18572
	15080	13812	14010	15955	9662	19762	16570	11714	2862	11347	12363	2885
	13476	19136	3340	13481	6845	8548	16230	15749	18141	2803	2874	2819
	2798	2851	2774	2772	2847	2769	2801	11161	14362	6202	8537	8518
	7037	8133	6764	827	279	4941	1712	818	19365	8356	7588	3017
	2235	2242	2245	2243	18869	6400	5637	915	8416	12444	510	12026
	11634	16063	16061	4419	4399	7500	7499	7479	7507	7501	7478	2530
	9859	1460	18194	11314	2685	4226	15542	19925	15520	16922	15560	5221
	8058	14961	984	19513	2729	2751	2748	2730	8932	313	12905	6008
	4112	8720
316:	5462	8702	15896	14144	15464	19725	9380	4042	545	16095	1526	8816
	16014	8322	3022	1553	2635	18516	10285	6421	5546	6489	8482	8478
	5585	16227	2190	10760	1465	2255	11395	7990	14793	10172	16192	7992
	12560	3462	14906	12272	16970	2134	2867	2758	7489	6672	10233	12166
	15802	1638	918	18996	2473	4032	8620	3162	3756	12149	13374	1308
	1307	11769	13333	10034	11874	12574	4659	9026	13327	2753	14426	2429
	11931	6246	16533	2250	3513	4318	4974	3869	15232	3820	4665	16608
	13406	16766	3671	4310	8986	18992	8995	19933	19932	8988	7545	3888
	1293	17163	11580	4168	4167	16335	4007	13342	13297	2864	18382	15220
	6521	15457	12531	12431	4213	7363	7483	7328	2936	8542	8532	1997
	2463	1996	7728	9652	15509	7851	12529	2192	8974	9761	13146	8836
	9376	9401	2717	11802	11806	2558	2553	18119	3929	423	19081	8210
	9326	5275	14503	3110	14226	5291	5571	4513	5138	6994	5137	17365
	1785	5345	11683	6635	7064	18861	13492	12133	805	10146	9632	15755
	16143	8100	13038	16967	19575	19594	3996	16196	1721	16725	14719	12580
	11240	5778	618	8966	8955	19713	9948	9946	3468	7884	16898	1191
	18670	7152	18629	2566	12157	3148	14714	9023	16483	14008	17781	13594
	7412	4148	15789	2191	19158	8063	16731	853	6697	3043	4675	3548
	5951	5342	3186	6258	12561	3976	16231	7192	3154	16854	9100	11130
	3448	9146	12098	14067	15841	18357	879	3661	7236	1239	14934	1964
	8301	13913	18880	2792	2960	8392	12600	19792	13251	1556	8938	11739
	11738	17186	14126	8704	6167	15076	7923	10995	8699	4137	19459	2768
	11490	2285	468	3256	3962	3526	6749	785	4270	18693	14414	12394
	15009	15406	7292	12653	1253	9468	10332	19444	7625	16015	5343	16491
	5692	14537	13107	10267	5745	11145	6063	13844	12413	7513	1555	19155
	8411	14705	6809	12611	16041	16179	16590	16403	14059	5845	15694	17067
	9812	2968	9811	11761	5358	7512	10371	10369	6536	5488	6872	5893
	5937	18324	3446	15225	17313	18360	16566	17334	12104	16784	4519	11690
	1160	2536	14367	18839	10104	11824	2933	10654	19865	12293	12274	12271
	7670	10692	7530	2086	7632	7453	5948	6608	10756	17250	3010	4463
	3520	2896	3517	10296	1847	11132	11288	6317	16569	19515	4801	16530
	3438	12214	12211	3078	7475	6795	4591	15447
317:	2231	3383	14866	16544	7418	9107	4315	9446	19269
318:	4830	2716	9062	17385	658	13025	18942	1913	7312	19788	1716	3538
	4143	17321	815	18938	9848	2344	5906	7890	446	6885	11380	759
	13452	4140	13032	458	17467	1500	8433	6737	15173	681	2424	16493
320:	1136	7051	5444	7050	15227	18825	10186	7331	7329	11868	7003	6249
	4750	4038	15916	19050	15491	14585	17723	15235	17636	18713	18024	13564
	3655	7523	2416
321:	15725	14852	5781	6187	9364	6675	12873	12050	2178	18054	18447	870
	12686	19055	13293	6047	3502	15480	6688	6643	16132	12103	18489	19742
	17678	2836	12364	8309	14776	7846	17350	17117	11028	7278	1841
322:	19529	18972	10678	6073	19135	18539	10466	12487	17080	14595	7367	8165
	4913	16318	11781
323:	9079	9955	19786	2490	2106	6931	4177	11411	14494	401	16768	10346
324:	11512	8262	6323	11449	12376	15280	6345	6342	6758	18459	5739	3469
	6326	6372	4249	12966	5720	5704	5725	5724	5756	5810	5785	5764
	5783	5788	5723	5727	11451	8412	19543	15504	16837	1442	3587	7395
	11317	13364	750	15231	7715	3807	7915	8643	10574	16564	2736	16086
	7850	13459	10318	9779	731	10396	10077	6470	798	7137	6756	6754
	14009	17356	13319	2236	18981	17018	6664	803	19647	1274	15145	18856
	9265	1809	1806	1811	9896	17998	8581	15642	5684	17295	7254	7960
	5636	8730	9734	9831	19625	7753	8179	7998	5300	6623	12543	16137
	18696	5777	1167	4524	1077	11970	3765	4567	14316	9593	16226	5513
	12102	3788	19164	15971	3631	12022	13285	9219	5808	2023	12867	12139
	14800	9148	9151	5703	19445	3505	1417	9937	12900	5835	8793	17627
	9980	10119	15928	3285	12749	534	19027	7116	10086	3784	18420	9372
	3809	16983	19749	4147	2561	14685	19010	5508	15932	1043	8298	15444
	13412	8608	15947	13976	8067	3584	19918	10353	11595	11284	14370	8853
	4973	8385	5589	1798	1207	509	7947	11095	15540	3437	7615	7612
	10821	10244	18075	7438	19651	8598	9205	7814	14515	14553	7875	9013
	4551	17654	6535	4686	15510	9928	14939	9130	13820	10857	11701	13734
	9606	633	7757	10685	964	7436	502	18371	10090	18370	5553	15365
	16452	3436	13450	1356	18559	15301	2361	18638	10471	17333	9747	13715
	12322	6368	6349	6775	6779	6773	6777	7269	6774	7262	6328	17232
	17226	17209	17212	17211	5741	6344	6348	6476	12159	4157	7574	8332
	6766	5759	5760	12701	2721	10839	17534	8451	14104	12537	13141	14902
	14903	4314	14916	4309	13007	10518	3401	7876	1104	8383	14730	14720
	12059	1106	6982	1031	15090	15093	6907	17634	8009	484	7723	1281
	5926	776	6614	7009	15697	12348	13028	2330	12655	15081	6495	13560
	18571	14791	19199	6442	7955	9241	7078	6798	10503	15218	8382	19172
	938	11115	6504	5016	5836	18241	7022	17216	3591	6384	2085	8286
	9708	14861	18111	4952	13581	14185	2853	14506	14516	6690	10730	1934
	2612	10612	11767	14612	3998	3009	16097	19296	15380	18005	12479	8182
	4663	14202	1064	1476	5622	11833	13437	12832	3282	17447	4447	5740
	1103	6324	5794	13746	19613	13540	3119
325:	16615	14754	4057	8715	5965	14877	1866	8104	17176	4366	14518	9167
	9065	10806	584	14195	17198	8201	4762	17268	2697	6174	2254	8981
	13193	12420	6622	8325	5839	16464	6347	11709	18411	6294	10583
326:	11367	14736	13964	6308	3036	11437	10175	5974	9622	2625	2363	10917
	11431	9207	5827	1582	5507
327:	10691	6846	18734	10944	7446	14465	1433	5628	16454	18586	10494	17172
	7904	9524	7196	17084	9159	445	5749
328:	1385	15829	19449	11943	5936	18464	6928	7520	1074	12457	797	5586
	11159	6704	13511	5517	4980	15756	19404	11162	11144	11682	8750	6782
	14571	796	7484	18881	2581	19645	16081	10523	17997	6738	18661	512
	13506	959	14965	17458	6459	1020	16188	3545	7761	17240	19190	3663
	4263	17715	14220
329:	8714	3717	5566	10565	1859	668	9746	12025	1427	7417	530	14121
	7988	11832	3990	15583	10329	7800	2906	9120	14177	14111	16467	2762
	4786	19733	3949	3152	8034	16589	3235	4766	6719	8060	10844	12009
	12714	15999	13202	19675	9263	15341	11412	11538	1175	13971	9989	3509
	7314	9081	5349	6882	4703	8235	2796	5908	10247	12378	16419	16844
	12799	14375	5098	6430	9862	12923	12774	18038	9506	4345	14983	1075
	17160	6513	3472	1454	13829	6880	6497	17891	11910	6204	10379	14721
	14990	10513	5022	5978	10881	5325	4900	11308	9150	18664	5881	12552
	6583	6062	13051	8792	15886	882	6896	2502	9724	4391	16217	12349
	5780	2148	13289	1827	6149	7304	5083	12091	2946	7643	17480	13408
	14394	7079	11009	5514	9020	19826	17411	8276	9974	16420	12687	6975
	522	5006	10709	7584	18456	15757	4215	10986	2166	5442	6286	9293
	9482	944	7759	18574	18169	6499	4685	7873	8584	17543	9884	7382
	7352	7061	17128	14603	3906	11674	10746	1503	13826	14262	12721	17380
	11192	13270	15052	9833	12894	19206	19502	19182	12719	6650	17024	11885
	12688	5510	1496	14710	18472	12216	4163	6189	13422	1597	8924	12843
	10721	10028	1759	14239	14549	4604	10040	7920	1403	10667	14918	7430
	13189	11618	14583	3524	4966	17459	839	13522	3834	6998	2999	6865
	7167	13043	1901	11888	1209	6472	14813	788	3885	3956	7158	18767
	16576	15007	18092	16591	6954	6172	3857	2555	2459	2669	18256	10830
	6666	8480	11442	12577	9271	14060	9245	14655	15710	17030	7711	7709
	3351	6487	4533	2082	17184	8698	14994	13896	7835	8142	814	14855
	3617	935	10433	15296	18619	17848	3434	4185	18019	521	13339	14025
	5754	12949	10467	9496	5158	19096	2135	576	993	12906	11309	11504
	5691	9623	12589	3192	8950	6480	10293	11074	16003	19115	966	1849
	14063	14904	657	1848	16007	14717	4142	16489	12381	2754	8869	14112
	8121	19313	10861	19088	3712	6558	7559	10653	9717	16715	18087	10084
	17845	19383	16584	14634	8560	18766	7631	18659	1540	9731	6883	17687
	15181	15549	6519	11663	486	13767	15635	13224	15907	5070	10188	9295
	9297	19656	13831	9250	18896	6462	10456	4816	6498	11564	14166	10726
	13462	2420	6005	5935	11870	8991	1892	3542	12006	14673	1339	15348
	11771	7607	19801	18944	7773	6641	18882	7567	18085	12565
330:	904	12835	3861	1482	5979	9709	13954	6123	6143	16986	17855	18668
	1909	5240	12004	9616	16459	11407	4107	15822	4812	9206	13190	986
	738	8516	7209	9218	9155	15166	1046	9972	9234	14160	10926	9007
	12402	13404	14565	1314
331:	14505	19187	14237	2004	13667	15330	8712	2983	10833	6141	8832	10908
	14924	5518	1621	10016	549	17499	11318	10215	16348	19205	15747	5139
	1782	19628	11628	15831	15263	14491	13634	7647	10268	16730	6410	1484
	1366	16382	14778	1874
332:	625	19038	18542	16881	3521	18558	315	11296	10579	14173	1169	11180
	14471	696	8335	11062	1469	7394	18110	7821	19076	5860	4906	11277
	2138	9025	2195	14684	8109	11176	9010	14713	5710	3301	16679	1176
	8811	8290	4792	18465	8807	17003	8983	9258	15527	15529	14912	17091
	17736	13621	7248	8229	8007	10799	19406	11113	13933	9338	9443	15873
	16790	5467	4303	11067	15230	15245	17641	9012	19616	6000	14666	2725
	14734	17332	12078	13496	13912	13472	10405	4049	4046	969	16147	12289
	7597	9272	835	17757	15353	2123	17605	2911	2934	13460	10176	18009
	1003	13239	12374	3372	17390	11981	14556	12247	14559	12268	14560	14577
	14558	12267	14576	12226	9387	2109	7841	18828	11137	19936	14986	3553
	4554	11929	5440	2805	16033	3934	3003	1594	10929	12343	16214	17420
	5606	4643	10415	14917	6601	15702	10449	19711	13552	1507	19835	1698
	9321	11696	2863	14802	17698	1498	17572	14405	13598	12043	2262	567
	1583	2618	3734	18793	19614	14449	16036	8175	19636	13279	17286	5486
	17542	9087	15980	2290	4878	11904	428	10747	9550	13537	3804	8971
	5843	3250	1514	10411	13008	5960	18175	14056	11021	12535	9436	8113
	10697	15175	7725	16718	13365	5679	15598	19442	6306	9803	17134	10762
	2355	13282	9344	18414	962	10707	13732	15247	16140	14944	8665	3901
	12556	13238	3850	16213	15720	5407	7133	17222	15571	4342	952	16118
	708	14879	298	7886	19403	2997	14894	498	14617	11730	18476	3794
	6451	16585	3917	3066	16371	13698	6380	5627	5614	3881	18493	6416
	12573	15178	11348	7043	8200	17880	11947	15915	7621	7041	8209	18926
	10681	10164	14294	3702	8567	18263	9236	1978	3932	19800	9976	13414
	8046	11624	1429	3768	17319	17779	7609	16512	630	3171	7227	3551
	9996	16499	19516	8855	13654	9117	3812	16292	19434	10320	5172	1982
	3126	14582	9645	1807	19478	2739	7519	5561	12941	2436	17861	14481
	4909	1899	3459	15199	19324	1922	19333	4083	1541	12260	11304	16432
	8512	4387	10025	6653	9022	6289	7641	9339	2859	11610	9145	8381
	8830	8028	17745	3541	482	2045	8753	3209	9163	2838	17263	2745
	9173	17072	5826	7351	9534	19739	19536	7374	17185	14542	17759	5670
	4857	6297	13448	7546	11655	18214	11988	3497	12119	18421	2238	9138
	8088	14015	6428	10563	6419	14637	4955	9470	6734	18494	1521	11999
	10694	19034	6803	18967	314	1819	14284	18428	15805	9706	10688	13439
	13112	10153	5875	3218	18791	2938	12193	15881	16805	14320	13237	2072
	12833	13687	15276	12110	9603	12516	3748	11719	10927	18595	13468	2719
	7437	2981	5840	16220	19699	10298	2127	3040	11867	4738	17635	17582
	9714	2444	10594	8326	16463	2150	4184	2007	11355	16573	10940	9501
	2168	14219	13085	7255	18034	15653	19511	18649	7840	8220	2272	4799
	9676	5395	14310	16236	8684	4645	2741	19834	12291	4917	15188	3607
	13304	10053	19863	17528	10883	622	943	12590	14403	18836	9723	10918
	19559	18677	1038	15537	16448	8891	10556	10553	922	3334	13996	16085
	16717	16720	16629	13354	3416	6712	18027	18028	18046	18045	15020	9679
	4373	2046	2280	16254	15252	11438	7044	432	17329	10580	2438	10575
	2435	14080	12466	13227	13331	3373	4354	1137	9112	9115	8626	9114
	14182	14180	17288	17285	17287	17309	18279	17281	19761	5134	13133	13134
	7591	7579	7578	7590	7622	7593	7619	8710	11257	10155	17393	5497
	7611	7613	3368	3371	3345	3347	11146	3346	19018	18562	4131	617
	15703	18059	11680	15149	6854	10204	14371	15844	16942	6398	16524	14919
	6019	6427	4568	2096	17412	15137	6371	442	13742	19059	13740	18548
	8545	8646	10441	19790	9654	2971	11164	12791	16684	7548	18131	4903
	4891	11339	14205	916	13861	14326	7296	7059	9757	15666	8359	2239
	6482	16408	10426	1600	4339	9125	15701	10147	6375	17364	694	5869
	11598	14712	1946	16250	7313	3680	8784	12459	4338	15013	3889	4760
	2323	2136	16586	3470	11223	13993	8676	4320	6154	958	7689	4997
	6877	12357	17840	14977	10169	1152	12474	11366	1049	3453	16153	10586
	7466	18725	19446	12713	6237	2331	1068	5956	14116	4295	5532	19458
	9119	2957	3739	1926	5245	13988	1520	18033	18302	19042	17337	4538
	4879	14267	1733	12961	6254	3681	7830	2074	16366	1543	11786	9016
	14318	19058	3564	8810	13044	12256	15026	4210	6152	6276	7015	19314
	14293	8809	10942	9006	4237	5540	4066	15205	2922	7718	9533	18546
	10937	14422	11800	15473	1522	19100	8937	2303	11886	7722	16621	18323
	896	9132	14097	11222	15658	13479	11450	7853	10321	18832	7922	19744
	6867	15240	11684	18174	1205	4611	5375	16992	2152	19112	4946	19596
	16620	19036	18565	19016	18568	18507	18505	18503	18502	5629	7080	13753
	1126	8534	2047	12554	18939	18572	15080	13812	14010	15955	6939	14155
	19762	16570	11714	12363	13544	7253	10903	17939	19714	11964	15759	1212
	3044	7880	13476	18635	9531	13481	6845	19136	3340	1470	15749	8548
	16230	16655	5529	5322	6804	2189	2803	2874	2819	2798	2851	2774
	2772	2847	2769	2801	11161	14362	279	18610	1712	3375	2235	2242
	2245	2243	18869	9808	16261	14527	6784	8618	13941	16063	16061	4419
	4399	7500	7499	7479	7507	7501	7478	5967	5140	7332	2530	9726
	5117	1829	3095	15542	19925	15520	16445	16918	16920	16451	15541	16209
	16922	15560	15559	11352	19040	2729	2751	2748	2730	6517	313	6884
	6008
333:	417	6257	8790	13789	16313	14699	10733	16574	8828	5654	11626	2210
	11562	3826	12856	1109
334:	8128	4221	17278	17046	18251	12696	6743	3779	8868	11244	10704	12642
	6965	16337	9523
335:	18958	12883	14235	19663	17014	1182	2397	1912	8514	1875	540	4252
	8725	6215	16237	4063	729	2593	724	8219	17866	6912	6447	18691
	2763	5446	2066	15270	17205	19591
336:	12057	12728	5689	13469	14260	4138	1692	19601	15628	9152	17930	16195
	2028	8172	17751	16934	16401
337:	3127	12366	13856	16497	1925	19818	4950	15674	5793	15496	15493	2188
	2206	5492	1835	6671	11106	1524	9890	1111
338:	5950	18742	15397	6802	19876	12654	8206	17804	1971	16998	18602	18337
	4016	5119	2979	4844	10291	19710	18136	5748	10284	7029	5412	5278
	11920	1908	6210	762	9715	12314	6273	6084	19740	8804	15410	16699
	8756	13731	3128	3144	10582	15564
339:	11402	410	12775	2861	5260	5387	19121	1001	11194	18533	19260	466
	4275	18650	7675	1919	4821	7323	16863	9487	12895	16977	15299	2339
	8707	2334	9658	18289	19688	13814	2715	19839	17620	11898	1578	12967
	4603	13211	930	19517	19938	15569	15063	8098	19557	8485	18333	16609
	14853	9045	18679	9143	17460	7537	13585	18153	18578	1495	6307	4430
	14054	6332	15889	17603	11064	948	2013	18684	6751	1586	17167	17443
	8350	2105	8487	9467	13324	1745	1746	5173	4505	11300	7665	13801
	6730	2418	12477	12495	19560	8467	6040	6166	5180	14885	7681	18736
	6363	10978
340:	6122	1518	18485	14501	10446	5892	19266	13779	16587	8559	9244	8689
	11841	16626	17021
341:	10333	3897	11487	9936	11989	10885	8445	11048	9005	17316	16689	13965
	11249	5452	8186	2457	10098
342:	3006	14845	848	12847	17916	1499	5698	15349	8481	2524	12033	2479
	19698	8954	6422	13377	17520
343:	6892	17589	9778	16830	14586	3829	7946	7552	7372	2949	6234	6869
	12368	9735	13309	2554	7769	4764	8126	18134	2656	2408	9463	16486
	19750	2406	15386	7481	14227	7803	18842	18844	4369	15153	18161	19455
	3782	7008	2599	4449	1837	2204	6481	10484	18534	15820	3300	18577
	14013	8365	13671	11914	3706
344:	4518	2816	7393	8749	3714	18587	12336	13565	834	14466	2212	18658
	3417	2925
345:	471	17836	1008	8569	18625	14950	11789	8572	1102	10998	17791
346:	16406	9039	13935	10414	14191	11070
347:	6068	14653	2218	9394
348:	8023	17381	14654	13733	18394	10569	4912	9979	1032	937	13915	10361
	1651	5040	10191	12530
349:	1648	10364	2132	11847	18514	11591	2053
350:	13558	19061	6182	985	13642	11733	7195	10366	10180	4120	16484	13042
	18195	12100	8507
353:	4575	1730	16597	3415	11245	13804	9868	8538	15670	7953	11687	14656
	1891
354:	19676	7455	15869	13573	16262	19202	4246	19397	2728	12796	9583	7861
	5152	12632	18294	12083	15810
355:	2962	6855	10045	10561	12389	12391	2422	18108	12563	12416	4416	11255
	8450	16833	11444	14745	12519	14732	12470	14479	1758
356:	8236	1667	6732	4010	6333	999	12332	2454	12092	2743	7397	3753
	12781	16704	6391	18035	3785	1321	8174	19017	8597	16697	12677	9178
	12405	17672	12838	19432	2856	7305	5899	5645	1905	926	4508	6831
	11594	13483	19577	13571	17403	19874
357:	18266	7571	19462	4882	3947	18198	19775	19771	19267	9864	15678	7599
	19000	3750	3851	12623	17165	17571	14922	2910	16737	8164	5433	14442
	13244	5939	1262	17766	9254	2064
358:	4219	1193	4372	8802	10387	9975	16842	12280	12281	12279	17094	11872
	2301	709	13862	19439	1073	17661	15857	5981	5996	10915	2469	18211
	13345	7869	8911	18950	8909	18952	8225	2963	8049	6934	2376	6638
	15632	8089	9727	17744	4332	3625	2818	3137	17921	3318	16305	764
	13749	12825	10113	546	11893	19759	13294	15366	11250	13771	8642	15051
	6891	15845	12578	4987	6516	18800	4333	18374	4360	14269	17370	9277
	18408	4808	4029	12225	19338	787	15279	9722	10304	1332	9721	3088
	11139	6037	16019	8571	5449	4424	5365	2467	3432	7502	1283	2362
	17765	12194	5682	9922	14289	9064	13109	2442	5695	13898	1251	6239
	13774	860	2056	6839	6840	612	11105	11286	2407	12769	13122	9044
	13743	3836	3447	8243	6953
359:	12802	5201	10302	17468	16751	7450	9413	19290	5280	19612	15469	15474
	13637	6949	3348	2194	4865	3207	16722	3441	14427	7198	3775	3802
	12181
360:	19570	9059	8115	3359	3370	16218	4471	2588	3190	1342	1327	7790
	7330	15451	1506	12550	3293	13170	13832	8062	11326	2156	5293	5104
	3243	672	10504	12238	7614	14171	3051	10249	4843	15762	12428
361:	3065	950	8348	12335	19427	14109	13455	16581	18561	19522	5471	7987
	13435	14619	9480	6424	5284	10400	10362	7024	1443	7277	15946	6856
	5638	955	2157	1720	9881	18969	15813	7701	12321	3920	8603	9215
	3907	11529	14014	6824	2329	8668	10607	9238	2565	4098	9193	16372
	18237	17803	19666	2639	7110	12353	660	13242	17052	10200	12815	11310
	17724	3805	1392	19435	12595	5074	10118	5073	8413	6434	8087	3649
	6611	4839	1944	2006	14636	15167	19849	3029	5269	18965	9850	11641
	4433	12460	3711	13980	19654	16418	4220	14462	14907	12618	7928	11377
	1861	16288	3333	15079	11178	5930	14221	15099	7106	13591	11342	6453
	9428	3138	4529	18919	10578	11967	3123	15132	7246	1399	19641	1877
	15059	10489	18120	12869	12088	11647	7054	2316	4238	4224	17713	13334
	18328	6713	10427	5017	1537	13171	3450	18694	3746	11425	11266	11206
	14036	8884	13754	3840	5694	12680	14821	15322	12298	15719	13691	13441
	2511	17203	3588	3365	11160	4803	13723	19123	12875	17986	9764	4606
	4403	4531	10348	11668	16400	16295	8960	3752	5598	15851	14513	7149
	15712	16423	6990	14475	12448	4402	11453	9183	3764	10933	3278	7656
	10372	17668	18799	2893	12854	5169	17911	17413	11409	13265	438	10127
	17867	12591	2654	6083	11279	17487	15623	19297	15212	13033	4207	10444
	16705	11619	8797	17292	14740	6849	19452	2484	14138	14897	9108	16156
	4383	5020	6543	6549	11168	3290	6961	16871	3270	15512	2895	14733
	15488	2322	14667	15731	18753	18312	12557	13197	3823	7435	13672	2033
	5162	8163	7655	6409	8511	14850	8444	8899	15067
362:	15752	17695	9990	5932	15211	8418	6238	13628	14659	485	972	7628
	19124	17248	10742	14839	7306	6162	2258	9285
363:	15587	7536	11706	19935	8746	17583	13021	11251	695	6660	14178	15828
	10282	7088	4536	3957	2909	10712	8289	1371	15458	19277	18642	4003
	15273	4301	13644	7311	11813	1680	735	15095	1457	1128	3122	9711
	3793	1155	18202	5222	16291	5429	8099	7324	11246	909	2980	6952
	4744	9471	15817	19001	6196	6665	2449	16000	14068	9442	4512	15890
	11197	18150	14278	19695	6747	10772	7524	8625	11005	5911	9704	1140
	3532	6565	12151	16037	13454	9169	3324	9517	19274	3692	4037	3896
	1082	8078	12333	17688	6926	9760	17178	8565	15303	18874	5274	3608
	11418	15790	13582	9809	13016	12263	17135	18105	2163	12533	9985	14786
	6012	15171	3012	7976	18090	4450	16120	10148	9586	1280	9405	9430
	10773	8152	17676	19007	13766	7476	16279	18231	12787	12805	1089	16681
	3955	2488	673	12058	8888	7696	12741	2767	7629
364:	18935	11480	10102	2128	611	16123	4822	16369	3396	5943	18617	12251
	9404	19062	5185	6458	17236	4412	6457	4432	11891	4444	17731	5068
	13351	18269	1188	18262	18243	18264	18245	15524	18268	7045	10216	15254
	15585	9962	6016	6018	5983	10695	4247	11334	2233	11399	14429	17303
	18326	5114	4325	13602	3559	12081	18081	5579	15294	19095	8364	12907
	14773	15870	10476	3925	8957	18029	8308	2927	19174	3169	6014	4048
	6676	17157	11935	4361	4103	7348	3589	16742	2320	8945	12971	1753
	2476	9137	14089	16990	18646	2973	10662	10632	13827	11754	1568	5396
	13363	13991	6017	6232	13824	9366	1808	7556	18911	16773	9136	4823
	14910	396	19794	17197	7423	9283	517	4437	17158	6128	6636	12989
	7092	2462	14623	6769	11837	19463	3305	13186	5079	2059	2342	5833
	5569	1515	5189	675	4488	17733	8803	17720	1127	7371	13624	3909
	4748	1217	8255	9253	16038	5961	6590	16131	19015	11099	19890	2161
	12546	10713	925	11622	813	16009	13081	15471	17703	8306	13620	1120
	7320	18557	7452	7470	13172	15551	3325	1780	2126	16004	10743	14145
	11018	15477	18982	10765	13029	15065	4084	8672	12316	10611	3273	11834
	6279	6327	17101	19914	19661	12771	7385	9866	8494	1768	15903	6534
	13088	18689	5562	9170	7829	3895	17666	16326	15100	13815	9286	8993
	6944	16914	7289	8901	17669	12277	14660	16867	11040	1316	3645	2585
	2770	4895	1589	399	6220	18600	18397	917	5160	14743	19767	11532
	1354	13972	3182	2310	15434	2040	5147	8430	14935	13335	16362	10812
	11006	12064	11533	16872	18830	15354	1960	11327	15506	19294	14823	1796
	7366	15566	5646	12469	10813	15208	13430	17355	4773	1940	14330	14354
	12329	4797	2889	595	9772	14913	17811	14573	7131	12710	6929	13194
	15743	17161	14900	19555	4173	17031	5984	7342	16617	8400	3525	3512
	3611	3679	3669	3674	10419	9782	9140	17719	6888	2353	8032	12136
	15475	934	10480	19362	17217	16756	19071	18554	12338	17276	2282	13612
	1421	5210	19246	14938	4414	10326	861	19438	16936	5565	10516	7011
365:	13631	2299	17761	900	706	18107	11275	3061	17510	586	1894	15867
	13092	19868	17581	6902	13125	3191	17566	7355	11590	2877	15662	10745
	10009	17671	18076	2217	2055	11552	10714	19648	16707	9924	9503	19883
	12972	8648	6287	14196	10126	7645	11125	1084	2706	1420	1739	11228
	2641	9918	13470	15676	15827	15328	968	809	8295	15038	16125	2326
	13641	2592	15180	7187	19519	8254	7001	15948	419	12496	7126	5605
	18193	15842	6741	17608	8770	10875	17317	16846	9813	6726	15155	11019
	16116	16028	16531	1857	17795	11282	12582	8230	14029	9438	10288	9360
	9453	5792	14648	3353	782	13500	19798	3960	2396	13841	6828	19659
	4186	5277	570	7389	8319	1775	5548	697	6933	4532	9927	7345
	5347	17289	12608	7808	2718	9932	14265	15975	13060	14443	6331	18432
	13873	12306	4371	15241	12122	15732	5458	13523	8923	17770	1063	17401
	17362	14436	7068	13174	12734	11814	1336	10705	3094	7480	832	4811
	15561	12242	19576	792	9707	13586	7094	13475	8199	5714	15555	18801
	1992	5212	10193	18728	19345	15911	1076	1019	2978	11483	2478	14400
	8170	1943	17567	15417	5317	11195	14991	18347	7672	19335	19822	11797
	13910	8435	16903	15659	6408	13305	6028	491	12784	19379	8454	3118
	1686	10876	13040	17491	11393	2077	3299	874	13487	11838	9664	10552
	2610	8066	13005	12361	6853	3757	2887	15577	10529	14247	16087	18789
	15976	16374	10771	4277	14521	12901	18215	11495	3853	4255	1629	12007
	2466	9883	11432	13572	8080	5273	3638	18449	18786	16514	12076	1511
	6812	17300	5015	8838	9304	10185	7905	11345	15952	766	13089	2956
	613	8059	1979	12501	8535	16930	3363	10502	9217	16331	12258	3097
	9416	10257	11596	2590	11883	5567	18847	4461	15605	10990	10192	17106
	6082	15250	19258	19683	6624	17570	5102	9195	8650	11158
366:	7895	6135	5404	5150	6602	19424	2002	11648	2102	11953	8198	18899
	10410	16171	8925	5672	16257	7833	4196	7416	8915	14363	1706	15695
	9308	7164	13079
367:	17228	9424	6076	17145	3914	10656	6091	14671	7138	1890	732	16240
	11984	11978	17029	18186	17451	1323	5667	18155	19103	8458	10719	1530
	1664	4884	11793	11316	1027	569	6663	18298	6906	1989	11679	8546
	17917	7183	4074	5267	8898	17813
368:	10139	8837	7747	7598	13605	1318	9532	7490	2370	12273	4264	14618
	8627	10651	13584	18749	7057	7233	408	12207	5515	14163	1226	1228
	5775	5424	18314	18275	10203	8674	1800	2693	19005	8477	14053	9051
	10777	4549	6597	9738	7370	13649	5856	10149	13783	12613	17815	11337
	9362	13027	15862	4287	18724	8169	1419	6285	15077	2147	2905	14574
	1177	13510	9181	7135	7290	2520	6455	4618	7286	7288	2679	551
	9787	11002	1701	8930	8928	8942	1097	13702	15267	12778	15439	18947
	17593	12250	12347	19108	15478	7855	9687	17776	17774	12927	17808	3508
	7592	10782	2475	17662	17990	268	18627	12803	4005	6216	13658	8061
	16885	5642	8881	8964	10019	2902	8544	10703	1548	4925	12994	10443
	7766	13011	12993	13010	17357	15189	1888	18221	17786	9563	9562	2386
	5156	3903	547	18001	12480	13049	8197	2495	17171	18977	12208	19466
	8339	10488	4385	19012	16077	8291	17408	5878	19014	8302	16024	16026
	16018	16022	4841	9827	9828	9829	8403	9847	9830	9842	4690	2646
	13874	9849	14131	17831	9865	8405	9851	9844	7012	8406	4019	499
	9951	11218	13124	538	13569
369:	6354	13600	13597	13402	8495	11462	4678	17651	2271	18508	9403	8227
	19337	18039	11414	13507	8796	16388	13135	10409	13151	13116	13153	14811
	13648	16385	17968	17992	18014	17987	17989	17964	17984	17963	18015	17961
	17934	17931	18010	17966	17884	17850	17851	17885	17905	17839	17878	17900
	17881	17854	17903	17360	12858	19170	13662	7781	19569	19153	13650	13595
	5154	17820	2688	17837	17834	17821	2707	2705	17822	17363	3754	3771
	3775	3751	3778	3747	17028
370:	13195	17260	15550	15298	16070	13528	18906	6667	3884	19731	16485	15332
	11024	4720	7335	10724	9903	16073	9800	2005	2226	17918	14439	11382
	1930	15821	6790	3307	2184	3678	6020	2483	16932	11759	18061	8407
	17553	9683	10479	3498	7938	11193	10141	10064	5340	13160	1065	1050
	1066	8697	5057	1948	19456	10401	4381	5626	3147	4807	15513	12411
	1085	17692	15803	9678	13378	594	3808	6353	1729	12195	14567	9675
	19783	10272	1789	7097	4737	17591	9334	11855	9329	14859	6302	14086
	8083	15671	12954	13947	17819	5460	14531	15105	16035	18528	15990	19316
	10591	3086	18057	7458	4476	12898	14681	5385	6706	12606	19781	4024
	18079	12117	14093	15233	18591	7496	19829	4740	7646	6599	7671	19185
	12054	10751	9600	19620	8234	8875	8090	5475	9292	16255	13529	6245
	17823	2300	18651	19369	988	5505	16473	15879	14117	9448	8299	7128
	1260	12844	6801	11913	12153	3878	16199	3426	6628	5890	3275	9071
	19553	19622	5677	2987	5813	8587	5410	7917	10123	11678	19390	5338
	10026	16740	6793	1954	9499	1886	18780	8529	17665	18483	19399	13823
	10599	10014	11713	13942	16536	6829	13457	12144	4468	15992	10473	13518
	2219	10524	420	421	14605	14570	6724	1269	15608	11671	5125	6124
	12849	7013	12911	427	17957	7104	18943	14199	10798	14689	13399	9608
	5287	17769	9135	11977	16390	8020	11559	16849	475	16412	9947	18271
	8288	16395	11055	10283	8052	3695	4102	6848	1576	5301	7717	994
	2477	9188	4330	6788	6219	12712	8475	8239	14804	13446	8249	3014
	11497	719	15957	17377	11889	19701	16334	9730	14899	17716	13795	14007
	15861	9886	16290	1777	16807	13147	5390	5656	16417	11861	16848	8738
	10094	18845	16925	17514	13485	17384	15049	13629	16050	15795	15733	10818
	17446	13946	5886	1298	18331	2672	1478	4124	15983	15934	3583	13711
	1676	19213	15367	19827	17298	12179	5594	17065	6157	13890	4408	9574
	11208	1762	15748	10092	2000	6115	8274	11506	15771	13770	17670	13724
	4250	18579	18707	18706	18338	11926	18667	19212	9804	19854	14597	7979
	5533	5584	7353	4291	17318	16669	687	880	11774	18653	11461	14110
	6979	18340	8989	12588	16340	12839	4132	12893	15176	12464	5250	13418
	19208	6505	2317	5276	12735	5595	3237	2548	769	11313	4897	4054
	13148	18282	8680	17055	9088	13069	7258	15620	4755	2281	4479	14873
	14276	18888	1613	2084	2083	2525	15057	8026	1769	6771	18311	14286
	18220	3288	3011	1673	18154	842	9128	10218	12711	4076	14589	13986
	9857	976	14336	10790	16144	19128	6185	10202	19394	10344	5962	15092
	13101	13349	1772	5842	17973	9011	7194	12615	6592	16662	6996	4557
	2794	9462	13636	1591	641	17482	711	1379	4654	19782	16899	4214
	17607	4609	14972	12409	6334	8398	2027	15897	1125	2319	5865	18872
	5227	19597	6259	16933	6862	12848	17576	9766	5970	13699	9543	10511
	17456
371:	8393	9015	13413	2587	10796	17631	11121	433	4963	9556	575	5319
	15391	10530	9031	4635	9004	9073	3306	11645	3981	19101	19141	19812
	10554	17490	13822	14492	17246	1376	15351	19391	12231	4365	5194	2111
	5408	17546	18797	5903	6042	1218	4848	19643	4899	6748	1341	2965
	13343	10436	3965	12507	16052	10080	14622	15172	1156	15431	19244	5027
	7279	4985	13179	18612	9230	6746	8566	5265	5208	3815	11698	18164
	13048	16879	4441	8300	11252	16442	505	453	12406	1723	6094	17550
	19037	5884	14923	4421	18481	6530	12498	17991	3134	8890	3350	7985
	7864	10001	10340	991	5289	19317	10465	7319	2659	5841	13312	1585
	8483	2167	16410	18310	17967	11530	8907	847	867	8377	12438	12935
	1630	8528	11657	16778	524	942	13396	13561	12813	15308	14883	14448
	5321	8706	4558	4612	4061	16274	14724	2777	3112	607	6814	11242
	4393	19491	1312	1238	437	18920	2620	5133	8260	9619	13014	8506
	10965	2373	14342	18448	19165	17471	5405	6607	1741	14959	15075	16265
	13994	1258	14304	8744	10343	19837	12926	7213	3174	10993	14602	10617
	7779	10187	9485	15515	19398	18257	19349	1202	15472	7901	12094	13651
	1646	14731	1180	12154	16047	18288	2248	8051	19670	7996	7995	3855
	1551	14194	18409	5592	11704	13409	18199	12052	7391	8999	15302	4964
	15580	16350	15735	14708	15312	6145	15699	6159	15221	18322	10493	501
	6886	13963	12295	8982	13436	16066	11980	4664	9057	9034	16666	15687
	2427	11094	13668	3320	14079	9090	11181	8003	4257	11638	6544	6957
	3999	12296	10381	2653	4772	4810	8751	10568	10162	5600	5649	15893
	14376	16307	17697	9785	10884	16276	6169	6176	1037	9196	8367	7783
	14533	9056	15139	19917	12676	3038	10673	5503	7023	15682	10198	898
	1297	5858	14777	6955	17092	945	3032	10231	16741	4106	11060	10054
	18270	12505	13314	1821	7784	11537	6572	3603	13635	5021	9052	7601
	6284	8815	10431	18883	18773	17616	11724	3501	16356	11013	14805	9024
	14557	7384	17109	7703	18849	14347	5364	6468	8936	3979	19281	10693
	13957	3977	13486	7380	17077
372:	4087	6477	5737	5639	1608	6099
373:	11821	15570	15362	17684	12417	16687	6089	15073	1359	9786	19736	8877
	10254	5707	15924	3173	5784	19735	9392	2552	15018	11302	10241	18422
	14812	5768	9824	19506	6981	10715	19649	3163	9998	2058	6405	6584
	7848	14615	15185	2020	16636	15445	11375	18820	10331	1794	6619	8395
	6787	15426	6658	7293	10195	2815	19672	5744	14420	15534	15370	14303
	15177	12323	14958	14797	11491	452	5665	6655	9310	16947	3284	15338
	17037	6312	3537	1320	3839	1070	13922	11570	9105	5536	15484	14763
	14487	9068	12682	9682	10905	17502	4201	4406	8085	7056	8194	19718
	4289	19086	2788	10609	1276	11826	13369	15331	5251	8761	16059	6055
	8829	15454	5769	404	6808	15129	1725	10525	13488	7737	8203	2464
	17537	3566	8056	4191	10305	16861	7136	8740	1328	6582	7383	9441
	7191	15884	17130	5403	5900	19080	12583	10011	3547	16174	11235	17910
	9121	5888	18291	17740	7117	12383	15552	8043	8998	8613	9367	10541
	17801	8616	10220	1388	8599	4600	15321	13524	873	3602	1369	11422
	7039	17579	16732	1166	9497	14498	11817	691	12683	11133	10036	10168
	9240	7540	16302	2752	10828	18058	9911	15729	17807	1719	19117	1924
	3905	6227	4868	10358	11763	18113	5209	8329	10740	6938	9635	14512
	17399	18121	12733	13966	5279	1953	1959	15567	18234	3818	5220	2744
	18925	13352	15418	7193	635	9142	8342	1452	16526	8583	14626	10595
	15517	3675	15449	18995	1550	12913	1574	10748	15207	689	10793	16997
	12087	3593	9495	12369	16764	13313	16678	1034	9702	12049	15046	15685
	1505	4817	6591	10004	17280	1057	2209	16242	6770	11621	11066	19348
	13114	5818	18606	11096	2735	11091	16599	13119	10928	11061	18170	18353
	3156	18093	8497	15157	14638	11736	1201	11263	18000	17999	11949	914
	4681	8852	14381	624	7004	5368	718	17732	11589	12259	1569	3028
	5910	8948	4159	8737	11933	15646	4723	2738	4789	376	8578	16888
	18857	6948	802	19507	5226	6977	6646	17557	2557
374:	16075	17346	1488	14198	4254	1650	2972	17330	12199	963	19510	17391
	5693	10970	15087	18216	17575	18987	17965	11155	16523	17488	9549	11549
	10008	17518	8820	12423	1502	16789	5728	716	10447	10464	16976	5052
	6412	5108	3314	6292	8733	16269	15372	10825	12586	10413	10380	12879
	10280	19593	3339	9629	3902	1154	10253	8577	7055	2366	3283	13877
	9422	4904	10226	1456	18827	6632	11525	14700	16759	2841	3514	3755
	17006	17415	7347	3067	13384	3558	2974	1846	3323	6629	3184	19561
	19703	15961	8827	2835	9303	1071	10179	14834	435	12500	4768	8437
	7517	2760	14250	12828	10786	1268	13055	13052	12497	16738	9520	19359
	10335	2636	2886	9647	6341	6642	7315	12387	19717	11540	13887	5657
	16407	18284	16813	16160	19921	18470	14030	12523	13879	8162	18330	4380
	12845	8696	15369	16716	14346	16690	4719	3277	5678	5680	10403	3623
	5774	2508	1744	19808	4467	13035	11579	7401	2215	4902	11210	11968
	15518	8984	3055	3245	18372	7421	8387	11179	7162	12630	5371	6163
	5316	14827	17771	590	15838	18775	7975	11865	15784	2067	15507	17191
	10843	14530	15228	9977	13811	16873	2501	8524	6493	9867	12072	8122
	14458	2666	15333	9399	4828	15607	12871	15613	1771	16671	7034	11542
	15229	10987	6960	14341	14525	7247	4763	19509	5283	18405	8785	16222
	14297	18434	14175	18636	10459	13065	17257	16561	1889	14928	6483	19913
	2456	1117	3838	6107	6860	7721	6402	1845	12187	10183	3985	7714
	6914	17425	9651	13259	791	15634	15360	10630	8039	9174	19497	1774
	12575	11290	11928	14723	17308	16624	18115	9049	7707	4914	15036	6484
	13266	7824	18137	7109	4623	13337	19831	7899	18876	14992	15343	4660
	14819	3465	1564	18556	19844	4349	19085	9971	13851	16931	2174	8283
	19354	11651	4757	15968	6959	10967	11188	17645	8006	6247	8959	10307
	13566	11039	13744	17853	863	11617	7364	10342	548	17035	13825	1142
	13781	9657	5941	1581	17069	12968	15470	16299	4281	15614	1937	4559
	14610	4696	13538	7697	3637	7603	11413	2179	7933	19467	8355	1987
	19608	967	15718	3480	8769	12950	16296	15497	18747	16455	2726	19646
	1232	9750	12105	12718	12852	5921	12836	3388	7411	16468	8997	11368
	1122	7595	14296	9278	19558	12461	14633	16726	17802	7427	18821	8484
	8671	7208	18639	13316	13956	4535	18240	4560	14766	13755	10837	5042
	14741	10899	6606	836	5977	3221	7913	9252	12441	5332	6266	4788
	11839	7268	17954	2356	3394	7563	11666	4588	13840	956	15465	19389
	17489	4423	4266	13131	10548
375:	16510	13467	14806	18916	13489	19287	6963	11609	12123	6168	14647	17406
	19726	10559	16928	16901	7555	12727	15736	13018	2908	998	11571	14927
	11630	8370	8372	13218	10996	17016	7958	9384	13045	13447	8144	1147
	15001	2276	2823	913	14592	3364	18232	14988	977	13106	11518	14539
	12112	17129	2915	10863	1580	4515	5798	8253	8082	416	19385	19171
	1100	16820	6199	4571	18783	1742	10182	13173	16635	17155	13139	6972
	5995	17971	7440	13865	9190	7070	17784	2010	723	15385	11201	10394
	4498	13493	13320	14352	678	13273	448	5709	11312	18597	11116	7510
	15024	12386	4804	16278	7734	1363	7445	19533	12959	1292	16198	16824
	12162	2158	10445	8558	12176	1883	5802	17888	7202	9357	9212	9289
	15706	15848	18962	11998	16594	7471	9904	6557	5534	7297	14395	12716
	14630	1709	8057	5106	13882	13240	3414	8391	13833	18908	11344	5879
	12334	16882	8131	13298	15056	11047	2998	9898	12476	14335	16430	17095
	2214	1606	8257	16665	13613	6921	7748	14323	15979	5733	12116	6947
	8474	13708	15492	18185	6299	13610	18148	14215	18714	2930	12129	15295
	15268	14650	18384	18143	3511	6197	6361	17944	17442	5229	2611	565
	16358	16357	15084	16089	1181	17594	9098	2241	19299	12023	7283	10377
	6819	4949	8736	13205	2694	19763	13769	19621	13407	9290	16521	1825
	10029	12822	2903	15953	15986	11911	13793	11546	2234	14881	9908	11523
	15533	17577	11068	17517	17721	17374	10696	12227	19181	13058	8185	15758
	4153	877	8465	19011	4495	6613	16284	8138	18523	4389	15763	3113
	17826	2647	1098	10483	10463	19364	11397	11401	11381	6868	15127	1474
	16902	5599	19363	17325	9309	19361	5037	4928	3787	16923	4146	16939
	19535	15773	19339	15539	15125	18484	4203	18687	9493	10628	16954	18751
	5417	19261	19262	19234	1714	19898	19336	19855	19291	19286	19880	19877
	19295	19895	19875	1295	10357	6086	10734	11846	6683	12722	18816	18796
	7791	4566	18951	15711	11715	3398	18259	16246	1255	3813	1431	15683
	7433	3767	5490	5324	10201	9680	13775	10924	11448	5618	3801	6703
	2837	11877	2626	19090	4080	8371	9573	3492	13188	3045	16273	5297
	2403	2365	6847	7359	12126	16429	13888
376:	11821	15570	15362	17684	12417	16687	6089	1359	9786	19736	8877	10254
	5707	15924	3173	5784	19735	9392	2552	11302	15018	18422	14812	5768
	9824	19506	10715	19649	3163	9998	2058	6584	7848	14615	15185	2020
	15445	16636	11375	18820	10331	1794	6619	8395	15426	6787	6658	7293
	10195	2815	19672	5744	14420	15534	15370	14303	15177	12323	14958	14797
	11491	452	5665	6655	9310	16947	3284	15338	17037	6312	3537	1320
	3839	8693	1070	13922	11570	9105	5536	5454	15484	14763	14487	9068
	12682	9682	10905	17502	4201	4406	8085	8086	7056	8194	4289	2788
	19086	10609	17621	1276	11826	13369	15331	5251	8761	8829	15454	5769
	6808	15129	1725	10525	13488	7737	8203	2464	17537	3566	8056	4191
	16861	7136	8740	1328	6582	7383	9441	7191	15884	17130	5403	5900
	19080	10011	3547	16174	11235	17910	5888	18291	17740	7117	12383	15552
	8043	8613	9367	10541	17801	13465	8616	10220	1388	8599	4600	15321
	13524	873	3602	1369	11422	7039	16732	1166	9497	14498	11817	691
	12683	11133	10036	10168	9240	7540	16302	2752	10828	18058	9911	15729
	17807	1719	19117	1924	3905	6227	4868	10358	11763	18113	5209	8329
	10740	6938	14512	17399	18121	12733	13966	5279	1953	15567	3818	2744
	18925	13352	15418	7193	635	9142	8342	1452	16526	8583	14626	10595
	15517	3675	15449	18995	1550	12913	1574	10748	15207	689	10793	12087
	9495	12369	16764	13313	16678	1034	9702	12049	15046	9046	15685	1505
	4817	6591	10004	17280	18300	1057	2209	13541	16242	6770	17146	11621
	11066	2735	11091	11096	19348	10928	13114	5818	18353	3156	18093	15157
	11263	11949	914	4681	7472	8852	624	7004	5368	718	17732	12259
	3028	1569	5910	8948	4159	5738	8737	15646	4723	4789	12067	16888
	18857	373	6948	802	5226	6977	6646	17557	2557
377:	8393	9015	13413	2587	10796	17631	11121	634	433	4963	9556	575
	5319	15391	10530	431	9031	4635	9004	9073	3306	11645	3981	19141
	19101	19812	10554	16183	13822	14492	17246	1376	15351	19391	12231	4365
	5194	2111	5408	17546	18797	3789	5903	1218	4848	19643	6748	4899
	1341	2965	13343	10436	3965	12507	16052	10080	14622	1156	15431	5027
	7279	4985	13179	3070	3071	18612	2494	9230	6746	8566	5265	5208
	3815	11698	12148	18164	13048	16879	8300	4441	9892	11252	16442	505
	453	12406	1723	6094	17550	19037	5884	16347	17111	14923	4421	18481
	6530	12498	17991	3134	8890	3350	7985	7864	10001	10340	991	5289
	19317	10465	7319	2659	5841	13312	1585	8483	2167	16410	18310	17967
	11530	8907	847	867	8377	12935	12438	1630	8528	11657	18567	17817
	16778	524	942	2110	13396	13561	12813	15308	14883	14448	5321	8706
	4558	4612	4061	16274	14724	2777	607	3112	6814	11242	4393	19491
	1312	1238	437	18920	2620	5133	9619	8260	13014	8506	10965	3168
	2373	14342	18448	19165	17471	5405	6607	1741	14959	15075	16265	13994
	1258	14304	8744	10343	19837	12926	7213	3174	10993	14602	10617	7779
	10187	9485	15515	19398	18257	19349	1202	15472	7901	12094	13651	1646
	14731	1180	12154	16047	18288	7639	2248	8051	7996	7995	3855	1551
	14194	18409	5592	11704	8432	13409	18199	12052	7391	8999	15302	4964
	15580	16350	15735	14708	15312	6145	15699	6159	15221	18322	10493	501
	6886	13963	12295	8982	13436	16066	11980	4664	9057	9034	16666	15687
	2427	11094	13668	3320	14079	9090	11181	8003	4257	11638	6544	6957
	3999	12296	10381	2653	4772	4810	8751	10568	10162	5600	5649	15893
	14376	16307	17697	9785	10884	16276	6169	6176	1037	9196	8367	7783
	14533	9056	15139	19917	12676	3038	10673	5503	7023	15682	10198	898
	1297	5858	14777	6955	17092	945	3032	16741	4106	11060	10054	18270
	12505	13314	1821	7784	11537	6572	3603	13635	5021	9052	7601	6284
	8815	10431	18883	18773	17616	11724	3501	16356	11013	14805	14557	7384
	17109	7703	14347	18849	5364	8936	3979	19281	10693	13957	3977	7380
	13486	3384
378:	13086	19930	5574	17164	15382	2809	1565	9389	817	831	5123	14538
	9536	1834	1833	13082	18812	19542	12765	13660	1610	1609	15422	15421
	1627	13773	5445	16272	10838	19127	19142	9628	10532	7251	8084	9179
	10060	3233	3234	10382	15456	12062	12060	8373	598	11569	18041	16210
	3876	5308	5310	4105	19053	17469	5866	18520	18518	17299	16592	13763
	1649	2826	1088	5244	17633	8002	6325	8510	8509	17342	10265	8523
	11182	2722	2708	14181	17828	10012	8552	6241	10590	5676	10274	8661
	3295	6576	8917	12079	16538	11322	11319	15823	16815	16795	13353	14432
	2003	1985	7807	7667	1870	12245	15011	15010	7081	7327	7326	3951
	11419	11420	5668	5402	2091	2088	10840	16194	9002	9003	8127	8141
	1440	6106	6104	17126	17005	4082	9591	9590	1915	6725	10684	7300
	3317	3316	9330	19487	19488	14152	14153	2122	1914	1911	10665	14295
	14309	2813	2489	17345	11755	18173	14628	16281	4570	426	12610	17201
	17057	16060	16058	9609	987	9610	15673	921	1086	18306	11336	2498
	17087	17085	12034	12035	14678	14675	1138	544	16271	12318	12812	4308
	19378	14598	14599	7369	18594	7648	17481	19008	14345	5166	7493	13726
	13725	8384	3400	16200	7902	8410	8409	13137	16958	16957	15723	12257
	12253	15978	12973	4460	4459	13017	1570	1078	2531	15107	15091	8965
	13199	13198	19203	19218	2645	523	5806	17244	17241	9229	13301	12697
	16182	14543	17648	7956	541	7604	12160	14921	4194	19094	17637	18276
	18272	14246	14245	6265	11350	6263	11351	1447	9984	5779	16703	16701
	8272	8270	15033	637	11702	3382	13215	6987	17734	17485	17484	8448
	11578	16065	14275	12748	19807	2279	9116	2296	2779	13325	13222	5611
	2538	12956	15346	10824	1158	4300	18255	6983	15168	4680	19799	12284
	6950	16092	3445	2799	10239	3660	12422	2797	18788	11987	9273	10359
	2829	13626	19734	592	14588	3599	6112	6093	6110	572	5844	11779
	4108	3093	11517	11539	455	10947	11127	13214	3927	11199	12891	15461
	15460	7465	5719	5718	3367	13458	13456	1149	14244	6969	12138	9126
	1661	15396	5031	16825	1467	3689	6716	6528	6526	8496	18877
379:	16324	10027	16399	19626	19600	476	10737	6503	2637	5207	7908	18645
	13757	8204	17032	1213	10125	9070	5372	15085	10478	7338	18023	9641
	11812	14642	18106	9561	2651	1666	5437	14594	5439	12210	6002	12290
	5485	6003	14353	14349	14351	10819	8846	9429	2453	10112	16543	3140
	1146	1124	1121	15438	2354
380:	15042	683	16630	18404	5663	2482	10670	11782	18811	18709	11700	19144
	6755	9799	13818	6126	2455	8789
381:	17923	18890	5500	4771	6745	5378	3236	1210	11603	19433	19342	15300
	17953	11454	9261	12841	11591	5415	5453	5430	8278	14564	6761	17927
	18569	14781	10539	6214	3620	3528	17899	8748	12221	5504	5502	9489
	6425	8958
382:	19132	10614	8139	10836	16040	11545	7879	2100	1703	14547
383:	1189	16611	3586	4641	19633	17705	17150	8849	3552	7294	2029	6153
	18596
384:	741	1633	5923	8765	16673	14609	3698	6449	2951	2093	11563	614
	14593	14762	6397	7731	14290	17038	4311	14640
385:	11357	13113	2101	9359	2343	5986	9703	12857	8854	17190	4889	13808
	12572	3386	7845	17983	4794
386:	1016	16046	10106	6272	16847	10542	15062	5576	4893	5448	15407	7831
	16578	18278	2702	7215	9961	6250	3252	18599	16433	3226	17007
387:	7223	8111	7867	1322	9479	3291	18674	5132	12538	3515	18852	16518
	14682	5419	9781
388:	13217	19387	1319	13219	1333	2384	4322	7600	13280	5924	13688	2181
	16877	16122	5091	927	6100	3686
389:	11269	4678	13073	1755	1754	8654	3662	17371	3891	3973	2187	15246
	18462	4802	3390	619	643	13183	4014	1542	10000	19057	6229	1262
	14305	14256	17590	5708	4877	3775	8462
390:	17182	3673	6158	5731	8195	15335	7264	7267	5346	10728	11675	2337
	19110	7961	8491	12046	10224	6729	18918	18228	3153	8723	3309	3313
	6222	15956	4632	12325	6835	6387	2622	4496	9897	10860	8044	10003
	13262	13236	6403	5331	4640	1303	4602	1304	18435	16053	18480	17646
	17644	17675	16363	4781	3269	8610	10852	3582	5717	9242	9340	9346
	13271	1345	3780	11086	14006	2264	5062	5146	16786	891	1282	11639
	2472	9621	19732	15665	10462	16384	18138	9510	19888	19119	4931	18991
	13945	18937	13580	18401	13863	1936	4639	10245	760	13679	14995	17389
	5259	10385	9763	15381	10901	10886	10888	12415	15437	8840	7871	4684
	12209	17107	11203	16435	8624	13410	8530	10422	11650	15110	13639	12675
	13689	19825	4662	13277	5625	9279	3369	5976	1377	2144	4086	18044
	16889	6767	18418	11859	8188	6500	16529	16528	13234	14259	18077	11012
	10071	11825	16181	6479	3800	9154	19377	924	923	7361	4574	14380
	13895	17168	14901	17969	13735	17599	10270	4099	18973	3523	14337	8378
	4490	18286	5203	2114	13850	9109	9096	7060	4161	6561	1211	9640
	13790	19180	14796	5771	15355	14027	6282	12681	16502	4842	4262	4930
	15500	16490	14450	18361	15929	1171	7624	13836	15083	19541	1490	8022
	9426	19755	13700	10435	8346	13719	8681	19368	10906	19116	1509	3865
	12853	3125	5889	8316	18315	9077	436	1977	8354	5002	5025	16553
	5171	17152	9352	17908	9960	9963	4907	2079	14164	3744	3102	18375
	4128	4129	14794
391:	5830	10547	4780	6670	1560	15309	12977	11581	15243	10500	7299	19550
	4614	11584	18988	17503	13880	18201	7124	6036	17359	15414	19074	13696
	4180	810	12806	13939	1259	2434	12029	5666	8718	751	18491	15717
	1795	4475	1738	627	5732	18253	8660	11107	16309	12868	4299	14698
	14679	14502	8248	11990	9758	16167	9385	10043	10430	16906	12099	5791
	16810	13759	8562	2921	8889	10315	14077	2682	18457	14406	12241	10194
	5832	13548	15304	4676
392:	18452	9432	5303	14233	9970	7637	14980	15265	7581	3016	10412	19809
393:	19384	19382	682	6463	1021	6039	6041	6057	7898	7896	13532	13533
	8037	9356	16887	16866	7150	7148	11880	11903	7084	7083	3381	13816
	13817	12463	4530	2311	2312	10624	10623	15054	2256	2257	14016	14018
	8801	6140	6142	8800	6138	8357	8375	8360	3666	3668	11044	11022
	9473	9494	5001	5000	4831	4836	18327	19232	5812	5815	9999	7941
	7942	749	3079	7426	7451	8001	7999	8005	19635	4051	15885	12205
	2860	786	784	15576	15575	12310	12307	2306	3461	7640	10242
394:	12840	13577	8132	1535	5905	3211	3797	12558	6843	17949	15017	9231
	1310	17041	6367	18764	8461	15888	6866	6864	7983	8980	10079	15489
	11102	9933	7325	1749	8441	7720	17727

Example 3 Consensus Sequence Build

ClustalW program is selected for multiple sequence alignments of an amino acid sequence of SEQ ID NO: 198 and its homologs, through SEQ ID NO: 394 and its homologs. Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty. Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment. The consensus sequence of SEQ ID NO: 227 and its 25 homologs were derived according to the procedure described above and is displayed in FIG. 1.

Example 4 Identification of Amino Acid Domain by Pfam Analysis

This example illustrates the identification of amino acid domain by Pfam analysis.
The amino acid sequence of the expressed proteins that were shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software in the appended computer listing. The Pfam protein families for the proteins of SEQ ID NO: 198 through 394 are shown in Table 16. The Hidden Markov model databases for the identified patent families are also in the appended computer listing allowing identification of other homologous proteins and their cognate encoding DNA to enable the full breadth of the invention for a person of ordinary skill in the art. Certain proteins are identified by a single Pfam domain and others by multiple Pfam domains. For instance, the protein with amino acids of SEQ ID NO: 207 is characterized by two Pfam domains, i.e. “TFIIS_M” and “TFIIS_C”. See also the protein with amino acids of SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”. In Table 16 “score” is the gathering score for the Hidden Markov Model of the domain which exceeds the gathering cutoff reported in Table 17.

TABLE 16

PEP
SEQ		Pfam domain
ID NO	GENE ID	name	begin	stop	score	E-value

198	CGPG106	CMAS	69	306	−149.8	5.00E−05
198	CGPG106	Methyltransf_11	131	229	109.1	1.20E−29
198	CGPG106	Methyltransf_12	131	227	54.6	3.10E−13
200	CGPG117	SNF5	14	252	66.9	5.90E−17
202	CGPG1288	Cupin_3	57	131	130.8	3.40E−36
204	CGPG1458	Phosphorylase	180	958	1454.7	0
205	CGPG1542	LMBR1	11	488	715.1	4.40E−212
206	CGPG170	Metallophos	54	249	140.4	4.50E−39
207	CGPG1828	TFIIS_M	206	327	203.5	4.50E−58
207	CGPG1828	TFIIS_C	338	376	83.3	7.00E−22
209	CGPG2217	ORMDL	14	156	356.8	3.20E−104
210	CGPG2292	Tim17	52	186	16.1	4.20E−05
210	CGPG2292	SAM_1	192	253	13.8	0.019
212	CGPG2499	Glycolytic	4	339	588.8	4.50E−174
213	CGPG2653	WD40	135	172	40.3	6.20E−09
213	CGPG2653	WD40	235	271	40.7	4.60E−09
213	CGPG2653	WD40	276	313	38	3.00E−08
214	CGPG2813	HLH	204	251	34.1	4.60E−07
215	CGPG3002	eIF-4B	5	451	−50.3	1.90E−10
216	CGPG3154	C2	14	102	101.8	1.80E−27
216	CGPG3154	C2	198	272	57.8	3.20E−14
217	CGPG3235	B_lectin	66	176	156.8	5.30E−44
217	CGPG3235	S_locus_glycop	189	315	227	3.90E−65
217	CGPG3235	PAN_2	332	402	103.9	4.20E−28
217	CGPG3235	Pkinase_Tyr	488	757	75.6	2.10E−20
217	CGPG3235	Pkinase	488	757	109	1.30E−29
218	CGPG3274	Allene_ox_cyc	74	253	401.6	1.10E−117
219	CGPG3275	Auxin_inducible	50	155	135.6	1.30E−37
220	CGPG3363	zf-AN1	21	58	10.5	0.0033
220	CGPG3363	zf-AN1	103	148	12.4	0.0019
221	CGPG3367	TFIIA	9	375	556	3.40E−164
222	CGPG3375	Tryp_alpha_amyl	33	111	55.2	1.90E−13
224	CGPG3534	Ank	42	74	13.6	0.31
224	CGPG3534	Ank	75	107	36.8	6.90E−08
224	CGPG3534	Ank	108	140	1.6	17
224	CGPG3534	Pkinase_Tyr	160	413	202.4	9.90E−58
224	CGPG3534	Pkinase	160	413	198.5	1.40E−56
225	CGPG3638	Ribonuclease_T2	30	219	367.3	2.20E−107
226	CGPG3918	Metallophos	60	255	159.1	1.10E−44
229	CGPG3972	14-3-3	8	243	501.1	1.20E−147
230	CGPG3990	zf-C3HC4	259	299	47.3	4.60E−11
231	CGPG3994	Pkinase	554	802	207	4.10E−59
231	CGPG3994	Pkinase_Tyr	554	802	233	5.90E−67
232	CGPG4026	CMAS	59	320	−174.5	0.003
232	CGPG4026	Methyltransf_11	122	220	96.3	8.10E−26
232	CGPG4026	Methyltransf_12	122	218	37	5.90E−08
232	CGPG4026	Sterol_MT_C	229	358	301.1	1.80E−87
233	CGPG4048	Pkinase	21	275	342.1	8.80E−100
233	CGPG4048	Pkinase_Tyr	21	275	65.1	1.20E−19
233	CGPG4048	NAF	304	364	104.5	2.80E−28
234	CGPG4052	DUF298	127	242	222.4	9.20E−64
236	CGPG4058	Asp	99	437	−128.4	4.60E−06
237	CGPG4069	adh_short	31	218	−16.7	4.80E−05
239	CGPG4088	SWIB	324	399	96.4	7.80E−26
241	CGPG4121	Cyclin_N	58	190	133.7	4.80E−37
241	CGPG4121	Cyclin_C	192	314	44.3	3.70E−10
242	CGPG4122	zf-C3HC4	100	141	27.6	4.10E−05
243	CGPG4140	p450	48	484	140.8	3.30E−39
244	CGPG4154	zf-CCCH	90	115	22.6	0.0012
245	CGPG4311	FBPase	70	395	347.9	1.50E−101
247	CGPG4369	BolA	10	79	92.1	1.60E−24
248	CGPG442	2-Hacid_dh	85	394	140.9	3.20E−39
248	CGPG442	2-Hacid_dh_C	187	362	293	5.20E−85
248	CGPG442	ACT	551	621	39.9	8.10E−09
249	CGPG4454	p450	45	447	−35.5	2.10E−09
250	CGPG4456	p450	75	502	103.6	5.30E−28
252	CGPG4588	Auxin_inducible	1	102	151.5	2.00E−42
253	CGPG4765	DUF868	28	304	175.5	1.20E−49
255	CGPG4912	WD40	243	281	31	3.80E−06
256	CGPG4926	WD40	42	79	22.8	0.0012
256	CGPG4926	WD40	126	163	25.8	0.00014
257	CGPG4967	Asp	71	424	−94.7	4.70E−08
258	CGPG4977	Usp	3	157	85.3	1.80E−22
259	CGPG5001	adh_short	5	181	6.4	1.40E−06
260	CGPG5025	adh_short	30	212	5.7	1.50E−06
261	CGPG5041	DUF26	77	132	84.3	3.40E−22
261	CGPG5041	DUF26	188	242	100.7	4.00E−27
261	CGPG5041	Pkinase	333	558	60.3	5.70E−15
262	CGPG5116	ArfGap	15	137	174.1	3.20E−49
262	CGPG5116	C2	182	261	101.4	2.40E−27
263	CGPG5144	p450	61	535	157.9	2.40E−44
264	CGPG5171	p450	35	496	137	4.60E−38
265	CGPG5194	DUF1191	25	308	616.2	2.60E−182
268	CGPG5221	p450	30	499	135.4	1.40E−37
269	CGPG5269	PCI	297	401	108.9	1.30E−29
270	CGPG5404	Peptidase_S10	68	480	683.3	1.70E−202
271	CGPG5432	MtN3_slv	12	99	145.4	1.40E−40
271	CGPG5432	MtN3_slv	133	219	140.4	4.40E−39
272	CGPG5518	Ribosomal_S8e	1	238	325.9	6.30E−95
273	CGPG5535	WD40	335	371	28.8	1.80E−05
273	CGPG5535	WD40	434	471	37.1	5.50E−08
273	CGPG5535	WD40	476	513	47.2	5.20E−11
273	CGPG5535	WD40	518	555	35.6	1.60E−07
273	CGPG5535	WD40	567	604	36.1	1.10E−07
273	CGPG5535	WD40	621	658	34.9	2.50E−07
273	CGPG5535	WD40	663	706	35.5	1.60E−07
274	CGPG5540	ESCRT-III	21	207	265.7	8.40E−77
275	CGPG5568	AA_permease	69	521	−67.1	0.00013
276	CGPG5577	SMC_N	21	1049	−2.8	1.50E−11
277	CGPG5587	Thioredoxin	78	199	0.1	0.00015
278	CGPG5594	Histone	27	100	99.6	8.80E−27
279	CGPG5633	PGAM	91	277	153.2	6.30E−43
280	CGPG5640	Aminotran_1_2	30	385	362.1	8.00E−106
281	CGPG5646	iPGM_N	2	363	876.8	9.10E−261
281	CGPG5646	Metalloenzyme	373	488	173.3	5.40E−49
282	CGPG5656	Gln-synt_N	17	99	85.7	1.30E−22
282	CGPG5656	Gln-synt_C	105	358	528.1	8.50E−156
283	CGPG5659	Aminotran_3	35	361	321.7	1.20E−93
284	CGPG5661	PK	1	345	702	3.80E−208
284	CGPG5661	PK_C	355	469	115.4	1.50E−31
285	CGPG5684	Glycolytic	4	339	578.6	5.60E−171
286	CGPG5694	TIM	4	246	465.9	4.50E−137
287	CGPG5704	NDK	2	136	329	7.30E−96
288	CGPG5714	NDK	4	138	266.4	5.20E−77
289	CGPG5721	Rib_5-P_isom_A	47	215	354.4	1.70E−103
290	CGPG5728	zf-CCCH	80	106	39.3	1.20E−08
291	CGPG5757	Sad1_UNC	203	330	195.6	1.00E−55
292	CGPG5764	Actin	11	140	36.4	6.60E−08
293	CGPG5783	TFT	272	442	71.5	2.40E−18
294	CGPG5791	AA_permease	114	588	704.9	5.40E−209
295	CGPG5799	Aa_trans	206	601	453	3.60E−133
296	CGPG5856	Pkinase	79	353	159.5	8.00E−45
296	CGPG5856	Pkinase_Tyr	79	353	149.4	9.00E−42
297	CGPG5927	AAA	58	248	252.4	8.70E−73
297	CGPG5927	AAA	322	510	290.8	2.40E−84
298	CGPG5941	PfkB	42	336	86.2	9.00E−23
299	CGPG5957	CBM_20	86	178	23	5.70E−07
300	CGPG5967	DUF822	2	147	307.1	2.90E−89
301	CGPG6040	LEA_3	1	88	178.7	1.30E−50
302	CGPG607	PurA	28	275	44.4	5.80E−12
303	CGPG6178	DUF1336	53	267	427.9	1.30E−125
304	CGPG6185	UQ_con	7	148	197.9	2.10E−56
305	CGPG6306	APC8	1	161	401.5	1.10E−117
305	CGPG6306	TPR_1	339	372	34.7	2.90E−07
305	CGPG6306	TPR_2	339	372	23.8	0.00058
305	CGPG6306	TPR_2	373	406	23.7	0.00062
305	CGPG6306	TPR_1	373	406	34.1	4.40E−07
305	CGPG6306	TPR_2	407	440	22	0.0019
305	CGPG6306	TPR_1	407	440	24.3	0.00039
306	CGPG6318	MFS_1	31	496	35.3	1.90E−07
306	CGPG6318	PTR2	92	484	208.6	1.30E−59
307	CGPG6326	Kelch_1	34	79	45	2.40E−10
307	CGPG6326	Kelch_2	34	79	43.5	6.80E−10
307	CGPG6326	Kelch_1	152	198	26.7	7.70E−05
307	CGPG6326	Kelch_2	152	198	32.1	1.80E−06
307	CGPG6326	Kelch_2	203	249	20.2	0.0067
307	CGPG6326	Kelch_1	204	248	7.9	0.45
308	CGPG6370	Gp_dh_N	3	151	326	5.80E−95
308	CGPG6370	Gp_dh_C	156	313	362	8.80E−106
309	CGPG6429	ADH_N	25	135	159.4	8.40E−45
309	CGPG6429	ADH_zinc_N	166	305	105.1	1.90E−28
310	CGPG6440	PK	1	345	789.4	1.90E−234
310	CGPG6440	PK_C	357	471	167.5	3.10E−47
310	CGPG6440	PEP-utilizers	486	575	134	3.60E−37
311	CGPG6516	Aldedh	19	478	778.3	4.10E−231
312	CGPG6653	LRRNT_2	23	66	49.3	1.20E−11
312	CGPG6653	LRR_1	71	93	12.1	1.4
312	CGPG6653	LRR_1	95	117	10.5	2.8
312	CGPG6653	LRR_1	119	142	13	0.93
312	CGPG6653	LRR_1	144	166	19.5	0.011
312	CGPG6653	LRR_1	168	190	10.6	2.7
312	CGPG6653	LRR_1	192	214	8.8	5.7
312	CGPG6653	LRR_1	289	311	17.4	0.046
312	CGPG6653	LRR_1	313	335	10.8	2.4
312	CGPG6653	LRR_1	337	359	10.7	2.5
312	CGPG6653	LRR_1	361	384	12	1.5
312	CGPG6653	LRR_1	409	431	10.4	2.9
312	CGPG6653	LRR_1	457	479	11.9	1.5
312	CGPG6653	LRR_1	481	503	10.4	3
312	CGPG6653	LRR_1	505	527	10.5	2.8
312	CGPG6653	LRR_1	529	551	11.1	2.2
312	CGPG6653	LRR_1	553	575	9.3	4.7
312	CGPG6653	LRR_1	577	598	11.1	2.1
312	CGPG6653	Pkinase	695	966	134.8	2.20E−37
312	CGPG6653	Pkinase_Tyr	695	966	134.5	2.60E−37
313	CGPG6712	PGAM	91	277	154.6	2.30E−43
314	CGPG6737	PGAM	92	253	173	6.80E−49
315	CGPG6747	FBPase	106	429	448.6	7.20E−132
316	CGPG6796	Alpha-amylase	14	452	199.5	7.00E−57
318	CGPG6810	GH3	12	561	1261.5	0
320	CGPG6953	Ank	121	153	50.4	5.60E−12
321	CGPG7121	L51_S25_CI-B8	20	93	108.9	1.40E−29
322	CGPG7163	Prenylcys_lyase	149	500	788	5.20E−234
324	CGPG7206	Aldo_ket_red	14	298	389.4	4.80E−114
325	CGPG7225	Subtilisin_N	48	125	84.3	3.30E−22
326	CGPG7267	DUF588	34	164	154.6	2.40E−43
327	CGPG7272	DUF1005	50	254	524.8	8.50E−155
328	CGPG7281	FA_hydroxylase	86	229	361.9	9.30E−106
329	CGPG7308	CoA_binding	1	100	−10.8	0.04
329	CGPG7308	NAD_Gly3P_dh_N	4	147	−0.6	2.00E−06
329	CGPG7308	F420_oxidored	5	251	282.5	7.50E−82
330	CGPG7316	Anti-silence	1	155	419.9	3.20E−123
331	CGPG7371	Response_reg	29	157	94.8	2.30E−25
332	CGPG7457	PfkB	114	408	146.6	6.00E−41
335	CGPG7636	LSM	13	81	78.2	2.40E−20
338	CGPG7804	FAR1	62	279	364.9	1.20E−106
338	CGPG7804	SWIM	556	589	37.3	4.90E−08
339	CGPG7823	Rotamase	104	188	91.8	1.90E−24
339	CGPG7823	Rhodanese	203	298	44.1	4.40E−10
340	CGPG7828	DnaJ	12	81	66.1	1.00E−16
340	CGPG7828	zf-CSL	96	174	25.2	0.00021
343	CGPG7986	F-box	48	96	31.2	3.40E−06
343	CGPG7986	LRR_1	189	216	9	5.4
343	CGPG7986	LRR_1	428	451	8.2	7.7
343	CGPG7986	LRR_1	561	584	8.4	6.9
345	CGPG8015	zf-CCHC	18	35	24.2	7.90E−05
349	CGPG8083	Tryp_alpha_amyl	28	105	37.6	3.90E−08
350	CGPG8106	BURP	56	280	380	3.20E−111
353	CGPG8152	PAP_fibrillin	9	124	45.9	1.20E−10
354	CGPG8166	PBD	118	166	49.9	7.80E−12
355	CGPG8377	Oleosin	30	109	55	2.20E−13
356	CGPG8976	Ceramidase_alk	50	795	1545.5	0
357	CGPG8987	FH2	439	839	552.9	3.10E−163
358	CGPG9013	NAD_binding_1	234	350	138.6	1.50E−38
359	CGPG9080	EGF_CA	315	357	42.2	1.60E−09
359	CGPG9080	Pkinase	433	716	120.3	4.90E−33
359	CGPG9080	Pkinase_Tyr	433	704	113.1	7.40E−31
360	CGPG9081	DUF676	30	247	319	7.50E−93
361	CGPG9130	MMR_HSR1	266	369	71.9	1.80E−18
362	CGPG9133	PPR	125	159	13.2	0.19
362	CGPG9133	PPR	161	195	2.8	3.2
362	CGPG9133	PPR	196	230	22.7	0.0012
362	CGPG9133	PPR	232	266	42.1	1.70E−09
362	CGPG9133	PPR	267	301	29.4	1.20E−05
362	CGPG9133	PPR	302	336	49.4	1.10E−11
362	CGPG9133	PPR	337	371	32.9	1.00E−06
362	CGPG9133	PPR	372	407	7.9	0.8
362	CGPG9133	PPR	408	442	49.7	8.80E−12
362	CGPG9133	PPR	443	477	22.6	0.0013
362	CGPG9133	PPR	478	512	37.8	3.40E−08
362	CGPG9133	PPR	513	547	42.9	1.00E−09
362	CGPG9133	PPR	548	582	30.2	6.70E−06
362	CGPG9133	PPR	583	617	38.4	2.20E−08
363	CGPG9134	HD	91	232	46.2	1.00E−10
364	CGPG9137	RmaAD	65	337	122.2	1.30E−33
365	CGPG9141	Pantoate_transf	40	306	402.9	4.40E−118
366	CGPG9145	Lung_7-TM_R	168	423	385.2	8.80E−113
367	CGPG9147	DNA_pol_E_B	178	389	249.9	5.00E−72
368	CGPG9148	p450	36	502	128.8	1.40E−35
369	CGPG9155	Pkinase	86	347	56.9	6.10E−14
369	CGPG9155	Pkinase_Tyr	86	351	73.7	2.90E−20
370	CGPG9163	Na_H_Exchanger	12	378	280.1	3.80E−81
370	CGPG9163	TrkA_N	416	531	118.8	1.50E−32
371	CGPG9170	Complex1_30 kDa	90	158	103.8	4.70E−28
371	CGPG9170	Complex1_49 kDa	298	537	2.8	6.10E−13
373	CGPG9183	HTH_11	1	56	72.4	1.30E−18
373	CGPG9183	BPL_LipA_LipB	84	182	94.5	2.90E−25
373	CGPG9183	BPL_C	275	322	43.3	7.80E−10
374	CGPG9186	DHBP_synthase	8	203	370.6	2.20E−108
374	CGPG9186	GTP_cyclohydro2	208	366	−2.3	3.80E−10
375	CGPG9205	NTP_transferase	4	288	421	1.50E−123
375	CGPG9205	MannoseP_isomer	299	465	350.8	2.00E−102
375	CGPG9205	Cupin_2	380	450	55.3	1.80E−13
376	CGPG9207	HTH_11	6	59	50.9	3.90E−12
376	CGPG9207	BPL_LipA_LipB	83	180	104.5	2.80E−28
376	CGPG9207	BPL_C	271	317	46.5	8.50E−11
377	CGPG9219	Complex1_30 kDa	107	175	99.8	7.60E−27
377	CGPG9219	Complex1_49 kDa	311	537	−11.6	5.90E−12
378	CGPG9220	GDC-P	3	443	700.9	8.30E−208
379	CGPG9230	Peptidase_S10	88	488	657.2	1.20E−194
381	CGPG9238	Tryp_alpha_amyl	36	114	56	1.20E−13
382	CGPG9259	Mit_rib_S27	14	93	135.3	1.50E−37
383	CGPG9271	NPH3	215	418	189.9	5.60E−54
384	CGPG9275	ETC_C1_NDUFA5	35	91	112.8	9.00E−31
386	CGPG9283	DUF1195	6	161	180.3	4.30E−51
387	CGPG9309	MAP65_ASE1	38	575	52.8	1.10E−12
389	CGPG9322	Pkinase	103	383	170	5.40E−48
389	CGPG9322	Pkinase_Tyr	103	383	158.7	1.30E−44
390	CGPG9335	Sugar_tr	7	726	210.6	3.30E−60
390	CGPG9335	MFS_1	11	685	107.7	3.00E−29
391	CGPG9341	RMMBL	531	573	35.9	1.20E−07
392	CGPG9344	TPR_2	531	564	23.9	0.00051
393	CGPG9345	UPF0261	5	432	483.7	2.00E−142
394	CGPG976	Glyco_transf_8	216	533	401.3	1.30E−117

TABLE 17

Pfam domain	accession	gathering
name	number	cutoff	domain description

14-3-3	PF00244.9	25	14-3-3 protein
2-Hacid_dh	PF00389.19	11.2	D-isomer specific 2-hydroxyacid
			dehydrogenase, catalytic domain
2-Hacid_dh_C	PF02826.7	−82.2	D-isomer specific 2-hydroxyacid
			dehydrogenase, NAD binding domain
AAA	PF00004.18	12.3	ATPase family associated with various
			cellular activities (AAA)
AA_permease	PF00324.10	−120.8	Amino acid permease
ACT	PF01842.13	0	ACT domain
ADH_N	PF08240.2	−14.5	Alcohol dehydrogenase GroES-like domain
ADH_zinc_N	PF00107.16	23.8	Zinc-binding dehydrogenase
APC8	PF04049.3	−19.8	Anaphase promoting complex subunit 8/
			Cdc23
Aa_trans	PF01490.7	−128.4	Transmembrane amino acid transporter
			protein
Actin	PF00022.8	−30	Actin
Aldedh	PF00171.11	−209.3	Aldehyde dehydrogenase family
Aldo_ket_red	PF00248.10	−97	Aldo/keto reductase family
Allene_ox_cyc	PF06351.2	25	Allene oxide cyclase
Alpha-amylase	PF00128.12	−93	Alpha amylase, catalytic domain
Aminotran_1_2	PF00155.10	−57.5	Aminotransferase class I and II
Aminotran_3	PF00202.10	−207.6	Aminotransferase class-III
Ank	PF00023.18	0	Ankyrin repeat
Anti-silence	PF04729.4	25	Anti-silencing protein, ASF1-like
ArfGap	PF01412.8	−17	Putative GTPase activating protein for Arf
Asp	PF00026.13	−186.1	Eukaryotic aspartyl protease
Auxin_inducible	PF02519.4	−15	Auxin responsive protein
BPL_C	PF02237.6	16	Biotin protein ligase C terminal domain
BPL_LipA_LipB	PF03099.8	−0.2	Biotin/lipoate A/B protein ligase family
BURP	PF03181.5	−52	BURP domain
B_lectin	PF01453.14	28.2	D-mannose binding lectin
BolA	PF01722.7	23	BolA-like protein
C2	PF00168.18	3.7	C2 domain
CBM_20	PF00686.9	−3	Starch binding domain
CMAS	PF02353.10	−177.9	Cyclopropane-fatty-acyl-phospholipid
			synthase
Ceramidase_alk	PF04734.3	25	Neutral/alkaline non-lysosomal ceramidase
CoA_binding	PF02629.8	−12.8	CoA binding domain
Complex1_30kDa	PF00329.8	−3	Respiratory-chain NADH dehydrogenase, 30 Kd
			subunit
Complex1_49kDa	PF00346.8	−108	Respiratory-chain NADH dehydrogenase, 49 Kd
			subunit
Cupin_2	PF07883.1	16.6	Cupin domain
Cupin_3	PF05899.2	4.4	Protein of unknown function (DUF861)
Cyclin_C	PF02984.8	−13	Cyclin, C-terminal domain
Cyclin_N	PF00134.13	−14.7	Cyclin, N-terminal domain
DHBP_synthase	PF00926.10	−116	3,4-dihydroxy-2-butanone 4-phosphate
			synthase
DNA_pol_E_B	PF04042.5	−47.5	DNA polymerase alpha/epsilon subunit B
DUF1005	PF06219.2	25	Protein of unknown function (DUF1005)
DUF1191	PF06697.2	25	Protein of unknown function (DUF1191)
DUF1195	PF06708.1	25	Protein of unknown function (DUF1195)
DUF1336	PF07059.2	−78.2	Protein of unknown function (DUF1336)
DUF26	PF01657.7	0	Domain of unknown function DUF26
DUF298	PF03556.6	25	Domain of unknown function (DUF298)
DUF588	PF04535.2	25	Domain of unknown function (DUF588)
DUF676	PF05057.4	−60.7	Putative serine esterase (DUF676)
DUF822	PF05687.3	25	Plant protein of unknown function (DUF822)
DUF868	PF05910.2	25	Plant protein of unknown function (DUF868)
DnaJ	PF00226.19	−8	DnaJ domain
EGF_CA	PF07645.4	24.5	Calcium binding EGF domain
ESCRT-III	PF03357.10	−35.4	ESCRT-III complex subunit
ETC_C1_NDUFA5	PF04716.3	25	ETC complex I subunit conserved region
F-box	PF00646.21	13.6	F-box domain
F420_oxidored	PF03807.6	−34.5	NADP oxidoreductase coenzyme F420-
			dependent
FAR1	PF03101.4	0	FAR1 family
FA_hydroxylase	PF04116.2	−64.1	Fatty acid hydroxylase
FBPase	PF00316.10	−170.3	Fructose-1-6-bisphosphatase
FH2	PF02181.13	−98.3	Formin Homology 2 Domain
GDC-P	PF02347.5	−306.2	Glycine cleavage system P-protein
GH3	PF03321.3	−336	GH3 auxin-responsive promoter
GTP_cyclohydro2	PF00925.11	−49	GTP cyclohydrolase II
Gln-synt_C	PF00120.14	−124	Glutamine synthetase, catalytic domain
Gln-synt_N	PF03951.9	9	Glutamine synthetase, beta-Grasp domain
Glyco_transf_8	PF01501.9	−43.2	Glycosyl transferase family 8
Glycolytic	PF00274.9	−174.5	Fructose-bisphosphate aldolase class-I
Gp_dh_C	PF02800.9	−64.1	Glyceraldehyde 3-phosphate dehydrogenase,
			C-terminal domain
Gp_dh_N	PF00044.12	−74.2	Glyceraldehyde 3-phosphate dehydrogenase,
			NAD binding domain
HD	PF01966.11	18	HD domain
HLH	PF00010.15	8.2	Helix-loop-helix DNA-binding domain
HTH_11	PF08279.1	11.3	HTH domain
Histone	PF00125.13	17.4	Core histone H2A/H2B/H3/H4
Kelch_1	PF01344.14	7.8	Kelch motif
Kelch_2	PF07646.4	14	Kelch motif
L51_S25_CI-B8	PF05047.5	25	Mitochondrial ribosomal protein L51/S25/
			CI-B8 domain
LEA_3	PF03242.3	25	Late embryogenesis abundant protein
LMBR1	PF04791.5	−116.3	LMBR1-like membrane protein
LRRNT_2	PF08263.2	18.6	Leucine rich repeat N-terminal domain
LRR_1	PF00560.21	7.7	Leucine Rich Repeat
LSM	PF01423.12	13.7	LSM domain
Lung_7-TM_R	PF06814.3	25	Lung seven transmembrane receptor
MAP65_ASE1	PF03999.2	−134.8	Microtubule associated protein (MAP65/ASE1
			family)
MFS_1	PF07690.5	23.5	Major Facilitator Superfamily
MMR_HSR1	PF01926.11	31.2	GTPase of unknown function
MannoseP_isomer	PF01050.8	−70	Mannose-6-phosphate isomerase
Metalloenzyme	PF01676.7	−14.4	Metalloenzyme superfamily
Metallophos	PF00149.17	22	Calcineurin-like phosphoesterase
Methyltransf_11	PF08241.1	17.1	Methyltransferase domain
Methyltransf_12	PF08242.1	21.4	Methyltransferase domain
Mit_rib_S27	PF08293.1	25	Mitochondrial ribosomal subunit S27
MtN3_slv	PF03083.5	−0.8	MtN3/saliva family
NAD_Gly3P_dh_N	PF01210.12	−44	NAD-dependent glycerol-3-phosphate
			dehydrogenase N-terminus
NAD_binding_1	PF00175.10	−3.9	Oxidoreductase NAD-binding domain
NAF	PF03822.4	4.5	NAF domain
NDK	PF00334.9	−59.9	Nucleoside diphosphate kinase
NPH3	PF03000.4	25	NPH3 family
NTP_transferase	PF00483.12	−90.5	Nucleotidyl transferase
Na_H_Exchanger	PF00999.10	−67.9	Sodium/hydrogen exchanger family
ORMDL	PF04061.4	25	ORMDL family
Oleosin	PF01277.7	−27	Oleosin
PAN_2	PF08276.2	−4.9	PAN-like domain
PAP_fibrillin	PF04755.2	25	PAP_fibrillin
PBD	PF00786.17	12.2	P21-Rho-binding domain
PCI	PF01399.15	25	PCI domain
PEP-utilizers	PF00391.12	10	PEP-utilising enzyme, mobile domain
PGAM	PF00300.12	−3	Phosphoglycerate mutase family
PK	PF00224.10	−244	Pyruvate kinase, barrel domain
PK_C	PF02887.5	−44	Pyruvate kinase, alpha/beta domain
PPR	PF01535.11	0	PPR repeat
PTR2	PF00854.12	−50	POT family
Pantoate_transf	PF02548.5	−93	Ketopantoate hydroxymethyltransferase
Peptidase_S10	PF00450.11	−198	Serine carboxypeptidase
PfkB	PF00294.13	−67.8	pfkB family carbohydrate kinase
Phosphorylase	PF00343.9	−601.1	Carbohydrate phosphorylase
Pkinase	PF00069.14	−70.8	Protein kinase domain
Pkinase_Tyr	PF07714.5	65	Protein tyrosine kinase
Prenylcys_lyase	PF07156.3	−164.1	Prenylcysteine lyase
PurA	PF04845.3	25	PurA ssDNA and RNA-binding protein
RMMBL	PF07521.1	18.5	RNA-metabolising metallo-beta-lactamase
Response_reg	PF00072.12	4	Response regulator receiver domain
Rhodanese	PF00581.9	25	Rhodanese-like domain
Rib_5-P_isom_A	PF06026.4	25	Ribose 5-phosphate isomerase A
			(phosphoriboisomerase A)
Ribonuclease_T2	PF00445.8	−53	Ribonuclease T2 family
Ribosomal_S8e	PF01201.11	25	Ribosomal protein S8e
Rotamase	PF00639.10	4	PPIC-type PPIASE domain
RrnaAD	PF00398.9	−73.3	Ribosomal RNA adenine dimethylase
SAM_1	PF00536.18	11.3	SAM domain (Sterile alpha motif)
SMC_N	PF02463.8	−95.8	RecF/RecN/SMC N terminal domain
SNF5	PF04855.3	25	SNF5/SMARCB1/INI1
SWIB	PF02201.9	−7	SWIB/MDM2 domain
SWIM	PF04434.7	10	SWIM zinc finger
S_locus_glycop	PF00954.11	−12.7	S-locus glycoprotein family
Sad1_UNC	PF07738.2	−20.4	Sad1/UNC-like C-terminal
Sterol_MT_C	PF08498.1	30.5	Sterol methyltransferase C-terminal
Subtilisin_N	PF05922.6	26.1	Subtilisin N-terminal Region
Sugar_tr	PF00083.13	−85	Sugar (and other) transporter
TFIIA	PF03153.4	25	Transcription factor IIA, alpha/beta subunit
TFIIS_C	PF01096.9	15	Transcription factor S-II (TFIIS)
TFIIS_M	PF07500.3	7.4	Transcription factor S-II (TFIIS), central
			domain
TIM	PF00121.8	−97	Triosephosphate isomerase
TPR_1	PF00515.16	7.7	Tetratricopeptide repeat
TPR_2	PF07719.5	20.1	Tetratricopeptide repeat
TPT	PF03151.7	−15.3	Triose-phosphate Transporter family
Thioredoxin	PF00085.9	−25.7	Thioredoxin
Tim17	PF02466.8	2.7	Tim17/Tim22/Tim23 family
TrkA_N	PF02254.7	4.7	TrkA-N domain
Tryp_alpha_amyl	PF00234.10	−4	Protease inhibitor/seed storage/LTP family
UPF0261	PF06792.1	25	Uncharacterised protein family (UPF0261)
UQ_con	PF00179.16	−30	Ubiquitin-conjugating enzyme
Usp	PF00582.16	36.1	Universal stress protein family
WD40	PF00400.20	21.5	WD domain, G-beta repeat
adh_short	PF00106.14	−17	short chain dehydrogenase
eIF-4B	PF06273.1	−205.7	Plant specific eukaryotic initiation factor 4B
iPGM_N	PF06415.3	−263.4	BPG-independent PGAM N-terminus
			(iPGM_N)
p450	PF00067.11	−105	Cytochrome P450
zf-AN1	PF01428.6	0	AN1-like Zinc finger
zf-C3HC4	PF00097.13	16.9	Zinc finger, C3HC4 type (RING finger)
zf-CCCH	PF00642.14	0	Zinc finger C-x8-C-x5-C-x3-H type (and
			similar)
zf-CCHC	PF00098.12	17.9	Zinc knuckle
zf-CSL	PF05207.3	2.9	CSL zinc finger

Example 5. Plasmid Construction for Transferring Recombinant DNA

This example illustrates the construction of plasmids for transferring recombinant DNA into the nucleus of a plant cell which can be regenerated into a transgenic crop plant of this invention. Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. DNA of interest, i.e. each DNA identified in Table 1 and the DNA for the identified homologous genes, are cloned and amplified by PCR prior to insertion into the insertion site the base vector.
A. Corn Transformation Vector
Elements of an exemplary common expression vector, pMON93093 are illustrated in Table 18. The exemplary base vector which is especially useful for corn transformation is illustrated in FIG. 2 and assembled using technology known in the art. The DNA of interest are inserted in a expression vector at the insertion site between the intron 1 of rice act 1 gene and the termination sequence of PinII gene.

TABLE 18

pMON93093

			Coordinates of
			SEQ ID NO:
function	name	annotation		19940

Agro	B-AGRtu.right border	Agro right border	11364-11720
transforamtion		sequence, essential for
		transfer of T-DNA.
Gene of	E-Os.Act1	upstream promoter	19-775
interest		region of the rice actin
expression
		1 gene
cassette	E-CaMV.35S.2xA1-B3	duplicated35S A1-B3	788-1120
		domain without TATA
		box
	P-Os.Act1	promoter region of the	1125-1204
		rice actin 1 gene
	L-Ta.Lhcb1	5′ untranslated leader	1210-1270
		of wheat major
		chlorophyll a/b binding
		protein
	I-Os.Act1	first intron and flanking	1287-1766
		UTR exon sequences
		from the rice actin 1
		gene
	T-St.Pis4	3′ non-translated region	1838-2780
		of the potato proteinase
		inhibitor II gene which
		functions to direct
		polyadenylation of the
		mRNA
Plant	P-Os.Act1	Promoter from the rice	2830-3670
selectable		actin	1 gene
marker	L-Os.Act1	first exon of the rice	3671-3750
expression		actin	1 gene
cassette	I-Os.Act1	first intron and flanking	3751-4228
		UTR exon sequences
		from the rice actin 1
		gene
	TS-At.ShkG-CTP2	Transit peptide region	4238-4465
		of Arabidopsis EPSPS
	CR-AGRtu.aroA-	Synthetic CP4 coding	4466-5833
	CP4.nat	region with dicot
		preferred codon usage.
	T-AGRtu.nos	A 3′ non-translated	5849-6101
		region of the nopaline
		synthase gene of
		Agrobacterium
		tumefaciens Ti plasmid
		which functions to
		direct polyadenylation
		of the mRNA.
Agro	B-AGRtu.left border	Agro left border	6168-6609
transformation		sequence, essential for
		transfer of T-DNA.
Maintenance	OR-Ec.oriV-RK2	The vegetative origin of	6696-7092
in E. coli		replication from
		plasmid RK2.
	CR-Ec.rop	Coding region for	8601-8792
		repressor of primer
		from the ColE1
		plasmid. Expression of
		this gene product
		interferes with primer
		binding at the origin of
		replication, keeping
		plasmid copy number
		low.
	OR-Ec.ori-ColE1	The minimal origin of	9220-9808
		replication from the E. coli
		plasmid ColE1.
	P-Ec.aadA-SPC/STR	romoter for Tn7	10339-10380
		adenylyltransferase
		(AAD(3″))
	CR-Ec.aadA-SPC/STR	Coding region for Tn7	10381-11169
		adenylyltransferase
		(AAD(3″)) conferring
		spectinomycin and
		streptomycin resistance.
	T-Ec.aadA-SPC/STR	3′ UTR from the Tn7	11170-11227
		adenylyltransferase
		(AAD(3″)) gene of E. coli.

B. Soybean Transformation Vector

Plasmids for use in transformation of soybean are also prepared. Elements of an exemplary common expression vector plasmid pMON82053 are shown in Table 19 below. This exemplary soybean or canola transformation base vector illustrated in FIG. 3 is assembled using the technology known in the art. Recobminant DNA of interest, i.e. each DNA identified in Table 1 and the DNA for the identified homologous genes, is cloned and amplified by PCR prior to insertion into the insertion site the base vector at the insertion site between the enhanced 35S CaMV promoter and the termination sequence of cotton E6 gene.

TABLE 19

pMON82053

			Coordinates of
			SEQ ID NO:
function	name	annotation	19941

Agro	B-AGRtu.left	Agro left border sequence, essential	6144-6585
transforamtion	border	for transfer of T-DNA.
Plant	P-At.Act7	Promoter from the arabidopsis actin	6624-7861
selectable		7 gene
marker	L-At.Act7	5′UTR of Arabidopsis Act7 gene
expression	I-At.Act7	Intron from the Arabidopsis actin7
cassette		gene
	TS-At.ShkG-	Transit peptide region of Arabidopsis	7864-8091
	CTP2	EPSPS
	CR-AGRtu.aroA-	Synthetic CP4 coding region with	8092-9459
	CP4.nno_At	dicot preferred codon usage.
	T-AGRtu.nos	A 3′ non-translated region of the	9466-9718
		nopaline synthase gene of
		Agrobacterium tumefaciens Ti
		plasmid which functions to direct
		polyadenylation of the mRNA.
Gene of	P-CaMV.35S-enh	Promoter for 35S RNA from CaMV	1-613
interest		containing a duplication of the −90 to
expression		−350 region.
cassette	T-Gb.E6-3b	3′ untranslated region from the fiber	688-1002
		protein E6 gene of sea-island cotton;
Agro	B-AGRtu.right	Agro right border sequence, essential	1033-1389
transformation	border	for transfer of T-DNA.
Maintenance	OR-Ec.oriV-RK2	The vegetative origin of replication	5661-6057
in E. coli		from plasmid RK2.
	CR-Ec.rop	Coding region for repressor of primer	3961-4152
		from the ColE1 plasmid. Expression
		of this gene product interferes with
		primer binding at the origin of
		replication, keeping plasmid copy
		number low.
	OR-Ec.ori-ColE1	The minimal origin of replication	2945-3533
		from the E. coli plasmid ColE1.
	P-Ec.aadA-	romoter for Tn7 adenylyltransferase	2373-2414
	SPC/STR	(AAD(3″))
	CR-Ec.aadA-	Coding region for Tn7	1584-2372
	SPC/STR	adenylyltransferase (AAD(3″))
		conferring spectinomycin and
		streptomycin resistance.
	T-Ec.aadA-	3′ UTR from the Tn7	1526-1583
	SPC/STR	adenylyltransferase (AAD(3″)) gene
		of E. coli.

C. Cotton Transformation Vector

Plasmids for use in transformation of cotton are also prepared. Elements of an exemplary common expression vector plasmid pMON99053 are shown in Table 20 below and FIG. 4. Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. Each recombinant DNA coding for a protein identified in Table 1 is amplified by PCR prior to insertion into the insertion site within the gene of interest expression cassette of one of the base.

TABLE 20

			Coordinates
			of SEQ ID
function	name	annotation	NO: 19942

Agro	B-AGRtu.right border	Agro right border sequence,	11364-11720
transforamtion		essential for transfer of T-
		DNA.
Gene of interest	Exp-CaMV.35S-	Enhanced version of the 35S	7794-8497
expression	enh + ph.DnaK	RNA promoter from CaMV
cassette		plus the petunia hsp70 5′
		untranslated region
	T-Ps.RbcS2-E9	The 3′ non-translated region	67-699
		of the pea RbcS2 gene which
		functions to direct
		polyadenylation of the
		mRNA.
Plant selectable	Exp-CaMV.35S	Promoter and 5′ untranslated	730-1053
marker		region of the 35S RNA from
expression		CaMV
cassette	CR-Ec.nptII-Tn5	Neomycin	1087-1881
		Phosphotransferase II gene
		that confers resistance to
		neomycin and kanamycin
	T-AGRtu.nos	A 3′ non-translated region of	1913-2165
		the nopaline synthase gene of
		Agrobacterium tumefaciens
		Ti plasmid which functions to
		direct polyadenylation of the
		mRNA.
Agro	B-AGRtu.left border	Agro left border sequence,	2211-2652
transformation		essential for transfer of T-
		DNA.
Maintenance in	OR-Ec.oriV-RK2	The vegetative origin of	2739-3135
E. coli		replication from plasmid
		RK2.
	CR-Ec.rop	Coding region for repressor	4644-4835
		of primer from the ColE1
		plasmid. Expression of this
		gene product interferes with
		primer binding at the origin
		of replication, keeping
		plasmid copy number low.
	OR-Ec.ori-ColE1	The minimal origin of	5263-5851
		replication from the E. coli
		plasmid ColE1.
	P-Ec.aadA-SPC/STR	romoter for Tn7	6382-6423
		adenylyltransferase
		(AAD(3″))
	CR-Ec.aadA-SPC/STR	Coding region for Tn7	6424-7212
		adenylyltransferase
		(AAD(3″)) conferring
		spectinomycin and
		streptomycin resistance.
	T-Ec.aadA-SPC/STR	3′ UTR from the Tn7	7213-7270
		adenylyltransferase
		(AAD(3″)) gene of E. coli.

Example 6. Corn Plant Transformation

This example illustrates the production and identification of transgenic corn cells in seed of transgenic corn plants having an enhanced agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and/or improved seed compositions as compared to control plants. Transgenic corn cells are prepared with recombinant DNA expressing each of the protein encoding DNAs listed in Table 1 by Agrobacterium-mediated transformation using the corn transformation vectors pMON93093 as disclosed in Example 6. Corn transformation is effected using methods disclosed in U.S. Patent Application Publication 2004/0344075 A1 where corn embryos are inoculated and co-cultured with the Agrobacterium tumefaciens strain ABI and the corn transformation vector. To regenerate transgenic corn plants the transgenic callus resulting from transformation is placed on media to initiate shoot development in plantlets which are transferred to potting soil for initial growth in a growth chamber followed by a mist bench before transplanting to pots where plants are grown to maturity. The plants are self fertilized and seed is harvested for screening as seed, seedlings or progeny R2 plants or hybrids, e.g., for yield trials in the screens indicated above.
Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. The transgenic plants and seeds having the transgenic cells of this invention which have recombinant DNA imparting the enhanced agronomic traits are identified by screening for nitrogen use efficiency, yield, water use efficiency, cold tolerance and improved seed composition.

Example 7. Soybean Plant Transformation

This example illustrates the production and identification of transgenic soybean cells in seed of transgenic soybean plants having an enhanced agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and/or improved seed compositions as compared to control plants. Transgenic soybean cells are prepared with recombinant DNA expressing each of the protein encoding DNAs listed in Table 1 by Agrobacterium-mediated transformation using the soybean transformation vectors pMON82053 disclosed in Example 7. Soybean transformation is effected using methods disclosed in U.S. Pat. No. 6,384,301 where soybean meristem explants are wounded then inoculated and co-cultured with the soybean transformation vector, then transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots.
The transformation is repeated for each of the protein encoding DNAs identified in Table 1.
Transgenic shoots producing roots are transferred to the greenhouse and potted in soil. Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. The transgenic plants and seeds having the transgenic cells of this invention which have recombinant DNA imparting the enhanced agronomic traits are identified by screening for nitrogen use efficiency, yield, water use efficiency, cold tolerance and improved seed composition.

Example 8. Canola Transformation

This example illustrates plant transformation useful in producing the transgenic canola plants of this invention and the production and identification of transgenic seed for transgenic canola having enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
Tissues from in vitro grown canola seedlings are prepared and inoculated with overnight-grown Agrobacterium cells containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette. Following co-cultivation with Agrobacterium, the infected tissues are allowed to grow on selection to promote growth of transgenic shoots, followed by growth of roots from the transgenic shoots. The selected plantlets are then transferred to the greenhouse and potted in soil. Molecular characterization are performed to confirm the presence of the gene of interest, and its expression in transgenic plants and progenies. Progeny transgenic plants are selected from a population of transgenic canola events under specified growing conditions and are compared with control canola plants. Control canola plants are substantially the same canola genotype but without the recombinant DNA, for example, either a parental canola plant of the same genotype that is not transformed with the identical recombinant DNA or a negative isoline of the transformed plant
Transgenic canola plant cells are transformed with recombinant DNA from each of the genes identified in Table 1. Transgenic progeny plants and seed of the transformed plant cells are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil as reported in Example 9.

Example 9. Selection of Transgenic Plants with Enhanced Agronomic Trait(s)

This example illustrates identification of nuclei of the invention by screening derived plants and seeds for an enhanced trait identified below.
Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants will not exhibit an enhanced agronomic trait. Populations of transgenic seed and plants prepared in Examples 6 and 7 are screened to identify those transgenic events providing transgenic plant cells with a nucleus having recombinant DNA imparting an enhanced trait. Each population is screened for enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and heat, increased level of oil and protein in seed using assays described below. Plant cell nuclei having recombinant DNA with each of the genes identified in Table 1 and the identified homologs are identified in plants and seeds with at least one of the enhanced traits.

A. Selection for Enhanced Nitrogen Use Efficiency

Transgenic corn plants with nuclei of the invention are planted in fields with three levels of nitrogen (N) fertilizer being applied, i.e. low level (0 pounds per acre N), medium level (80 pounds per acre N) and high level (180 pounds per acre N). Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used as the N source and apply by broadcast boom and incorporate with a field cultivator with rear rolling basket in the same direction as intended crop rows. Although there is no N applied in the low level treatment, the soil should still be disturbed in the same fashion as the treated area. Transgenic plants and control plants can be grouped by genotype and construct with controls arranged randomly within genotype blocks. For improved statistical analysis each type of transgenic plant can be tested by 3 replications and across 4 locations. Nitrogen levels in the fields are analyzed before planting by collecting sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples are analyzed for nitrate-nitrogen, phosphorus (P), potassium (K), organic matter and pH to provide baseline values. P, K and micronutrients are applied based upon soil test recommendations.
Transgenic corn plants prepared in Example 6 and which exhibit a 2 to 5% yield increase as compared to control plants when grown in the high nitrogen field are selected as having nuclei of the invention. Transgenic corn plants which have at least the same or higher yield as compared to control plants when grown in the medium nitrogen field are selected as having nuclei of the invention. Transgenic corn plants having a nucleus with DNA identified in Table 3 as imparting nitrogen use efficiency (LN) and homologous DNA are selected from a nitrogen use efficiency screen as having a nucleus of this invention.

B. Selection for Increased Yield

Many transgenic plants of this invention exhibit improved yield as compared to a control plant. Improved yield can result from enhanced seed sink potential, i.e. the number and size of endosperm cells or kernels and/or enhanced sink strength, i.e. the rate of starch biosynthesis. Sink potential can be established very early during kernel development, as endosperm cell number and size are determined within the first few days after pollination.
Much of the increase in corn yield of the past several decades has resulted from an increase in planting density. During that period, corn yield has been increasing at a rate of 2.1 bushels/acre/year, but the planting density has increased at a rate of 250 plants/acre/year. A characteristic of modern hybrid corn is the ability of these varieties to be planted at high density. Many studies have shown that a higher than current planting density should result in more biomass production, but current germplasm does not perform. well at these higher densities. One approach to increasing yield is to increase harvest index (HI), the proportion of biomass that is allocated to the kernel compared to total biomass, in high density plantings.
Effective yield selection of enhanced yielding transgenic corn events uses hybrid progeny of the transgenic event over multiple locations with plants grown under optimal production management practices, and maximum pest control. A useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Selection methods may be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more planting seasons, for example at least two planting seasons to statistically distinguish yield improvement from natural environmental effects. It is to plant multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, for example 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges. Transgenic events can be grouped by recombinant DNA constructs with groups randomly placed in the field. A pollinator plot of a high quality corn line is planted for every two plots to allow open pollination when using male sterile transgenic events. A useful planting density is about 30,000 plants/acre. High planting density is greater than 30,000 plants/acre, preferably about 40,000 plants/acre, more preferably about 42,000 plants/acre, most preferably about 45,000 plants/acre. Each of the transgenic corn plants and soybean plants with a nucleus of the invention prepared in Examples 6 and 7 are screened for yield enhancement. At least one event from each of the corn and soybean plants is selected as having at least between 3 and 5% increase in yield as compared to a control plant as having a nucleus of this invention.

C. Selection for Enhanced Water Use Efficiency (WUE)

The following is a high-throughput method for screening for water use efficiency in a greenhouse to identify the transgenic corn plants with a nucleus of this invention. This selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle. The primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment. The hydration status of the shoot tissues following the drought is also measured. The plant height are measured at three time points. The first is taken just prior to the onset drought when the plant is 11 days old, which is the shoot initial height (SIH). The plant height is also measured halfway throughout the drought/re-water regimen, on day 18 after planting, to give rise to the shoot mid-drought height (SMH). Upon the completion of the final drought cycle on day 26 after planting, the shoot portion of the plant is harvested and measured for a final height, which is the shoot wilt height (SWH) and also measured for shoot wilted biomass (SWM). The shoot is placed in water at 40 degree Celsius in the dark. Three days later, the shoot is weighted to give rise to the shoot turgid weight (STM). After drying in an oven for four days, the shoots are weighted for shoot dry biomass (SDM). The shoot average height (SAH) is the mean plant height across the 3 height measurements. The procedure described above may be adjusted for +/−˜one day for each step given the situation.
To correct for slight differences between plants, a size corrected growth value is derived from SIH and SWH. This is the Relative Growth Rate (RGR). Relative Growth Rate (RGR) is calculated for each shoot using the formula [RGR %=(SWH−SIH)/((SWH+SIH)/2)×100]. Relative water content (RWC) is a measurement of how much (%) of the plant was water at harvest. Water Content (RWC) is calculated for each shoot using the formula [RWC %=(SWM−SDM)/(STM−SDM)×100]. Fully watered corn plants of this age run around 98% RWC.
Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for water use efficiency. Transgenic plants having at least a 1% increase in RGR and RWC as compared to control plants are identified as having enhanced water used efficiency and are selected as having a nucleus of this invention. Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting drought tolerance improvement (DS) and homologous DNA are identified as showing increased water use efficiency as compared to control plants and are selected as having a nucleus of this invention.

D. Selection for Growth Under Cold Stress

Cold germination assay—Three sets of seeds are used for the assay. The first set consists of positive transgenic events (F1 hybrid) where the genes of the present invention are expressed in the seed. The second seed set is nontransgenic, wild-type negative control made from the same genotype as the transgenic events. The third set consisted of two cold tolerant and one cold sensitive commercial check lines of corn. All seeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide, Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan is applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the experiment.
Corn kernels are placed embryo side down on blotter paper within an individual cell (8.9×8.9 cm) of a germination tray (54×36 cm). Ten seeds from an event are placed into one cell of the germination tray. Each tray can hold 21 transgenic events and 3 replicates of wildtype (LH244SDms+LH59), which is randomized in a complete block design. For every event there are five replications (five trays). The trays are placed at 9.7 C for 24 days (no light) in a Convrion® growth chamber (Conviron Model PGV36, Controlled Environments, Winnipeg, Canada). Two hundred and fifty millilters of deionized water are added to each germination tray. Germination counts are taken 10th, 11th, 12th, 13th, 14th, 17th, 19th, 21st, and 24th day after start date of the experiment. Seeds are considered germinated if the emerged radicle size is 1 cm. From the germination counts germination index is calculated.
The germination index is calculated as per:
Germination index=(Σ([T+1−n _i]*[P _i −P _i-1]))/T
where T is the total number of days for which the germination assay is performed. The number of days after planting is defined by n. “i” indicated the number of times the germination had been counted, including the current day. P is the percentage of seeds germinated during any given rating. Statistical differences are calculated between transgenic events and wild type control. After statistical analysis, the events that show a statistical significance at the p level of less than 0.1 relative to wild-type controls will advance to a secondary cold selection. The secondary cold screen is conducted in the same manner of the primary selection only increasing the number of repetitions to ten. Statistical analysis of the data from the secondary selection is conducted to identify the events that show a statistical significance at the p level of less than 0.05 relative to wild-type controls.
Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for water use efficiency. Transgenic plants having at least a 5% increase in germination index as compared to control plants are identified as having enhanced cold stress tolerance and are selected as having a nucleus of this invention. Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting cold tolerance improvement (CK or CS) and homologous DNA are identified as showing increased cold stress tolerance as compared to control plants and are selected as having a nucleus of this invention.
E. Screens for Transgenic Plant Seeds with Increased Protein and/or Oil Levels
The following is a high-throughput selection method for identifying plant seeds with improvement in seed composition using the Infratec® 1200 series Grain Analyzer, which is a near-infrared transmittance spectrometer used to determine the composition of a bulk seed sample. Near infrared analysis is a non-destructive, high-throughput method that can analyze multiple traits in a single sample scan. An NIR calibration for the analytes of interest is used to predict the values of an unknown sample. The NIR spectrum is obtained for the sample and compared to the calibration using a complex chemometric software package that provides a predicted values as well as information on how well the sample fits in the calibration.
Infratec® Model 1221, 1225, or 1227 analyzer with transport module by Foss North America is used with cuvette, item #1000-4033, Foss North America or for small samples with small cell cuvette, Foss standard cuvette modified by Leon Girard Co. Corn and soy check samples of varying composition maintained in check cell cuvettes are supplied by Leon Girard Co. NIT collection software is provided by Maximum Consulting Inc. Calculations are performed automatically by the software. Seed samples are received in packets or containers with barcode labels from the customer. The seed is poured into the cuvettes and analyzed as received.

TABLE 21

Typical sample(s):	Whole grain corn and soybean seeds
Analytical time to run method:	Less than 0.75 min per sample
Total elapsed time per run:	1.5 minute per sample
Typical and minimum sample size:	Corn typical: 50 cc; minimum 30 cc
	Soybean typical: 50 cc; minimum
	5 cc
Typical analytical range:	Determined in part by the specific
	calibration.
	Corn - moisture 5-15%, oil 5-20%,
	protein 5-30%, starch 50-75%, and
	density 1.0-1.3%.
	Soybean - moisture 5-15%, oil
	15-25%, and protein 35-50%.

Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for increased protein and oil in seed. Transgenic inbred corn and soybean plants having an increase of at least 1 percentage point in the total percent seed protein or at least 0.3 percentage point in total seed oil and transgenic hybrid corn plants having an increase of at least 0.4 percentage point in the total percent seed protein as compared to control plants are identified as having enhanced seed protein or enhanced seed oil and are selected as having a nucleus of this invention.

Example 10. Cotton Transgenic Plants with Enhanced Agronomic Traits

Cotton transformation is performed as generally described in WO036911 and in U.S. Pat. No. 5,846,797. Transgenic cotton plants containing each of the recombinant DNA having a sequence of SEQ ID NO: 1 through SEQ ID NO: 197 are obtained by transforming with recombinant DNA from each of the genes identified in Table 1. Progeny transgenic plants are selected from a population of transgenic cotton events under specified growing conditions and are compared with control cotton plants. Control cotton plants are substantially the same cotton genotype but without the recombinant DNA, for example, either a parental cotton plant of the same genotype that was not transformed with the identical recombinant DNA or a negative isoline of the transformed plant. Additionally, a commercial cotton cultivar adapted to the geographical region and cultivation conditions, i.e. cotton variety ST474, cotton variety FM 958, and cotton variety Siokra L-23, are used to compare the relative performance of the transgenic cotton plants containing the recombinant DNA. The specified culture conditions are growing a first set of transgenic and control plants under “wet” conditions, i.e. irrigated in the range of 85 to 100 percent of evapotranspiration to provide leaf water potential of −14 to −18 bars, and growing a second set of transgenic and control plants under “dry” conditions, i.e. irrigated in the range of 40 to 60 percent of evapotranspiration to provide a leaf water potential of −21 to −25 bars. Pest control, such as weed and insect control is applied equally to both wet and dry treatments as needed. Data gathered during the trial includes weather records throughout the growing season including detailed records of rainfall; soil characterization information; any herbicide or insecticide applications; any gross agronomic differences observed such as leaf morphology, branching habit, leaf color, time to flowering, and fruiting pattern; plant height at various points during the trial; stand density; node and fruit number including node above white flower and node above crack boll measurements; and visual wilt scoring. Cotton boll samples are taken and analyzed for lint fraction and fiber quality. The cotton is harvested at the normal harvest timeframe for the trial area. Enhanced water use efficiency is indicated by increased yield, improved relative water content, enhanced leaf water potential, increased biomass, enhanced leaf extension rates, and improved fiber parameters.
The transgenic cotton plants of this invention are identified from among the transgenic cotton plants by agronomic trait screening as having increased yield and enhanced water use efficiency.

Example 11. Monocot and Dicot Plant Transformation for the Suppression of Endogeneous Protein

This example illustrates monocot and dicot plant transformation to produce nuclei of this invention in cells of a transgenic plant by transformation where the recombinant DNA suppresses the expression of an endogenous protein identified by Pfam, SNF5, LMBR1, TFIIS_M, TFIIS_C, or Glyco_transf_8. Corn callus and soybean tissue are transformed as describe in Examples 6 and 7 using recombinant DNA in the nucleus with DNA that transcribes to RNA that forms double-stranded RNA targeted to an endogenous gene with DNA encoding the protein. The genes for which the double-stranded RNAs are targeted are the native gene in corn and soybean that are homolog of the genes encoding the protein with an amino acid sequence of SEQ ID NO:200, 201, 205, 207, 211, and 394.
Populations of transgenic corn plants and soybean plants prepared in Examples 6 and 7 with DNA for suppressing a gene identified in Table 3 as providing an enhanced trait by gene suppression are screened to identify an event from those plants with a nucleus of the invention by selecting the trait identified in this specification.

Claims

We claim:

1. A plant cell nucleus with stably integrated, recombinant DNA comprising a promoter that is functional in plant cells and that is operably linked to DNA from a plant, bacteria or yeast that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name selected from the group of Pfam names consisting of L51_S25_CI-B8, iPGM_N, WD40, BPL_LipA_LipB, DUF676, AAA, S_locus_glycop, ArfGap, Rotamase, Metallophos, CMAS, Sugar_tr, LMBR1, RrnaAD, NAF, BolA, Pkinase, C2, FA_hydroxylase, p450, Complex1_30 kDa, Histone, DUF822, PEP-utilizers, PCI, ETC_C1_NDUFA5, 2-Hacid_dh, Tryp_alpha_amyl, PK_C, MAP65_ASE1, FBPase, SWIB, Ank, Ribosomal_S8e, 2-Hacid_dh_C, SMC_N, GTP_cyclohydro2, PfkB, ORMDL, ADH_zinc_N, SWIM, TrkA_N, HLH, GH3, SNF5, Ceramidase_alk, Ribonuclease_T2, Complex1_49 kDa, Gp_dh_C, Aldo_ket_red, zf-AN1, TFIIS_C, MFS_1, Thioredoxin, DUF1005, LEA_3, Sterol_MT_C, Gp_dh_N, TFIIS_M, PAN_2, BPL_C, DUF26, Aa_trans, ACT, ADH_N, NAD_binding_1, Auxin_inducible, B_lectin, Anti-silence, Response_reg, 14-3-3, LRRNT_2, GDC-P, zf-CCHC, NPH3, TPR_1, TFIIA, DHBP_synthase, UQ_con, TPR_2, TPT, F-box, adh_short, Cyclin_C, Na_H_Exchanger, AA_permease, MtN3_slv, TIM, NDK, Pantoate_transf, Allene_ox_cyc, Cyclin_N, Methyltransf_11, CBM_20, Methyltransf_12, Rhodanese, Glycolytic, Actin, Usp, eIF-4B, Glyco_transf_8, BURP, Alpha-amylase, F420_oxidored, EGF_CA, Kelch_1, PGAM, Aminotran_1_2, Kelch_2, UPF0261, CoA_binding, DUF868, Peptidase_S10, Lung_7-TM_R, Oleosin, Sad1_UNC, Gln-synt_C, LSM, NTP_transferase, Metalloenzyme, Prenylcys_lyase, Subtilisin_N, SAM_1, DUF298, ESCRT-III, DNA_pol_E_B, Aminotran_3, NAD_Gly3P_dh_N, Gln-synt_N, MMR_HSR1, DUF588, zf-CCCH, DnaJ, Pkinase_Tyr, Cupin_2, LRR_1, Cupin_3, zf-CSL, FAR1, HD, FH2, APC8, PTR2, MannoseP_isomer, Rib_5-P_isom_A, DUF1336, Phosphorylase, DUF1191, Asp, Mit_rib_S27, PAP_fibrillin, DUF1195, Aldedh, zf-C3HC4, PPR, PK, PurA, RMMBL, HTH_11, Tim17, and PBD wherein said Pfam gathering cutoff for said protein domain families are stated in Table 17; wherein said plant cell nucleus is selected by screening a population of transgenic plants that have said recombinant DNA and express said protein for an enhanced trait as compared to control plants that do not have said recombinant DNA in their nuclei; and wherein said enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced resistance to salt exposure, enhanced shade tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

2. The plant cell nucleus of claim 1 wherein said protein has an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 198 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO: 394 and homologs thereof listed in Table 2.

3. The plant cell nucleus of claim 1 wherein said protein is selected from the group of proteins identified in Table 1.

4. A plant cell nucleus with stably integrated, recombinant DNA to suppress the level of an endogenous protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by Pfam name in the group of Pfam names consisting of SNF5, LMBR1, TFIIS_M, TFIIS_C, and Glyco_transf_8, wherein the Pfam gathering cutoff for said protein domain families is stated in Table 17 wherein said plant cell nucleus is selected by screening a population of transgenic plants with said recombinant DNA and have the level of said endogenous protein suppressed for an enhanced trait as compared to control plants that do not have said recombinant DNA; and wherein said enhanced trait is selected from the group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced resistance to salt exposure, enhanced shade tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

5. The plant cell nucleus of claim 1 further comprising DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell.

6. The plant cell nucleus of claim 5 wherein the agent of said herbicide is a glyphosate, dicamba, or glufosinate compound.

7. A transgenic plant cell or plant comprising a plurality of plant cells with a plant cell nucleus of claim 1.

8. The transgenic plant cell or plant of claim 7 which is homozygous for said recombinant DNA.

9. A transgenic seed comprising a plurality of plant cells with a plant cell nucleus of claim 1.

10. The transgenic seed of claim 9 from a corn, soybean, cotton, canola, alfalfa, wheat or rice plant.

11. The transgenic corn seed of claim 10 wherein said seed can produce corn plants that are resistant to disease from the Mal de Rio Cuarto virus or the Puccina sorghi fungus or both.

12. A transgenic pollen grain comprising a haploid derivative of a plant cell nucleus of claim 1.

13. A method for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated recombinant DNA in a nucleus of claim 1, wherein said method for manufacturing said transgenic seed comprising:

(a) screening a population of plants for said enhanced trait and said recombinant DNA, wherein individual plants in said population can exhibit said trait at a level less than, essentially the same as or greater than the level that said trait is exhibited in control plants which do not express the recombinant DNA,

(b) selecting from said population one or more plants that exhibit said trait at a level greater than the level that said trait is exhibited in control plants,

(c) verifying that said recombinant DNA is stably integrated in said selected plants,

(d) analyzing tissue of said selected plant to determine the production or suppression of a protein having the function of a protein encoded by nucleotides having a sequence selected from the group consisting of one of SEQ ID NO:198-394; and

(e) collecting seed from said selected plant.

14. The method of claim 13 wherein plants in said population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells, and wherein said selecting is effected by treating said population with said herbicide.

15. The method of claim 14 wherein said herbicide comprises a glyphosate, dicamba, or glufosinate compound.

16. The method of claim 15 wherein said selecting is effected by identifying plants with said enhanced trait.

17. The method of claim 16 wherein said seed is corn, soybean, cotton, alfalfa, wheat or rice seed.

18. A method of producing hybrid corn seed comprising:

(a) acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA in a nucleus of claim 1;

(b) producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA;

(c) selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide;

(d) collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants;

(e) repeating steps (c) and (d) at least once to produce an inbred corn line;

(f) crossing said inbred corn line with a second corn line to produce hybrid seed.