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  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/13—Plant traits
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the invention is in the fields of plant breeding and molecular biology.
• the invention relates to the identification of corn loci conferring increased kernel oil concentration using genetic markers and the use of genetic markers as an aid to the identification and breeding of corn with increased kernel oil concentration.
• Corn is a major crop used as a human food source, an animal feed, and as a source of carbohydrate, oil, protein, and fiber. It is principally used as an energy source in animal feeds, or as a raw material for the recovery of starch, protein feed fractions, fiber, flaking grits, flour, and oil. Most commercial corn produced throughout the United States is produced from hybrid seed. The production of corn hybrids requires the development of elite corn inbreds that upon intermating produce agronomically superior hybrids. During the development of corn inbreds, plant breeders select for a number of different traits affecting agronomic performance. These traits include but are not limited to stalk strength, lodging, disease resistance, grain moisture and grain yield. Agronomic traits tend to be quantitatively measured with continuous rather than discrete distributions. It is theorized that quantitative traits are controlled by several genes with small and generally equivalent effects. Further, the observed phenotype is due partially to this genetic component and an environmental component.
• heritability of a trait is defined in the broad sense as the ratio of the genetic variance to the total phenotypic variance.
• Many agronomic traits display low heritability; i.e., the performance of parent plants is a poor predictor of offspring performance.
• traits with low heritability have small genetic variance components in comparison with observed variation.
• the impact on the plant breeder is that in breeding populations, the value of a plant's genetic composition is difficult to determine from agronomic trait measurements. In an attempt to maximize their discriminative abilities, breeders collect multiple measurements both from individuals related by descent and from many environments. This strategy is resource intensive because it involves the use of extensive trialing to make even small gains in plant improvement.
• Corn with increased kernel oil concentration is important because it possesses improved feeding value for poultry (Han Y. et al. (1987) Poultry Sci. (5_ :103-111) and livestock (Nordstrom, J.W. et al. (1972) J. An. Sci 3.5 (2,1:357-361). Grain from conventional corn hybrids typically contains 4% oil.
• a long-term recurrent selection program was initiated in the open-pollinated cv. Burr's White by C.G. Hopkins in 1896. This recurrently-selected population known as Illinois High Oil (IHO), has been selected for increased oil concentration for over ninety generations (Dudley, J.W. and R.J. Lambert.
• Kernel oil concentration can be phenotypically measured using a variety of analytical methods. Oil concentration displays a non-discrete distribution, common for quantitatively-inherited traits controlled by several loci. Kernel oil measurements select those breeding lines with the highest phenotypic expression. Unfortunately, the genetic potential for high oil is limited in most of these lines because it is impossible to discriminate between lines based upon their true genetic composition. This situation is further aggrevated when simultaneous selection for agronomic performance is practiced. It would therefore be advantageous to base selection upon the genotype of the plants in the population. Genetic markers, especially nucleic acid markers, may be used to advantage as an indirect selection method for complex quantitative traits. Genetic markers identifying alleles conferring increased oil would therefore be an advantageous tool for plant breeding programs developing elite high oil corn germplasm.
• a method for reliably and predictably breeding for corn with increased kernel oil concentration comprises a) using one or more genetic markers to select a corn plant from a corn breeding population by marker- assisted selection, wherein the genetic markers are selected from the group consisting of sl375, sl384, sl394, sl416, sl422, sl432, sl457, sl480, sl476, sl478, sl484, sl500, sl513, sl529, sl544, sl545, sl630, sl633, sl647, sl750, sl756, sl757, sl767, sl772, sl774, sl780, sl797, sl813, sl816, sl817, sl836, sl853, sl860, sl870, sl921,
• the present invention provides a method for the identification of and selection for genes controlling increased com kernel oil concentration. These oil alleles were initially identified in materials composed of or derived from the Alexho synthetic breeding populations. Further, the method facilitates the use of this high oil material in breeding programs with the objective of developing new high oil com germplasm.
• the method uses genetic markers to predict the oil breeding value of lines in a com breeding program. By indirect selection of oil loci using these markers, those lines with the greatest genetic potential for increased kernel oil concentration are chosen. According to the method, any type of genetic marker may be used to identify an association with kernel oil concentration. The method is only limited by the ability to measure polymorphism at a given marker locus. Those skilled in the art will recognize that the various genetic markers which may be used includes but is not limited to restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), simple sequence repeats (SSRs), AFLPs, various single base pair detection methods, allozymes, and phenotypic markers.
• RFLPs restriction fragment length polymorphisms
• RAPDs random amplified polymorphic DNAs
• SSRs simple sequence repeats
• AFLPs various single base pair detection methods
• allozymes and phenotypic markers.
• SSR markers useful in the practice of the instant method include si 375, si 384, sl394, sl416, sl422, sl432, sl457, sl476, sl478, sl480, sl484, sl500, sl513, S1529, sl544, sl545, sl630, sl633, sl647, sl750, sl756, sl757, sl767, sl772, sl774, sl780, sl797, sl813, sl816, sl817, sl836, sl853, sl860, sl870, sl921, sl922, S1925, sl931, sl933, sl939, sl946, sl949, s2054, s2055, s2057, s2058, s2097,
• An additional embodiment of the present invention are com plants and high oil com germplasm that are produced using the instant breeding method. DETAILED DESCRIPTION OF THE INVENTION
• Table 1 provides a brief description of the genetic markers that form a part of the instant invention. Each marker is defined by it's constituent nucleic acid primers (forward and reverse) that facilitate amplification of the specific marker locus of the com genome. Also indicated is the required identifier for each sequence. The identifiers listed in Table 1 correspond to those listed in the Sequence Listing (infra) as required by 37 C.F.R. ⁇ 1.821 et seq.
• ACAGCTAGCCAAGATCTGATT reverse 30 sl545 CGATACTAATGGAAGCCCTAA forward 31 ATGGCCCATTAAGTTTATCAC reverse 32
• AGCGGCATCTATGTTCTATG reverse 50 si 780 CCCAGTGCGAAGAGACTC forward 51 ACACCTGCTCTGCACCAC reverse 52 si 797 CTAACCCACGACGACCCT forward 53 GCATGAGTGCATGTGCAT reverse 54 sl813 CTGCCACATGCTTTTCTG forward 55 CTGTAAAGAAGCTGGTCTGGA reverse 56 sl816 TTCTCCTCATGGATGCGT forward 57 CTATTTGGAAGTATGGGCTTCA reverse 58 sl817 GAGGGCATCTATGTGCAAC forward 59
• GCGCGAGTGGAGTAGTAAG reverse 82 si 949 AAGATTATGCAGATGAGACACC forward 83
• Com Any variety, cultivar, or population of Zea mavs L. Elite. This term characterizes a plant or variety possessing favorable traits, such as, but not limited to high yield, good grain quality, and disease resistance. This enables its use in commercial production of seed or grain at a profit. The term also characterizes parents giving rise to such plants or varieties.
• High Oil Com Germplasm This term characterizes com plants which, when either self-pollinated or used as either the male or the female parent in a variety of outcrossing combinations, produce kernels with increased oil when compared to kernels produced by non-high oil germplasm.
• Examples of high oil com germplasm include but are not limited to open-pollinated varieties, hybrids, synthetics, inbred lines, races, and populations or com plants derived from one of the aforementioned.
• This term refers to a group of individuals from a common ancestry; a more narrowly defined group than a variety.
• Synthetic This term refers to a genetically heterogeneous collection of plants of known ancestry created by the intermating of any combination of inbreds, hybrids, varieties, populations, races or other synthetics.
• Inbred This term refers to a substantially homozygous individual, variety or line.
• Recombinant Inbreds A population of independently derived lines developed by repeated selfing each generation until complete homozygosity is approached. Each recombinant inbred is derived from a single F2 plant using a breeding method commonly referred to as single seed descent. Breeding. The art and science of improving a species of plant or animal through controlled genetic manipulation.
• Marker- Assisted Selection The use of genetic markers to identify and select plants with superior phenotypic potential. Genetic marker(s) determined previously to be associated with a trait locus or trait loci are used to uncover the genotype at trait loci by virtue of linkage between the marker locus and the trait locus. Plants containing desired trait alleles are chosen based upon their genotypes at linked marker loci.
• Alexho Synthetic Recurrently selected, high oil com germplasm developed by Denton Alexander at the University of Illinois. Alexho synthetic high oil com germplasm is composed of multiple synthetic populations defined by their cycle of advancement in the recurrent selection breeding program.
• Breeding Population A genetically heterogeneous collection of plants created for the purpose of identifying one or more individuals with desired phenotypic characteristics.
• Phenotype The observed expression of one or more plant characteristics.
• Phenotypic Value A measure of the expected expression of an allele at a trait locus.
• the phenotypic value of an allele at a trait locus is dependent upon its expressive strength in comparison to alternative alleles.
• the phenotypic value of an individual, and hence its phenotypic potential, is based upon its total genotypic composition at all loci for a given trait.
• Genetic Marker Any morphological, biochemical, or nucleic acid based phenotypic difference which reveals a DNA polymo ⁇ hism. Examples of genetic markers includes but is not limited to RPLPs, RAPDs, allozymes, SSRs, and AFLPs.
• Marker locus The genetically defined location of DNA polymo ⁇ hisms as revealed by a genetic marker.
• Trait Locus A genetically defined location for a collection of one or more genes (alleles) which contribute to an observed characteristic.
• Genotype The allelic composition of an individual at genetic loci under study.
• Restriction Fragment Length Polymo ⁇ hism A DNA-based genetic marker in which size differences in restriction endonuclease generated DNA fragments are observed via hybridization (Botstein, D. et al. 1980. Am. J. Hum. Genet. 32: 314-331.
• Random Amplified Polymo ⁇ hic DNA (RAPD).
• RAPD Random Amplified Polymo ⁇ hic DNA
• SSR Simple Sequence Repeat
• AFLP A DNA amplification-based genetic marker in which restriction endonuclease generated DNA fragments are ligated to short DNA fragments which facilitate the amplification of the restricted DNA fragments (Vos, P. et al. 1995. Nucleic Acids Res. 23:4407-4414). The amplified fragments are size separated and differences in amplification patterns observed.
• the present invention relates to the discovery of trait loci controlling kernel oil concentration through the use of genetic markers.
• oil measurements and marker-based genotypes were generated for members of the populations.
• the locations of oil concentration loci were determined in relation to markers genetically linked to these trait loci.
• Indirect selection of preferred oil alleles may now be practiced using the information at one or more linked genetic markers.
• Selected com plants comprise one or more alleles encoding a high oil phenotype. It is recognized that several different populations and population types could be used to locate trait loci of interest.
• Some of the population types include but are not limited to recombinant inbreds, backcrosses, F2's or their self- pollinated or intermated derivatives, and synthetics. Further, it is understood that an alternative to measuring phenotypic and genotypic variation within populations is the measurement of genotypes and phenotypes between populations. In this alternative the second population is a selected derivative of the first population, selection being either on the trait of interest (phenotypic selection) or on specific marker alleles (genotypic selection). It is also recognized by those skilled in the art that alternative statistical approaches may be used to determine a linkage relationship between marker loci and trait loci.
• SSR regions for each DNA sample were analyzed using the following protocol: 1. Ten ⁇ l of amplification cocktail (see Table 2) was added to 5 ⁇ l
• the DNA fragment flanked by sequences complementary to the primers present in the amplification cocktail was amplified by PCR (U.S. Patent No. 4,683,202 and U.S. Patent No. 4,683,195) using the following protocol: 1) 45 cycles of 50 sec at 95°C, 50 sec at 54°C and 80 sec at 72°C and 2) 1 cycle of 300 sec at 72°C;
• 10X Buffer is a pH 9.0 solution composed of 800 mM Tris-OH, 200 mM
• One hundred thirty three polymo ⁇ hic SSR marker loci were used to genotype the recombinant inbreds from the LH119wx x ASKC28wx cross and one hundred and three polymo ⁇ hic SSR marker loci were used to genotype the LH51 x ASKC28wx-derived population.
• twenty publicly available polymo ⁇ hic SSR loci with previously established chromosome locations and covering all ten maize chromosomes (available from Research Genetics, Huntsville, AL) were also mapped in both populations.
• Linkage data for significant marker loci was examined to determine both the number of trait loci present and their probable location.
• Significant marker loci on the same linkage group are either detecting the same trait locus or alternatively different trait loci.
• a determination of the number trait loci on a linkage group was made.
• Significant marker loci, on the same linkage group and uninterrupted by non-significant marker loci were declared to be detecting the same trait locus on the chromosome. If significant marker loci on the same chromosome were interrupted by non-significant marker loci then each significant region was declared to contain a trait locus resulting in multiple trait loci on the same chromosome.
• Each oil locus is defined by one or more linked marker loci.
• oil loci which were identified in one population were identified at the same location in the second population.
• an oil locus was found in one population, but not in the second population.
• the allele with a positive oil effect was found in LH51 and thus it would be unexpected to identify the same locus in the LH119wx x ASKC28wx population.
• the second case it was found that different ASKC28wx-derived marker alleles were segregating in the populations; therefore, each population was measuring the oil effect of a different ASKC28wx allele at the trait locus.
• an objective of a com breeding program could be the creation of new elite inbred lines which contain trait alleles conferring increased kernel oil concentration. These trait alleles would be introduced by the intermating of high oil germplasm with one or more elite com inbreds.
• the resultant hybrid could be self-pollinated to produce an F2 population for the pu ⁇ oses of initiating a conventional pedigree breeding program (Allard, R. W. (1960) Principles of Plant Breeding. John Wiley & Sons, Inc. New York. Pp 115-128).
• plant tissue would be collected from each F2 individual in the population and genotyped with the SSR marker loci listed in Table 1.
• Those F2 individuals with the highest frequency of SSR marker alleles derived from the high oil source would be selected and further culled based upon their agronomic fitness.
• those oil loci in a heterozygous state could become fixed for either the high oil or low oil allele. It is therefore likely that genotyping and selection of later generation materials would be practiced in order to further segregate breeding lines based upon their marker allele and hence oil allele composition.
• the resulting inbreds from the pedigree breeding program may not demonstrate sufficient agronomic competitiveness or sufficient kernel oil expression because an inadequate number of oil alleles was recovered. These new inbreds could therefore be used as parental material and new breeding projects initiated.
• the SSR markers could again be used for further selection of oil as described.
• selection methodology would be based upon the allelic composition of one or more marker loci which identify trait oil loci present in a population. Further selection would be performed by examination and selection of genotypes from individual plants, families, or their progeny. Various predictive models could be developed using genotypic information, which could generate various selection indices. These models would permit weighting the effect predicted by marker loci. This is because the predictive value of an individual marker locus is dependent upon its genetic distance from the corresponding trait locus as well as the expressivity of the trait locus. Selection strategies which combine phenotype-based and genotype-based selection may also be envisioned. The marker loci presented here are predictive of oil loci in Alexho synthetic populations.
• ASKC28wx represents the 28th oil breeding cycle of a genetically closed population
• earlier breeding cycles are composed of the same oil loci. It is expected that cycles differ simply in their allelic frequency at the identified oil loci. Therefore, in breeding populations derived from earlier Alexho cycles, the marker loci described in this invention will be useful in identification of oil loci and in prediction of oil concentration.
• KERNEL OIL CONCENTRATION It is important to identify com plants and lines which, when used as parents, have the greatest probability of producing offspring with superior performance. Transgressive segregant offspring of such parents would result from the crossing of parents with complementary sets of alleles conferring the high-oil phenotype.
• marker alleles which predict desired trait performance i.e., high oil
• By genotyping lines at those marker loci the value of those lines as parents is revealed.
• A-E For example, if one wanted to create an individual containing superior alleles at 5 separate oil loci (A-E), one could identify and cross a parent composed of desired alleles for locus A, B, and C with a parent composed of desired alleles at B, D, and E. These parents are complementary because they permit the recovery of progeny containing desired alleles at all 5 loci. Ideally, parents would be chosen which when combined ensure maximum complementation of loci, so that a high frequency of desired recombinants are recovered.
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO:10: CGGACGACGA CTGTGTTC 18
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 14: TGCTGCACTA CTTGAACCTA G 21
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 15: ACACAGAGAT GACAAAAGCA A 21
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO:20: CTTGTTGTAA TGGATGAGTG AG 22 (2; INFORMATION FOR SEQ ID NO:21-
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 23: ACAGATCTTG ACACGTACAT ACC 23
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 39: AACTGATGAA TACCTTCCCA G 21
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 43: AAGCACGGCC CAATAGAAT 19
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 48: TGATACTCTG GTGCATGTTC 20 (2) INFORMATION FOR SEQ ID NO: 49.
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO:51: CCCAGTGCGA AGAGACTC 18
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 60: GCTCAGAAGT TGCGTTTATG 20 (2) INFORMATION FOR SEQ ID N0:61.
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 66: CAGGCTTACC TAGCCTTCTC 20
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 70: GGAAGAACCA ATCCCATATC T 21
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 75: AGTGAGGAAA GAATATGCTG G 21
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 78: AATGGTACGG TTCAGGATG 19
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO:82: GCGCGAGTGG AGTAGTAAG 19
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 83: AAGATTATGC AGATGAGACA CC 22
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 90: CAAGGTAAAG TGACAAAGCA G 21
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 95: ATCTGAACAC TTGAGCAACA A 21
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 100: CTTCCTCGGT GTCAGACG 18 (2) INFORMATION FOR SEQ ID NO: 101:
• MOLECULE TYPE other nucleic acid
• SEQUENCE DESCRIPTION SEQ ID NO: 104: GGTAGGTGGG TAGGGGTT 18

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• 1998
  • 1998-03-17 ZA ZA9802250A patent/ZA982250B/xx unknown
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  • 1998-03-19 AU AU65751/98A patent/AU734755B2/en not_active Ceased
  • 1998-03-19 CA CA002280933A patent/CA2280933A1/en not_active Abandoned
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