CN106661610A - Markers linked to reniform nematode resistance - Google Patents

Abstract

This disclosure concerns methods and compositions for identifying cotton plants that have a reniform nematode resistance trait. Some embodiments concern molecular markers to identify, select, and/or construct reniform nematode resistant plants and germplasm, or to identify and counter select relatively susceptible plants. This disclosure also concerns cotton plants comprising a reniform nematode resistance trait that are generated by methods utilizing at least one marker described herein.

Description

Resist sex-linked mark with reniform nematode

Technical field

It relates to disease resistance in plants.In some embodiments, it relates to reniform nematode resistance, such as exist Reniform nematode resistance in cotton.In particular embodiments, it relates to for identifying reniform nematode resistance in biology Composition and method, for example, the molecular marker with reniform nematode resistance close linkage.Further embodiment is related to use In reniform nematode resistance trait is incorporated in host living beings body, for example by using with reniform nematode resistance close linkage point Sub- mark is incorporated into the composition in host living beings body and method.

Background

Cotton (Gossypium spp.) is a kind of important fiber and oilseed crop all over the world.In great majority Other produce cotton country, including the U.S., and cotton is as annual crop plantation, although its natural habit is perennial 's.Gossypium (Gossypium) includes about 50 known species, and they originate in America, Asia, Africa and Australia Arid and semi-arid lands.Fryxell(1992)Rheedea2:108-65.These species are according to its chromosome size and inter-species The chromosome pairing of hybridization (inter-specific cross) is segmented into multiple gene groups, including 8 diplonts Group and 1 tetraploid plant group (i.e. " AD " genome).Most of cotton fiber is by upland cotton (" upland cotton " (" Upland Cotton ")) produce, it is a species in tetraploid AD gene groups.And, although cotton planting is to a great extent Upper these high yield upland cotton cultigens of dependence, but they are compared with other plant groups, and heredity change is less, and is considered as Easily by pathogen and insect infection.Brubaker&Wendel(1994)Am.J.Bot.81:1309-26；Bowman et al. (1996)Crop Sci.36:577-81。

In recent years, many areas in the U.S. and other national output of cottons are subject to a kind of parasite --- reniform nematode The impact of (" RN ") (Rotylenchulus reniformis (Rotylenchulus reniformis)).Parasitism of the reniform nematode in cotton is related to And plasomidum is formed, enter seating in a restaurant or dining hall so as to providing nutrition for developmental female (developing female), and here occurring The event of point (feeding site) may determine neurological susceptibility degree of the cotton plant to nematode.Agudelo et al.(2005) J.Nematology 37:185-9；Rebois et al.(1975)J.Nematology 7:122-39.

The instrument for being available for resisting RN crop damages is little.For example, nematicide is such asAnd soil fumigant Such asHave been used for reducing adverse effect of the reniform nematode to output of cotton, but when using as indicated, these Nematicide only part is effectively.Host plant resistance will be the economically most viable means for managing reniform nematode infection, but It is resistant to RN without upland cotton cultigen.Robinson et al.(1999)Crop Sci.39:850-8；Koenning et al.(2004)Plant Dis.88:100-13；Usery et al.(2005)Nematropica 35:121-33； Weaver et al.(2007)Crop Sci.47:19-24。

Reniform nematode resistance is identified in wild diploid species, such as long calyx cotton (G.longicalyx) (Dighe et al.(2009)Crop Sci.49:1151-64) with nonirrigated farmland cotton (G.aridium) (Romano et al. , ibid) and amphidiploid phenotype (2009)：Print plus cotton (Inca Cotton) GB713 (Guti é rrez et al. (2011) TAG Theor.Appl.Genet.122:271-80).It is identified, by obtaining to the infiltration of nonirrigated farmland cotton from long calyx cotton The heredity of RN resistances is responsible for by single dominant gene.Robinson et al.(2007)Crop Sci.47:1865-77. In addition it is identified, the RN resistances penetrated into from tree cotton and nonirrigated farmland cotton (Rose＆Standley) Skovsted and obtain by two not Dominant gene at homogenic seat is responsible for heredity.Sacks&Robinson(2009)Field Crops Res.112:1-6.Identification The useful resources of RN resistances as much as possible are important.When the nematode population or population that run into breakthrough resistance, or develop When such colony or population, multiple resistance source may prove valuable resource.

It is time-consuming and difficulty a process that proterties (such as RN resistances) is penetrated in upland cotton from other derived genes, This is because for example, cotton heredity is complicated, is related to the difference of ploidy, and there is various genomes and two sub-gene group, wherein being permitted Mostly it is that incompatible or compatibility is low.Robinson(2007),Annu.Rev.Phytopathol.45:263-88； Percival et al. (1999), " Taxonomy and germplasm resources, " is embodied in Cotton:Origin, History,Technology,and Production.Smith&Cothren(eds.),New York,NY,John Wiley& Sons, the 33-63 page.Being additionally, since chromosome pairing difficulty causes the n plant survival rate of interspecific hybridization relatively low, obtains and has the phase The genetic stocks and probability of suitable offspring is then lower on agronomy is penetrated in prestige.Referring to Romano et al. (2009) TAG Theor.Appl.Genet.120:139-50.In the conceived case, proterties interested is passed through hexaploid by people Bridge system (hexaploid bridgingline) is penetrated in upland cotton from diploid species.See, for example, Robinson et (2007), al. ibid；Konan et al.(2007)Plt.Breed 126:176-81.

Plant breeding procedures are combined into the anticipant character from two or more cultigens or from extensive various source Breeding pond (breeding pool), cultigen is developed by selfing and the desired phenotype of selection from breeding pond.Target is single The improved combination of the anticipant character from Parental Germplasms is constituted in kind.These important proterties can include that higher seed is produced Amount, the resistance to disease and insect, more preferable stem and root, the tolerance to arid and heat, and preferably agronomy quality.At this Need many steps in the program of sample to develop the new cultigen comprising one or more anticipant character.Plant breeding first has to point The problem and weakness of existing germplasm are analysed and determined, program object is set up, and determines specific breeding objective.In any two germplasm In the case of possible incompatible or poor compatibility, particularly in the complicated plant of science of heredity is such as cotton, it is necessary to select tool There is the germplasm of the proterties for meeting program object.

Each procedure of breeding should include the periodic objective appraisal to procedure of breeding efficiency.Evaluation criterion is because of mesh And target and it is different, but the annual amount of acquisition (gain) (according to the comparison with appropriate criteria) obtained from selection should be included, The overall value of the breeding system being pushed into, and the success produced by per unit input (for example, annual, spend per dollar, etc.) The number of cultigen.Then, to propulsion breeding system likely, Object Industry region can represented with the time of more than 3 years Thoroughly detected under environment, and be compared with suitable standard.The time of NPD projects cultigen is selected from optimal strain The person of choosing；Those are still defective in terms of minority proterties to can serve as parent, to produce the new colony for further selecting.From Hybridization for the first time starts, and these processes finally guide list marketing and allocation step into, it usually needs 8-12.Therefore, new cultivation The exploitation planted is a time-consuming process, needs accurate perspective planning, the effective use of resource, and direction as few as possible Change.

Most difficult task is the excellent individuality of identification heredity in plant breeding.A kind of method of identification good plant is to see Examine the performance of its standard cultivar planted relative to other experimental plants and extensively.If single observation can not reach a conclusion, Repeated observation can be then utilized, preferably to estimate its genetic worth.This task is extremely difficult, because (for most Number proterties), real genotype value can be covered by plant trait that other mix or environmental factor.

Pattern that selection of the practitioner to breeding and system of selection is replicated depending on plant, the proterties to be improved it is heritable Type (such as F of the cultigen used in property, industry₁Hybrid cultivar, is sheerly cultigen, etc.), and the complexity of character inheritance Property.For highly heritable proterties, in single position, the selection of the superior individual plants of assessment is probably effective, but right In the low proterties of heredity, need to be selected according to the mean value obtained from the repeat assessment to corresponding plants family.It is popular System of selection generally include pedigree select, amendment pedigree select, mixing select and recurrent selection.Back cross breeding can be used for By one or several gene transfers favourable to height inherited characteristics in desired cultigen.Various recurrent selection techniques can For the quantitative inheritance proterties that improvement is controlled by many genes.

Breeder selects first and hybridizes two or more parent systems, is followed by selfing and the selection for repeating, and produces many New genetic make up.Breeder can pass through in theory hybridization, selfing and mutagenesis and produce billions of different genetic make ups. Such breeder can not be directly controlled process on a cellular level.Therefore, two breeders never develop Identical strain with identical proterties, or even closely similar strain.

Every year, plant breeder can be selected for being advanced to follow-on germplasm.By the germplasm various unique and different Geography, weather and edaphic condition under plant.Then during planting season or at the end of, further selected.Opened The cultigen for sending is not expected.This not predictability is that caused by the selection of breeder, the selection is in unique ring Occur in border, and cannot be controlled by DNA level (using the conventional procedure of breeding), can produce it is millions of not With possible genetic make up.Except may by it is very careless and substantially in the way of predict in addition to, the common breeder of this area The unpredictable final gained strain that he is developed.Similarly, same breeder can not be by using identical original Parent and identical selection technique produce twice identical cultigen.During excellent new cotton cultigen is developed, this Plant a large amount of consumings that unpredictability causes the resource of money and other side.

When mark assisted Selection (MAS) is available, it is possible to use mark assisted Selection provides time, cost and work Significant advantage in power (in select in progeny plants carry out Genotyping compared with).SNP (SNP) indicates Thing becomes the preferred mark for MAS in various crop improved plan, because they have high abundance, are easy to automatic Change, and have high flux Genotyping platform can use.However, in the cotton species of cultivation, it is the genome complexity of height, narrow Narrow hereditary basis, allotetraploid property, and the shortage of reference gene group hinders the exploitation of candidate SNP mark.

It is open

Sex-linked molecular marker is resisted to can be used to help the mark of reniform nematode resistance trait in cotton with reniform nematode Will thing assisted Selection.Disclosed herein is some specific marks, their identified reniform nematodes in cotton gene group resist Property QTL regions close to or within, they between parent upland cotton genotype and RN resistances source have polymorphism, wherein these Mark and reniform nematode resistant phenotype chain (for example, close linkage).In embodiments, resist with reniform nematode sex-linked Molecular marker can be selected from the group：The mark listed in table 2-4 and chain with any mark for listing in table 2-4 Mark.For example, sex-linked molecular marker is resisted to be selected from the group with reniform nematode：The mark listed in table 3-4, with And the mark chain with any mark for listing in table 3-4.These marks can reniform nematode is infected it is resistant Vegetable lamb and cultigen mark assisted Selection in provide brilliance effectiveness.

This document describes include in the first vegetable lamb of the identification comprising reniform nematode resistance or such vegetable lamb The method of germplasm.For example, add cotton (Inca Cotton) GB713 parents' comprising print is derived from using the identification of specific mark The reniform nematode resistance upland cotton of reniform nematode Resistance QTL or germplasm.In some instances, comprising the first of reniform nematode resistance Vegetable lamb or germplasm can be the neurological susceptibility ratios for infecting reniform nematode and/or damaging in first plant or the parent of germplasm Neurological susceptibility is low what is observed in plant or germplasm (that is, reducing) plant or germplasm.In some instances, comprising reniform nematode First vegetable lamb of resistance or germplasm can be to reniform nematode infect and/or damage neurological susceptibility ratio with first plant Or germplasm belongs to that neurological susceptibility what is observed in the specific conventional plant or germplasm of same species (for example, upland cotton) is low (that is, to drop It is low) plant or germplasm.Some embodiments of these methods can be included in the first vegetable lamb or germplasm and determine at least One resists sex-linked mark with reniform nematode.

Also describe the method for producing vegetable lamb or germplasm comprising reniform nematode resistance.Some enforcements of these methods Scheme can include resisting sex-linked mark from the first vegetable lamb or germplasm (such as island with reniform nematode by least one Cotton plant or germplasm) penetrate into the second vegetable lamb or germplasm (such as upland cotton plant or germplasm), may be included with producing The vegetable lamb of reniform nematode resistance or germplasm.The vegetable lamb or germplasm produced by preceding method is also included within specific reality In applying scheme.

Some embodiments include the method for producing Transgenic cotton plants.The example of such method may include one Individual or multiple exogenous nucleic acid molecules are incorporated in target vegetable lamb or its offspring, wherein at least one of described exogenous nucleic acid Comprising cotton gene group nucleotide sequence, the cotton gene group nucleotide sequence is chain with reniform nematode resistance with least one Mark it is chain；Or at least one of wherein described exogenous nucleic acid includes such nucleotide sequence, the nucleotides sequence Row can be chain with reniform nematode resistance with least one with another nucleotide sequence specific hybrid, another nucleotide sequence Mark it is chain.

Some embodiments include the system and kit for identifying the vegetable lamb that may include reniform nematode resistance. The instantiation of such system and kit may include one group of nucleic acid probe, each of which nucleic acid probe comprising can with it is another The nucleotide sequence of nucleotide sequence specific hybrid, another nucleotides sequence is listed in cotton and at least one and kidney shape line Worm resists sex-linked mark chain.The system and kit of the vegetable lamb of reniform nematode resistance may be included for identification Instantiation may include detector, and it is configured to detect one or more from the nucleic acid probe group or the letter of its amplicon Number output, thereby identification at least one resists the presence or absence of of sex-linked mark with reniform nematode.Specific example bag Such instruction is included, the explanation is by least one mark presence or absence of related to having reniform nematode resistance Connection.

By the detailed description of the multiple embodiments below with reference to accompanying drawing, aforementioned and further feature will be easier to expect.

Brief description

Fig. 1 includes the log of all samples in research (a-e)₁₀(X+1) frequency distribution of ratio.

Fig. 2 includes the generation mean analysis (generation-mean that the RN enumeration datas in phenotypic screen are carried out analysis)。

Fig. 3 includes the figure in the Shang QTL areas of chromosome 21, and black text shows peak LOD positions and chain mark.

Fig. 4 includes that the figure of mark-trait associations in QTL areas is represented, the LOD with RN resistance traits and % is explained.

Fig. 5 includes that SNP detects the flow chart description of pipeline (pipeline).

Sequence table

The standard letter abbreviation of the nucleotide sequence nucleotide base listed in subsidiary sequence table shows, such as 37C.F.R. Defined in § 1.822.Only show a chain of each nucleotide sequence, but it is to be understood that when shown chain is mentioned, Also include complementary strand.In subsidiary sequence table：

SEQ ID NO:1-72 shows the SNP mark chain with the main reniform nematode Resistance QTL on cotton chromosome 21 Will thing.

SEQ ID NO:73-78 shows the SSR mark chain with the QTL on cotton chromosome 21.

Describe in detail

I. the general view of many embodiments

Embodiment of the present invention is related to and tetraploid sea island cotton (Gossypium barbadense) genotype print plus cotton The specific molecular mark of the reniform nematode resistance close linkage of (Inca Cotton) GB 713.In some embodiments, this A little marks and its equivalent can be used to for reniform nematode resistance to penetrate into desired cotton species on agronomy from this source Or in cultigen (for example, to overcome cultivated cotton in shortage to the host resistance of reniform nematode), or in vegetable lamb or The proterties is identified in germplasm.For many reasons, expect to produce compared with conventional cultivation kind with increase to reniform nematode Infection and/or the cotton (for example, upland cotton) of the resistance damaged.It is also expected to reniform nematode resistance source as much as possible is identified, So as to for example, there is provided for breaking through the nematode population of resistance or the protection of population, and/or multiple lines are combined in single germplasm Worm Resistance QTL is providing improved resistance.Embodiments herein provides the strategy and process of high flux and cost effective, Program is penetrated into for designing and implementing RN resistances.

Some embodiments include that for example, method and composition is included from tetraploid sea island cotton genotype for identification Print adds the vegetable lamb of the reniform nematode resistance of cotton (Inca Cotton) GB 713, and/or carrying to can be predicted and determine this kidney The germplasm of the genotype of shape nematode resistance proterties.Some embodiments include the method for making such vegetable lamb and germplasm.This Class method can include, for example but be not limited only to, and penetrate into desired reniform nematode resistance markers allele, and/or heredity Method for transformation.In particular embodiments, including for example, by vegetable lamb obtained in preceding method and/or germplasm.For Select the vegetable lamb comprising reniform nematode resistance and/or carry genotype that is measurable and determining reniform nematode resistance trait The system and/or kit of germplasm are also the feature of some embodiments.

The vegetable lamb comprising reniform nematode resistance trait, including above-mentioned identification and selection are identified and selected using MAS Process can for produce pest resistance, the desired vegetable lamb of agronomy one efficient, eco-friendly approach is provided. Embodiment of the present invention provides some cotton marker gene seats and QTL interchromosomals every (chromosome Interval), they represent the isolating of statistically significant with reniform nematode resistance and (therefore, it is possible to predict and determine reniform nematode Resistance).Detection to these marks or the additional gene loci of (therefore of equal value with it) chain with these marks can be used for Mark aids in cotton breeding program, to produce plant and the germplasm of reniform nematode resistance.

Some embodiments provide for identification show reniform nematode resistance the first vegetable lamb or germplasm (for example, Strain or kind) method.In some instances, detect in the first vegetable lamb or germplasm with from tetraploid sea island cotton One or more marks of the reniform nematode resistance chain (such as close linkage) of genotype print plus cotton (Inca Cotton) GB 713 At least one of will thing seat (such as multiple mark seats) allele.In instances, marker gene seat can be selected From：Locus in table 2-4；The mark chain with any mark illustrated in table 2-4, including：DASCTP_4812_64； DASCTP_60046_472；DCTE1_261396_78；DASCTP_60046_46；DCTE1_278814_262；DCTE1_ 214911_694；DCTE1_233925_865；DCTE1_232591_126；DCTE1_365870_69；DASCTP_39375_ 356；DCTE1_240643_347；DCTE1_208529_965；DCTE1_271930_223；DASCTP_59246_191； DASCTP_28910_164；DASCTP_1656_527；DASCTP_51689_504；DCTE1_214869_124；DASCTP_ 8602_496；DASCTP_8602_418；DASCTP_10515_556；BNL3279；BNL4011；GH132；And with least one Other chain marks of aforementioned QTL marks.Marker gene seat can be selected from：The locus of table 2-4 and with table 2-4 Shown in the chain mark of any mark, except DASCTP_4812_64；DASCTP_60046_472；DCTE1_ 261396_78；DASCTP_60046_46；DCTE1_278814_262；DCTE1_214911_694；DCTE1_233925_865； DCTE1_232591_126；DCTE1_365870_69；DASCTP_39375_356；DCTE1_240643_347； DCTE1_ 208529_965；DCTE1_271930_223；DASCTP_59246_191； DASCTP_28910_164；DASCTP_1656_ 527； DASCTP_51689_504；DCTE1_214869_124；DASCTP_8602_496；DASCTP_8602_418； DASCTP_10515_556；BNL3279；BNL4011；And outside GH132.

In some instances, multiple marker gene seats can be selected or identified in same plant or germplasm.For example, table The marker gene seat that illustrates in 2-4 and the marker gene seat chain with any marker gene seat for illustrating in table 2-4 All combinations, may be embodied in and stay in the multiple marker gene seats for selecting in plant or germplasm or identifying.In some examples In, the mark to homeologous cotton gene group can be improved by following process (herein referred to as " HAPSNP pipelines ") The efficiency of detection, the process includes using sequence assembling program to produce the contig nucleotide sequence (contig) with high stringency, first The SNP of hypothesis is first detected, the gene frequency of haplotype information and locus in each genotype is generated；Using allele Paralog/homeologous the SNP from homologous SNP is distinguished with Haplotype frequencies, so as to strengthen the energy of identification high-quality SNP Power.This can the method according to disclosed in international patent application No.PCT/US13/020211 complete.

In some embodiments, reniform nematode resistant cotton plant is included from sea island cotton print plus cotton (Inca Cotton) the heterologous nucleic acids (for example, at least one gene) of the RN Resistance QTLs of GB 713, it can be given comprising the heterologous core The vegetable lamb of acid or germplasm are with RN resistances, or the RN resistances for improving the vegetable lamb comprising the heterologous nucleic acids or germplasm.According to The molecular marker of instantiation can be used to identify such nucleic acid and it be sequenced.

II. abridge

AFLP AFLPs

ASH allele specific hybridizations

CM centimorgans

EST ESTs

LG linkage groups

LOD probability logarithms (with 10 as bottom)

MAS mark assisted Selections

Amplifications of the NASBA based on nucleotide sequence

PCR PCRs

QTL quantitative trait locuses

RAPD Randomly amplified polymorphic DNAs

RFLP RFLPs

RIL recombinant inbred strains

RKN root-knot nematodes

RN reniform nematodes

RT-PCR reverse transcriptase-PCR

SNP SNPs

SSCP single strand conformation poly morphisms

SSR simple sequence repeats

III. term

As used in the application (including claim), unless separately expressly stated otherwise in the content, otherwise odd number and The term (for example, " one ", " one " and " being somebody's turn to do ") of singulative includes plural.Thus, for example, appellation " plant ", " plant Thing " or " plant " also indicate that multiple plants.Similarly, appellation " mark ", " mark " or " mark " Indicate multiple marks.And, based on context, term " plant " can be used for indicating the plant similar in heredity or Identical offspring.Similarly, term " nucleic acid " can refer to multiple copies of nucleic acid molecules.Similarly, term " probe " can refer to Many similar or identical probe molecules, term " mark " can refer to many similar or identical marks.

Number range includes limiting the numeral of the scope, and including each integer and non-integer in the range of the restriction Fraction.Unless otherwise stated, herein all of whole technologies and scientific terminology have and those of ordinary skill in the art The identical implication being commonly understood by.

For the ease of investigating each embodiment described in the disclosure, the explanation to particular term is provided below：

It is detached：" detached " biological components (such as nucleic acid or protein) are thin with the organism of the component Lock-in Other biological components (that is, other chromosomes and exchromosomal DNA and RNA, and protein) in born of the same parents are substantially separate, therewith Produce respectively, or therefrom purify, while causing the change of chemistry and function in the component.For example but it is not limited only to, can By fracture connection nucleic acid nucleic acid to be separated from chromosome with the chemical bond of remaining DNA in chromosome." separated " nucleic acid molecules and protein includes the nucleic acid molecules and the protein that purify by standard purification techniques.Term also includes passing through The nucleic acid and protein of recombinant expressed preparation in host cell, and the nucleic acid molecules of chemical synthesis, protein and peptide.

Nucleic acid molecules：As it is used herein, term " nucleic acid molecules " can refer to the polymerized form of nucleotides, it can be wrapped Include the sense and antisense chain of RNA, cDNA, genomic DNA, and above-mentioned synthesized form and mixed polymer.Nucleotides can refer to Ribonucleotide, deoxyribonucleotide, or the modified forms of any kind nucleotides.As it is used herein, " nucleic acid point Son " and " nucleic acid " and " polynucleotides " are synonymous.The term includes the single-stranded and double chain form of DNA.Nucleic acid molecules can be wrapped Nucleotides naturally occurring and/or through modification is included, they are bonded by naturally occurring and/or non-naturally occurring nucleotides It is connected together.

Nucleic acid molecules by chemistry or biochemical modification, or can contain non-natural or derivative nucleosides soda acid Base, this is that those skilled in the art readily recognize that.Such modification includes, for example, label, methylate, one or many Individual naturally occurring nucleotides is replaced with analog, and (for example, uncharged connecting key is modified between nucleotides：For example, methylphosphine Acid esters, phosphotriester, phosphoramidate, carbamate etc.；Charged connecting key：For example, thiophosphate, two thio phosphorus Acid esters etc.；Pendant side group moiety：For example, peptide；Intercalator：For example, acridine, psoralen, etc.；Chelating agent；Alkylating agent；Modify with passing through Connecting key：For example, α-different head nucleic acid etc.).Term " nucleic acid molecules " also includes any topological conformation, including single-stranded, double-strand, portion Divide duplex, triple repetitions, hairpin (hairpinned), ring-type and padlocked conformation.

Target group：As it is used herein, term " target group " can refer to plant population's (example of Genes location Such as, vegetable lamb colony).Target group generally obtains from the crossing controlled of parent genotype, and parent genotype can be by two Inbred strais is provided.With regard to the decision of Juvenile stage, for the Mating design of development and location colony, and mark type used, Each depend on gene to be positioned, the availability of mark and molecular linkage map.Plant parent in target group should just feel emerging Sufficiently variation is respectively provided with for the proterties of interest on nucleotide sequence and phenotypic level.Using the variation of parental nucleic acid sequence fixed Recombination event is tracked in position colony plant.

The availability of the polymorphic marker of offer information depends on the amount of variant nucleic acid sequence.Therefore, in specific parent Specific Information sign thing may not be identified in the hybridization of this genotype, the mark of even now there may be.

" genetic map " is the genetic linkage on one or more chromosomes (or linkage group) of given species between locus The description of relation, can be determined by analyzing and positioning colony.In some instances, genetic map can be shown as figure or table The form of lattice.Term " Genes location " (genetic mapping) can refer to is got the bid by using genetic marker, target group The separation of will thing and the standard genetic principle of recombination frequency are limiting the process of the linkage relationship of locus." genetic map position " Certain position (relative to the genetic marker of surrounding on same linkage group or chromosome) on genetic map is referred to, in given thing Specific mark is can be found that at the position in kind.Relatively, " physical map of genome " is referred in given species and indicated Absolute distance (for example, with base-pair or with adjacent genetic fragment tolerance that is detached and overlapping) between thing.The thing of genome Reason figure not necessarily reflects the actual recombination frequency observed in test cross of certain species between the difference of physical map.

Hybridization：As it is used herein, term " hybridization " (or " hybridization ") is referred to produce offspring (for example, by pollination Cell, seed and plant) Gamete Fusion.This term both include sexual hybridization (that is, one plant is pollinated by another) or Including selfing (that is, self-pollination for example uses the pollen and ovule from same plant).

Backcrossing：Backcrossing methods can be used to that nucleotide sequence is incorporated in plant.Baclccrossing techniques have been widely used tens of Year, for new proterties to be incorporated in plant.Jensen, N. edit .Plant Breeding Methodology, John Wiley&Sons,Inc.,1988.In typical backcrossing scheme, original cultigen interested (recurrent parent) is treated with carrying Second cultigen (nonrecurrent parent) hybridization of transfer gene of interest.Then, the filial generation obtained by the hybridization is close with samsara again This hybridization, repeats this process until obtaining such plant, wherein not only from the gene being transferred of nonrecurrent parent, wheel The substantially all of expectation Morphological and physiological characteristics for returning plant are also recovered in the plant changed.

Penetrate into (introgression)：As it is used herein, term " infiltration " is referred to the equipotential base at genetic locus Because being delivered in genetic background.In some embodiments, the infiltration of the specific allelic form at locus can be with this The form of sample occurs：The allelic form is delivered at least one by the sexual hybridization between two parents of same species In individual offspring, parent described in wherein at least one has the specific allelic form in its genome.It is specific comprising this The offspring of allelic form can repeatedly be returned with the strain with expectation genetic background.Can select in backcross progeny should Specific allelic form, so as to produce new kind, wherein the specific allelic form has been fixed on the hereditary back of the body Jing Zhong.In some embodiments, the infiltration of specific allelic form can occur in following forms：Two donor gene groups Between restructuring (for example, fusion chloroplaset in), wherein at least one donor gene group in its genome have the spy Fixed allelic form.Infiltration can be related to the transmission of specific allelic form, and it can be, for example but be not limited only to, The selected allelic form of mark allele；QTL；And/or transgenosis.

Germplasm：As it is used herein, term " germplasm " refer to ownership or from single plant or plant population (for example, Plant lines, kind, family) genetic stocks, and from derived from plant or flora clone.Germplasm can be organism or thin The part of born of the same parents, or it can be from organism or cell separation (for example, detached).Usually, germplasm can provide tool The genetic stocks being made up of specific molecular, it is the basis of plant genetic quality.As it is used herein, " germplasm " refer to it is specific The cell of plant；Seed；The tissue (tissue of new plant for example, can be grown) of specified plant；It is non-seed with specified plant Partly (for example, leaf, stem, pollen and cell).

As it is used herein, term " germplasm " and " genetic stocks " are synonymous, and can be used for finger and can breed plant The seed (or other vegetable materials) of thing." Germplasm Bank " can refer to different seeds or the organized set of other genetic stocks (wherein each genotype is uniquely identified), can therefrom turn out known cultigen, it is possible to produce new cultigen. In embodiments, the germplasm used in method described herein or plant is from cotton strain or kind.Specific real In example, germplasm is the seed of cotton strain or kind.In specific example, germplasm is the nucleic acid from cotton strain or kind Sample.

Gene：As it is used herein, term " gene " (or " genetic elements ") can refer to losing with functional meaning The genomic dna sequence of biography.Term " gene " can also be used to refer to, for example but be not limited only to, by heritable genomic dna sequence The cDNA and/or mRNA of coding.

Genotype：As it is used herein, term " genotype " refers to that individual (or group of individuality) has at one or more Gene effect at body locus.One or more bases that the genotype of the group of individual or individuality is inherited by the individuality from its parent Because the allelic form at seat is defined and is described.Term " genotype " can be additionally used in refer to individuality at individual gene seat, it is many At individual locus, or the Gene effect at all locus of its genome." haplotype " is individuality in multiple genetic locis The genotype at place.In some instances, the genetic loci described in haplotype can be physics and genetic linkage；I.e. each gene Seat may be located on same chromosome sections.

Breeding (elite)：As it is used herein, term " breeding " is referred to based on remarkable agronomy performance by seed selection With the kind or cultigen for selecting.Breeding cotton strain includes, for example but is not limited only to, DP 555BG/RR, DP 445BG/RR, DP 444BG/RR,DP 454BG/RR,DP 161B2RF,DP 141B2RF,DP 0924B2RF,DP 0935B2RF,DP 121RF, and DP 174RF (Deltapine)；ST5599BR, ST5242BR, ST4554B2RF, ST4498B2RF, and ST5458B2RF(Stoneville)；FM9058F, FM9180B2F, FM1880B2F, and FM1740B2F (Fiber-Max)； PHY485WRF, PHY375WRF, and PHY745WRF (PhytoGen)；And MCS0423B2RF, and MCS0508B2RF (Cotton States)。

Quantitative trait locus：The specific dye related to specific quantity phenotype can be positioned in the genome of organism Colour solid locus (or interval).Each such locus is referred to as " quantitative trait locus " or QTL.It is as used herein , term " quantitative trait locus " refers to such DNA sequence dna section, and it is quantitative that it has been identified as being discussed further below The basic DNA sequence dna (for example, gene, non-coding sequence, and/or intergenic sequence) of shape or phenotype：The quantitative character or phenotype There is variation in degree, and can be two or more DNA sequence dnas (for example, between gene, non-coding sequence, and/or gene with attribution Sequence) or the interaction between its expression product and its environment.Therefore, term " quantitative trait locus " includes having at least The polymorphic genetic locus of two allele, these allele are under at least one genetic background (for example, at least one In individual breeding population or offspring) different impacts can be produced to the expression of phenotypic character.In practice, can be with Molecular Identification QTL, to help gene location group in containing participate in specified quantity proterties (such as RN resistances) sequence region.

As it is used herein, term " QTL intervals " can refer to the DNA section chain with the basal gene of QTL proterties.QTL Interval is usual, but necessarily, more than QTL itself.QTL intervals can include the DNA sections positioned at the sides of QTL 5 ' and/or 3 '.

Kinds of experiments normal form is had been developed for for identifying and analyzing QTL.See, for example, Jansen (1996) Trends Plant Sci.1:89.The report major part with regard to QTL positioning in crop species delivered is based on double parents Using.Referring to Lynch and Walsh (1997) Genetics and Analysis of Quantitative Traits, Sinauer Associates,Sunderland.Generally, these normal forms are related to one or more pairs of parents, and parent can be with Be, for example, from independent a pair of two inbreeding strains, or be belonging to different inbreeding strains or be multiple have relationship or without relationship Parent, each of which shows the feature of different phenotypic characters relevant interested.Generally, this experimental program is related to And 100-300 separation offspring derived from the single hybridization from two divergence (divergent) inbred strais, two of which divergence is closely Hand over system to be selected to for example maximize phenotype and molecular marker difference between strain.To parent and separation offspring Multiple marker gene seats carry out Genotyping, and one kind is estimated to several quantitative characters (for example, RN proterties).So Afterwards, the statistically significant correlation between genotype value and phenotypic variation degree is identified in generation after isolation, as QTL.

The strong point or weakness of this experimental program are decided by the use of closed crossing, because the F of gained₁Parent has phase Same chain phase (form of treaty of the allele in parent's generation).Therefore, in F₁After plant selfing, all of separation F₂Afterwards , all there is information in generation, and linkage disequilibrium is maximized, and chain phase is known, only two QTL allele, and The frequency of (in addition to backcross progeny) each QTL allele is 0.5.

It is known to those skilled in the art it is many for determine mark whether with QTL (or another mark) genetic linkage Statistical method, these methods are included but are not limited to, standard linear model (for example, ANOVA or regression plot；Haley and Knott(1992)Heredity 69:315)；With likelihood method (for example, it is desirable to maximize algorithm；Lander and Botstein(1989)Genetics 121:185-99；Jansen(1992)Theor.Appl.Genet.85:252-60； Jansen(1993)Biometrics49:227-31；Jansen(1994)“Mapping of quantitative trait loci by using genetic markers:An overview of biometrical models, " it is embodied in J.W.van Ooijen and J.Jansen (editor), Biometrics in Plant breeding:applications of molecular markers,pp.116-24,CPRO-DLO Metherlands；Jansen(1996)Genetics 142: 305-11；With Jansen and Stam (1994) Genetics 136:1447-55).

Statistical method example includes：Single-point analysis of markers；Interval mapping (Lander and Botstein (1989) Genetics 121:185)；Composite interval mapping；Penalized regression analysis (penalized regression analysis)；It is multiple Miscellaneous pedigree analysis (complex pedigree analysis)；MCMC is analyzed；MQM analyzes (Jansen (1994) Genetics 138:871)；HAPLO-IM+ is analyzed；HAPLO-MQM is analyzed；With HAPLO-MQM+ analyses；Bayesian MCMC；Ridge regression；Identity- Parentage analysis (identity-by-descent analysis)；Return with Haseman-Elston, any of which is suitable for In the content of specific embodiments of the present invention.Can be used in instantiation identification and Mapping of QTL complicated breeding population can Statistical method is selected to be described in United States Patent (USP) 6,399,855 and pct international patent disclose No.WO0149104A2.It is all this A little methods are all computation-intensives, are generally performed with the help of the computer based system comprising specific software.Properly Statistics bag can obtain from various public and industry sources, and be well known by persons skilled in the art.

Mark：Although the specific dna sequence of coded protein is typically fully conservative between species, other Region of DNA domain (for example, noncoding DNA and introne) tend to develop and accumulate polymorphism, therefore in the individuality of same species Between be probably it is different.Genome mutation can be any source, for example, variation be likely due to the insertion of DNA, disappearance, Caused by the presence of repetition, repetition DNA element, point mutation, recombination event and transposable element and sequence.These regions may be contained Useful molecular genetic mark.Usually, in offspring the polymorphism proterties of detached any otherness heredity (including nucleic acid Polymorphism) all it is potential mark.

As it is used herein, term " mark " and " molecular marker " is referred to be used as ginseng when linked gene seat is identified The product (for example, protein) of the nucleic acid of examination point or its coding.Therefore, mark can be referred to for identification with specific etc. The gene or nucleic acid of the plant of position gene.Mark can be described as the variation at given genomic locus.Genetic marker can To be short dna sequence, such as single base is to changing the sequence around (SNP or " SNP "), or length dna sequence Row, such as microsatellite/simple sequence repeats (" SSR ")." mark allele " or " mark allelic form " is referred to The version of mark present in concrete individuality.As it is used herein, term " mark " can refer to the clone of chromosomal DNA Sections, or can with/DNA molecular complementary with clone's sections of chromosomal DNA can also be referred to.The term also refers to and genome mark The complementary nucleotide sequence of will thing sequence, such as nucleic acid primer and probe.

Mark can be described as, for example, the specific polymorphic genetic of the specific location in the genetic map of organism Element.Genetic map can be that the figure of genome (or a part for genome, such as individual chromosome) is represented, wherein chromosome The distance between upper terrestrial reference (landmark) and terrestrial reference are measured with the recombination frequency between terrestrial reference and terrestrial reference.Hereditary terrestrial reference can be with Be various known polymorphic markers any one, for example but be not limited only to：Simple repeated sequence (SSR) mark；Limit Property fragment length polymorphism (RFLP) mark processed；With SNP (SNP) mark.As an example, SSR marks Will thing can stem from the nucleic acid (for example, EST (EST)) of genome or expression.

Other marks include, for example but are not limited only to, EST；AFLP (AFLP) (Vos et al. (1995)Nucl.Acids Res.23:4407；Becker et al.(1995)Mol.Gen.Genet.249:65；Meksem et al.(1995)Mol.Gen.Genet.249:74)；Randomly amplified polymorphic DNA (RAPD)；With isodynamic enzyme mark.Same work Enzyme mark can serve as genetic marker, for example come track the isodynamic enzyme mark chain with specific first mark or Other kinds of mark.Isodynamic enzyme is the enzyme of various ways, they on amino acid sequence (therefore, in its code nucleic acid sequence On row) it is different from each other.Some isodynamic enzymes are polymerases, containing the subunit being slightly different.Other isodynamic enzymes be it is polymeric or Monomer, but scaled off from protoenzyme at the different loci of protoenzyme (pro-enzyme) amino acid sequence.Isodynamic enzyme Can be characterized and be analyzed on protein level or nucleic acid level.Therefore, in a particular embodiment, it is as herein described any It is used equally to analyze isodynamic enzyme mark based on the method for nucleic acid.

" genetic marker " is included in colony the allele for having polymorphism, and the allele in the colony can pass through One or more analysis method (such as rflp analysis, aflp analysis, isodynamic enzyme analysis of markers, snp analysis and ssr analysis) is entered Row detection and differentiation.Term " genetic marker " can also refer to and can be used as joining when genetic linkage locus (such as QTL) is identified The genetic locus (" marker gene seat ") of examination point.These marks are also referred to as " QTL marks ".

Above-mentioned physically target property (and the method for detecting them) is different, but all these marks are all Can according to polynucleotides length and/or sequence in addition physics distinguish (and and any special sign thing multiple equipotentials Gene is mutually distinguished).Have been established for many methods for being used for detection molecules mark and appraisal mark thing allele.This area There is diversified code known to technical staff for detecting this variability, and these codes are intended to what is detected for them Polymorphism type is typically special.These codes include, for example but are not limited only to, PCR amplifications and single-strand conformation polymorphism (SSCP) detection, for example, by electrophoresis；With autonomous lasting sequence replicating (3SR) (see Chan and Fox (1999) Reviews in Medical Microbiology 10:185-96)。

Molecular marker technology can typically improve the plant breeding efficiency by MAS.With desired phenotypic character (for example QTL) show that the molecular marker allele of linkage disequilibrium is useful to select desired proterties to provide in plant population Instrument.Realizing the key components of MAS methods is：Intensive (having information) molecular marker is created in plant germplasm Genetic map；At least one QTL is detected based on the statistical correlations between mark and phenotypic variation degree；Based on qtl analysis result Define a series of specific useful mark allele；It is used for and/or is extrapolated to educating for current series with by these information In planting germplasm, the selection to make based on mark determines to provide possibility.

The hereditary variability that can be observed in the different groups of same species (such as cotton), such as in target group really Fixed hereditary variability.Although genetic map may morph between the colony of same species, from colony Genetic map and polymorphic marker's information typically still can be used to belong to multiple colonies of different subspecies, with realize identification and/or Select the plant comprising the proterties chain with the mark and/or germplasm, and plant of the counter-selection comprising bad proterties and/or kind The purposes such as matter.

Two kinds of mark used in concrete MAS schemes described herein is SSR marks and SNP marks. SSR marks include any kind of molecular heterogeneity for causing nucleotide sequence length variation.Exemplary SSR marks are short DNA section (for up to hundreds of base-pairs), it is made up of two or three base-pair sequences of multiple tandem sequence repeats.These are heavy Complex sequences produces adjustable length high polymorphism region of DNA due to fidelity of repair poor (for example, due to polymerase sliding) Domain.SSR seems to be random distribution in genome, and general flank has conservative region.SSR marks could also be from RNA sequence (in forms such as cDNA, part cDNA or EST) and Genomic material.

The heterogeneity of SSR marks makes them be highly suitable as molecular genetic mark.For example, SSR genome mutations Degree is hereditary, and is multiple alleles, codominance and repeatable detection.The increasingly complicated detection technique based on amplification The heterogeneity for being extended to the nucleotide sequence between detection sample of (for example, the technology of PCR-based) provides various sensitive Method.Probe (for example, nucleic acid primer) can be designed to hybridize with the flank conserved region of SSR, and probe can be used to expand Variable SSR areas.The different size of amplicon produced from SSR areas has distinctive and reproducible size.It was observed that Two homologues, different size of SSR amplicons from a certain individual or Different Individual of plant population are limited SSR mark allele.Simply by the presence of at least two SSR mark equipotential bases that can produce PCR primer of different sizes Cause, the SSR just can be used as mark.

Chain (no) balance：As it is used herein, term " linkage equilibrium " refers to the shape of two mark independent separates State, the i.e. mark random combine in offspring.The mark for showing linkage equilibrium is considered as that chain (no matter not whether they Positioned at same chromosome).As it is used herein, term " linkage disequilibrium " refers to two marks in nonrandom mode point From state；(therefore according to definition, the interval in same linkage group is less than the recombination frequency that i.e. mark has less than 50% 50cM).In some instances, the mark for showing linkage disequilibrium is considered as chain.

Chain, close linkage and pole close linkage：As it is used herein, chain between gene or mark can refer to Gene or mark in following phenomenon, wherein certain chromosome to be had significantly be delivered to together in follow-on individuality Probability.Therefore, a mark can be measured and/or represented with another mark or the chain of gene with recombination frequency.Two Individual gene or mark are each other closer to this probability is closer to " 1 ".Therefore, term " chain " can refer to one or more bases (this is from the independent assortment when mark/gene is located on coloured differently body with the probability more than 0.5 for cause or mark It is expected) transmit together with another gene.When the presence of gene is contributed to individual phenotype, the mark with the gene linkage Will thing can be referred to as chain with the phenotype.Therefore, term " chain " can refer between mark and gene, or mark and table Relation between type.

When two chain marks or gene separated from one another on chromosome, Relative Hereditary distance (is determined by hybridization frequency It is fixed, and measured with centimorgan (cM)) typically proportional to physical distance (being measured with base-pair).The definition of 1 centimorgan is：Show Two genetic markers of 1% recombination frequency (that is, two marks occur a hybridisation events in per 100 cell divisions) The distance between.Usually, mark and another mark or gene are closer to (no matter the distance between they are with losing Pass distance metric still measured with physical distance), they it is chain must be tightr.Due to the restructuring between chromosome distance and proterties The frequency of event is generally proportionate, so there is an approximate physical distance to be associated with recombination frequency.

As it is used herein, term " chain " can refer to or many separated by the genetic distance less than big 50cM Individual gene or mark.Therefore, two " chain " genes or mark can be separated by less than about 45cM；Less than about 40cM；It is little In about 35cM；Less than about 30cM；Less than about 25cM；Less than about 20cM；Less than about 15cM；Less than about 10cM；Less than about 5cM.

As it is used herein, term " close linkage " can refer to one or more bases of mutual distance within about 35cM Cause or mark.Therefore, two " close linkage " genes or mark can separate less than 36cM；Less than 35cM；It is less than 34cM；Less than about 33cM；Less than about 32cM；Less than about 31cM；Less than about 30cM；Less than about 29cM；Less than about 28cM；Less than about 27cM；Less than about 26cM；Less than about 25cM；Less than about 24cM；Less than about 23cM；Less than about 22cM；Less than about 21cM；Less than about 20cM；Less than about 19cM；Less than about 18cM；Less than about 17cM；Less than about 16cM；Less than about 15cM；Less than about 14cM；Less than about 13cM；Less than about 12cM；Less than about 11cM；Less than about 10cM；Less than about 9cM；Less than about 8cM；Less than about 7cM；Less than about 6cM；Less than about 5cM；Or even less genetic distance.

As it is used herein, term " pole close linkage " can refer to one or many of mutual distance within about 5.0cM Individual gene or mark.Therefore, two " pole close linkage " genes or mark can be separated by less than 6.0cM；It is less than 5.5cM；Less than 5.0cM；Less than about 4.5cM；Less than about 4.0cM；Less than about 3.5cM；Less than about 3.0cM；Less than about 2.5cM； Less than about 2.0cM；Less than about 1.5cM；Less than about 1.0cM；Less than about 0.5cM.

Certain mark is with coding to the gene of the contributive polypeptide of certain phenotype closer to (either according to genetic distance Or physical distance tolerance), the special sign thing gets over close linkage with the phenotype.In view of foregoing teachings will recognize, with certain The chain mark of gene or phenotype includes those and the gene or the mark of phenotype close linkage, and those are extremely tight therewith Chain mark.In some embodiments, certain mark with to the contributive gene of RN resistant phenotypes closer to (no matter It is to measure according to genetic distance or physical distance), the mark gets over close linkage with RN resistant phenotypes.Therefore, RN in cotton The genetic marker of the chain of resistant phenotype, close linkage and pole close linkage can be used to MAS programs identify comprising RN resistances The cotton variety of (compared with parental breed and/or at least one specific conventional cultivation kind), individuality of the identification comprising RN resistances Vegetable lamb, and by (for example, " AD " genomic cotton, such as upland cotton) in this proterties breeding to other cotton varieties with raising The resistance for RN being infected and/or being damaged.

In some embodiments, the linkage relationship between molecular marker and phenotype can be expressed as " probability " or " adjust Whole probability (adjusted probability) ".In this linguistic context, probable value is " a certain phenotype and special sign thing equipotential base Therefore be random presence or absence of particular combination " statistics likelihood.Therefore, probability score is lower, phenotype and spy Determine the possibility that mark allelic form will isolate bigger.In some instances, probability score can be described as " aobvious Write " or " not notable ".In specific example, the probability score of random combine is considered as 0.05 (p=0.05 (5% probability)) It is " notable " instruction for isolating.However, in other examples, notable probability can any be less than the general of 50% (p=0.5) Rate.For example, notable probability can be less than 0.25；Less than 0.20；Less than 0.15；And/or less than 0.1.

In some embodiments, the chromosome 18 that the mark chain with RN resistant phenotypes can be illustrated from table 2-4 Select with 21 cotton QTL marks.In some embodiments, the mark chain with RN resistant phenotypes can from table The distance of the QTL marks illustrated in 2-4 is selected in mark within about 10cM.Therefore, the mark chain with RN resistant phenotypes The distance of the QTL marks (the QTL marks for for example, illustrating in table 3-4) illustrated in will thing and table 2-4 can be for example 10cM；9cM；8cM；7cM；6cM；5cM；4cM；3cM；2cM；1cM；0.75cM；0.5cM；Within 0.25cM；Or it is less.For example, Mark may be located between the two QTL marks illustrated in table 2-4.

Plant breeder can advantageously use molecular marker, be shown with expectation phenotype (such as RN resistances) by identification The mark allele for isolating probability (showing as linkage disequilibrium) of statistically significant is identifying desired individuality.It is logical Molecular marker or molecular marker cluster that identification is isolated with quantitative character are crossed, therefore breeder identifies QTL.By identification The mark allele (or from expectation allele of multiple marks) related to phenotype is expected to selection, plant breeding Person can quickly select phenotype by selecting suitable molecular marker allele (i.e. MAS).It is placed in dividing on genetic map Sub- mark is more, and the figure instructs that the potential use of MAS is bigger, and the genetic background that can be used during MAS is more.

Mark group：As it is used herein, " group " of mark or probe is referred to can be used for identification comprising property interested The particular combination of the individual mark of shape or probe (or the data got by it).In some embodiments, with RN resistances The chain group mark thing of phenotype can be used to identify the vegetable lamb comprising RN resistances.Corresponding to mark group or the number of probe groups Can be stored in electronic media according to (or the data got using these marks or probe).Each mark in mark group Will thing may be useful to Characters Identification, and the indivedual marks selected from the group, and including some in these marks But it is not all, of subgroup, it is also possible to which effectively identification includes the individuality of proterties interested.

Allele：As it is used herein, term " allele " is referred to occurs in two kinds of specific gene seat or more One kind in various different nucleotide sequences.For example, a kind of allele possibly be present on a chromosome, and another kind etc. Position gene possibly be present on another homologue, just as example on the coloured differently body of heterozygous individual, or occur As occurring in the different homozygosis or heterozygous individual of colony.In some embodiments, it is specific etc. at specific gene seat Position gene may be chain with the desired phenotype of agronomy (for example, RN resistances).In some embodiments, it is specific at locus Allele may be that identification expects that the plant (for example, RN susceptible plants) of phenotype creates conditions not comprising agronomy, so as to It is enough to remove those plants from the procedure of breeding or sowing.Mark allele can together be separated with favourable phenotype, from And the benefit of plant of the identification comprising the phenotype is provided." allelic form of chromosome segment " may refer to such dyeing Body section, it includes mark allelic nucleotide sequence, and the sequence contributes to, or chain in being physically located at the dye The particular phenotype at one or more genetic locuses on colour solid sections.

" gene frequency " can refer to that allele is present at certain locus in plant, strain or strain colony Frequency (being represented with ratio or percentage).Therefore, for allele " A ", genotype is " AA, " " Aa, " " AB, " or " aa " The gene frequency that has of dliploid individuality be respectively 1.0,0.5,0.5, or 0.0.Gene frequency in strain can With by estimating being averaging from the gene frequency of the individual specimen of the strain.Similarly, in strain colony etc. Position gene frequency can be averaging to calculate by the gene frequency of the strain to constituting the colony.For with Finite Number Mesh is individual or colony of strain, gene frequency can be expressed as the individual or strain containing the allele (or it is any its Its specific cluster) counting.

If certain mark and certain linkage of characters, and the presence of the mark allele is the anticipant character or proterties The instruction that form will occur in the plant comprising the allele, then the mark allele is related to the proterties " just ". If certain mark and certain linkage of characters, and the presence of the mark allele is that the anticipant character or proterties form will not The instruction occurred in the plant comprising the allele, then the mark allele is related to the proterties " negative ".

" homozygosis " is individual to only have a form of allele (for example, in two homologous dyeing at given locus There is the diplont that identical allele is copied) in body at the specific gene seat of each.If deposited at locus In more than one allelic form, then individuality is that " heterozygosis " (for example, has the first equipotential of a copy at locus The dliploid individuality of the second allelic form of gene forms and a copy).Term " homogenieity " refer to colony into Member has identical genotype (that is, identical gene frequency) at one or more specific gene seats interested.Phase Instead, term " heterogeneity " refers to that the genotype of individuality in colony at one or more specific gene seats interested is different.

Appraisal mark thing equipotential base can be carried out using any technology that can be used in characterizing the nucleotide sequence at locus Cause.Method for the detection of mark allele includes, for example but is not limited only to that (for example, mark expands method for identifying molecules Increase the amplification and detection of son).For example, the allelic form of SSR marks or SNP marks can pass through the skill based on amplification Art is detected.In the typical detection method based on amplification, the part quilt of marker gene seat or marker gene seat Amplification (uses, for example, PCR, LCR and transcription, using from the detached nucleic acid of vegetable lamb interested as amplification template), and right The amplicon of gained amplification mark is detected.In some embodiments, it is possible to use plant RNA is used as anti-for expanding The template answered.In some embodiments, it is possible to use plant genome DNA is used as the template for amplified reaction.At some In enforcement, QTL marks are SNP marks, and detected allele is SNP mark allele, the side of detection Method is allele specific hybridization (ASH).In some instances, QTL marks are SSR marks, and be detected etc. Position gene is SSR mark allele.

Stabilizing annealing of the ASH technologies based on short, single strand oligonucleotide probes and the single-stranded target nucleic acid of complete complementary.Can be with The isotope or heterotope mark being attached to by detection on probe realizes detection.For every kind of polymorphism, can design Two or more different ASH probes, make them have identical DNA sequence dna in the region in addition to pleomorphism site.Each Probe can be homologous with an allelic sequences perfection, so that the scope of probe can distinguish all known optional equipotentials Gene order.When each probe and target DNA suitable probe design and hybridization conditions under hybridize when, between probe and target DNA Single base mismatch can prevent hybridization.So, only one of which alternate probe can hybridize with the target sample homologous with allele.It is right For two allele for heterozygosis or heterologous sample can the probe alternative with two hybridize.

ASH marks can be used as dominant mark, wherein by the hybridization of only one probe whether determining only one etc. The presence or absence of position gene.Alternative allele can be inferred by the disappearance for hybridizing.In instances, ASH probes and target point Son can be RNA or DNA molecular；Outside the sequence with probes complementary, target molecule can include the nucleotides of any length；Visit Pin can be designed to hybridize with any bar chain of DNA target；And the size of probe can be changed to meet different hybridization conditions Needs.

Amplification variable sequence refers to the plant of the nucleotide residue variability that height is shown between the member of same species Genome amplification sequence.All biologies are respectively provided with variable genome sequence, and each organism (in addition to clone) The variable sequence group being had is different.Once particular variable sequence is identified, its presence can be used to predict phenotypic character. In some examples, the DNA from plant can serve as template, be expanded using the primer of DNA variable sequence flanks.Variable sequence Row can be amplified and subsequently be sequenced.

Alternatively or additionally means, autonomous training sequence replicates (self-sustained sequence Replication) may also be used for identifying genetic marker.Autonomous training sequence is replicated and refers to a kind of nucleic acid amplification method, its The target nucleic acid sequence for using substantially is replicated in vitro under conditions of constant temperature in vitro by index, wherein using participating in reverse transcription disease Three kinds of enzymatic activitys that poison is replicated：Reverse transcriptase；RNase H；The RNA polymerase relied on DNA.Guatelli et al.(1990) Proc.Natl.Acad.Sci.USA87:1874.Retroviral rna replicon strategy is simulated by using cDNA intermediates, This reaction accumulates the cDNA and RNA copy of original target.

The data of the mark allele that representative is detected can be sent to (for example, electronics transmission；With by infrared Line, the transmission of wireless or optical propagation) in computer or computer-readable medium, for analyzing or storing.

For example, can by amplimer or amplimer pair with from the first vegetable lamb or the detached genomic nucleic acids of germplasm Mixing, wherein the primer or primer pair are complementary to or partial complementarity is at least a portion of marker gene seat, and the primer Or primer pair can use cotton gene group nucleic acid to cause the DNA that archaeal dna polymerase drives to be polymerized as template.The primer or primer To (for example, as SEQ ID NO:Primer or primer pair that one of 27 and its equivalent provide) use archaeal dna polymerase Extend in DNA polymerisations with templet gene group nucleic acid, produce at least one amplicon.

" position clone " refers to a kind of specific Cloning processes, wherein by target nucleic acid with mark connecing on genome Closely it is identified and isolated from target nucleic acid.For example, genomic nucleic acids clone can include the complete of two approximating chromosomal regions Portion or a part.If mark can be used to identify genomic nucleic acids clone from genomic library, standard side can be used Method, for example, be subcloned and/or be sequenced to identify and/or separate the subsequence of the clone near the mark.

Locus：As it is used herein, term " locus " is referred to correspond to measurable feature (for example, on genome Proterties) or polymorphism position.Locus (for example, SNP locus) is determined by the probe of the DNA hybridization being contained within locus Justice.

Mark assistant breeding：As it is used herein, term " mark assistant breeding " can refer to directly using MAS pair The method that one or more proterties (for example, RN resistances) carry out breeding.In current practice, plant breeder attempt identify with The proterties of the chain easy detection of agronomy anticipant character, such as pattern, kind skin profile or isoenzyme variant.Then, plant educates Kind person in detached breeding population by track the easy detection proterties separation tracking the Agronomic character.However, These only few linkage relationships are available for used in plant breeding.

Mark assistant breeding provides a kind of time and the efficient process of cost for improveing plant cultivars.Using Several examples of mark assistant breeding are directed to use with isodynamic enzyme mark.See, for example, Tanksley and Orton edited. (1983)Isozymes in Plant Breeding and Genetics,Amsterdam:Elsevier.One example is kind Isodynamic enzyme mark related to nematode pests resistant gene in eggplant.The resistance is controlled by the gene for being named as Mi, positioned at tomato The 6th chromosome, and with a kind of Acid Phosphatase Isozymes Aps1 poles close linkage.It is indirect using Aps1 isodynamic enzyme marks Mi genes are selected to provide advantage, you can clearly to determine the separation of colony by standard electrophoretic techniques；The isodynamic enzyme mark Thing can give a mark in seedling tissue, so as to maintain plant to maturation；And isodynamic enzyme mark allele is aobvious altogether Property cause to distinguish homozygote and heterozygote and be possibly realized.See Rick (1983), be embodied in Tanksley and Orton, ibid.

Probe：In some embodiments, the presence of mark can be detected by using nucleic acid probe in plant. Probe can be DNA molecular or RNA molecule.Rna probe can be synthesized by methods known in the art, such as using DNA point Subtemplate.Probe can include all or part of nucleotide sequence of mark, and the extra neighbour from Plant Genome Connect nucleotide sequence.This is herein referred to as " adjacent probe ".The referred to as former mark of the extra adjacent nucleotide sequence " on Trip " or " downstream ", this depends on being located at 5 ' or 3 ' sides of former mark from the adjacent nucleotide sequence of plant chromosome, such as Conventional understanding.Ordinary skill will recognize that, obtain extra adjacent nucleotide sequence to be included in mark Interior process can almost indefinitely repeat (only by the length limitation of chromosome), thereby other along Chromosome Identification Mark.

Sequence oligonucleotide probe can be prepared synthetically or by clone.Suitable cloning vector is this area skill Art personnel are well-known.Oligonucleotide probe can be mark or unmarked.There are many technologies to can be used for labeling nucleic acid point Son, including for example but being not limited only to：By nick translation；It is random to cause；Use terminal deoxy transferase tailing；Or similar method Radioactive label is carried out, wherein nucleotides such as radioactivity used³²P is marked.Other labels that can be used include, example As but be not limited only to：Fluorogen (for example, FAM and VIC)；Enzyme；Zymolyte；The co-factor of enzyme；Enzyme inhibitor；Etc..Or, make But it is the alternative means of the label for using itself or detectable signal being provided together with other reactants, it is possible to use with acceptor With reference to part, wherein acceptor labeled (for example, being marked with above-mentioned label) provides so as to itself or together with other reagents Detectable signal.See, for example, Leary et al. (1983) Proc.Natl.Acad.Sci.USA 80:4045-9.

Probe can contain the nucleotide sequence not adjoined with original logo thing；This probe is herein referred to as " non-neighboring Connect probe ".The sequence of non-adjacent probe is substantial access on genome with the sequence of former mark, so that the non-adjacent probe It is chain in heredity with homologous genes or proterties (for example, RN resistances).For example, in some embodiments, non-adjacent probe with The distance of the QTL marks (the QTL marks for for example, illustrating in table 3-4) illustrated in table 2-4 can be in about 10cM；9cM；8cM； 7cM；6cM；5cM；4cM；3cM；2cM；1cM；0.75cM；0.5cM；It is within 0.25cM or less.

Probe can be the exact copies of the mark to be detected.Probe can also be included, or by biological with theme The nucleic acid molecules for cloning the substantially the same nucleotide sequence composition of sections of (such as cotton) chromosomal DNA.As made herein , term " substantially the same " can refer to more than 85% identical nucleotide sequence.For example, substantially the same nucleotides sequence Row can be with reference sequences 85.5%；86%；87%；88%；89%；90%；91%；92%；93%；94%；95%； 96%；97%；98%；99% or 99.5% is identical.

Probe can also be and the exact copies of detected mark (" DNA target ") " can specific hybrid " or " specificity The nucleic acid molecules of complementation ".Term " can specific hybrid " or " complementary specificity " refer to the complementation of sufficient degree, so as in nucleic acid Occur to stablize and special combination between molecule and DNA target.Nucleic acid molecules need not be mutual with the target sequence 100% of specific hybrid Mend.When the complementation that there is sufficient degree, so as to avoid the nucleic acid and non-target sequences under conditions of specific binding is expected, for example When there is non-specific binding under stringent hybridization condition, nucleic acid molecules just can specific hybrid.

Cause the hybridization conditions of specific Stringency with the essence of selected hybridizing method and the composition of hybrid nucleic acid sequence Change with length.Usually, ionic strength (the particularly Na of hybridization temperature and hybridization buffer⁺And/or Mg⁺⁺Concentration) will be certainly The stringency of fixed hybridization, although scavenging period can also affect stringency.Hybridization conditions needed for regard to obtaining concrete Stringency Calculating be known to persons of ordinary skill in the art, and be discussed in the following documents：Sambrook et al. (editor) Molecular Cloning:A Laboratory Manual, second edition, the 1-3 volume, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, the 9th and 11 chapters；(compile with Hames and Higgins Volume) Nucleic Acid Hybridization, IRL Press, Oxford, 1985.With regard to nucleic acid hybridization more detailed description May refer to guidance, for example Tijssen, " Overview of principles of hybridization and the Strategy of nucleic acid probe assays, " it is embodied in Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, portion Point I, the 2nd chapter, Elsevier, NY, 1993；With Ausubel et al., editor, Current Protocols in Molecular Biology, the 2nd chapter, Greene Publishing and Wiley-Interscience, NY, 1995.

As it is used herein, " stringent condition " includes following condition, wherein only between hybrid molecule and DNA target Mispairing just can hybridize when being less than 50%." stringent condition " includes more specific horizontal stringency.Therefore, it is as used herein , " medium strict " condition is the condition that molecule of the sequence mismatch more than 50% will not hybridize；" high strict " condition is that sequence is wrong With the condition that will not hybridize more than 20%；" high strict " condition is the condition that sequence mismatch will not hybridize more than 10%.

Representational non-limiting hybridization conditions are presented herein below：

High stringency (detection sequence homogeneity is at least 90% sequence)：65 DEG C of hybridization 16 in 5x SSC buffer solutions Hour；Room temperature is cleaned 2 times, every time 15 minutes in 2x SSC buffer solutions；Clean 2 times with 65 DEG C in 0.5x SSC buffer solutions, 20 minutes every time.

High stringency (detection sequence homogeneity is at least 80% sequence)：In 5x-6x SSC buffer solutions 65-70 DEG C it is miscellaneous Hand over 16-20 hours；Room temperature is cleaned 2 times in 2xSSC buffer solutions, each 5-20 minutes；With 55-70 DEG C in 1x SSC buffer solutions Cleaning 2 times, every time 30 minutes.

Medium stringency (detection sequence homogeneity is at least 50% sequence)：The room temperature hybridization in 6x SSC buffer solutions 16-20 hours；- 55 DEG C of room temperature is cleaned 2 times in 2x-3x SSC buffer solutions, each 20-30 minutes.With regard to institute discussed above There is probe, probe can include extra nucleotide sequence, such as promoter；Transcription signal；And/or carrier sequence.It is discussed above Any probe be used equally to marks of the gene linkage for defining other and participating in cotton RN resistances, the so mark of identification Can be of equal value with the exemplary mark thing named in the disclosure, therefore within the scope of the invention.

Sequence iden：As it is used herein, term " sequence iden " or " homogeneity " are in two nucleic acid or polypeptide Can refer in the content of sequence when by two sequence alignments to realize maximum phase response in specific comparison window, two sequences Middle identical residue.

As it is used herein, term " Percent sequence identity " can refer to by comparing two in comparison window most The numerical value that the sequence (for example, nucleotide sequence) of good comparison determines, the wherein Sequence in comparison window and reference sequences (its not Containing addition or disappearance) compare and comprising adding or (that is, breach) may be lacked, to realize the optimal comparison of two sequences.Percentage Than by being calculated as below：It is determined that the position number of the identical nucleotides for all existing in the two sequences or amino acid residue is to produce The number of matched position, is then multiplied by 100 divided by the total number of positions in comparison window with the number of matched position by the result, Draw Percent sequence identity.

Method for comparing comparative sequences is well-known in the art.Various programs and alignment algorithm are for example following It is described in document：Smith and Waterman(1981)Adv.Appl.Math.2:482；Needleman and Wunsch (1970)J.Mol.Biol.48:443；Pearson and Lipman(1988)Proc.Natl.Acad.Sci.U.S.A.85: 2444；Higgins and Sharp(1988)Gene 73:237-44；Higgins and Sharp(1989)CABIOS 5: 151-3；Corpet et al.(1988)Nucleic Acids Res.16:10881-90；Huang et al.(1992) Comp.Appl.Biosci.8:155-65；Pearson et al.(1994)Methods Mol.Biol.24:307-31； Tatiana et al.(1999)FEMS Microbiol.Lett.174:247-50.What sequence alignment method and homology were calculated It is discussed in detail and may refer to, for example Altschul et al. (1990) J.Mol.Biol.215:403-10.

Basic Local Alignment Search Tool (the BLAST of American National Biotechnology Information center (NCBI)^TM；Altschul et Al. (1990)) can from it is multiple source obtain, including American National Biotechnology Information center (Bethesda, MD) and interconnection On the net, it is used in combination with various sequence analysis programs.Description with regard to how to determine sequence iden using this program, can be with In internet BLAST^TM" help " (help) one save in obtain.For the comparison of nucleotide sequence, it is possible to use the BLAST^TM (Blastn) " Blast 2sequences " function of program, using the default BLOSUM62 matrixes for being set as default parameters.When When making to be estimated in this way, will show that bigger percentage is same with the nucleotide sequence that reference sequences have bigger similitude One property.

External source：" external source " molecule is the nucleotide sequence and/or genomic locations with regard to polynucleotides, and the amino of polypeptide It is non-natural to particular system (for example, germplasm, kind, breeding kind, and/or plant) for acid sequence and/or cellular localization Molecule.In embodiments, external source or heterologous polynucleotide or polypeptide can artificially be added to biosystem (for example, plant Cell, plant gene, specified plant species or kind, and/or plant chromosome) in molecule, and not the concrete biology System is intrinsic.Therefore, certain nucleic acid being appointed as into " external source " can represent the nucleic acid from non-naturally occurring source, or Person can represent that the nucleic acid has non-natural configuration, genetic locus or element arrangements.

Relatively, for example, " natural "/" intrinsic " (native) or " endogenous " (endogenous) nucleic acid is such nucleic acid (such as gene), the nucleic acid elements of normal presence on the chromosome being located in nature except the nucleic acid or other genetic stocks Outside, the nucleic acid does not contain other nucleic acid elements.Endogenous gene transcript by native chromosomal locus nucleotide sequence Coding, is artificially added in cell.

Restructuring：Term " restructuring " refer to be changed by human intervention material (for example, recombinant nucleic acid, recombination, Recombination of polynucleotide, and/or recombinant polypeptide).For example, the arrangement of each several part of recombinant molecule or element may not be natural row Arrange, and/or the primary sequence of recombinant molecule has certain different from its intrinsic sequence.The change of material can be produced in it The recombined material removed in natural environment or state or from its natural environment or state.If the core of the ORFs of nucleic acid Nucleotide sequence removed from its natural surroundings and is cloned in any kind of artificial nucleic acid (such as carrier), then nucleic acid ORFs is restructuring.Produce recombinant molecule (particularly recombinant nucleic acid) code and reagent be in this area it is general, And it is using being that this area is conventional.Term " restructuring " also refers to the cell comprising recombined material or organism (example herein Such as, the plant comprising recombinant nucleic acid and/or plant cell).In some instances, recombinant organisms are genetically modified organisms.

" introduce/import " as it is used herein, term indicate by heterologous or exogenous nucleic acid it is indexable to it is intracellular when, be Finger is merged into nucleic acid using the available any method in any this area intracellular.This term includes nucleic acid introducing method, bag Include for example but be not limited only to, transfect；Conversion；And transduction.

As it is used herein, term " carrier " is to refer to a near few nucleic acid segment to be transferred to intracellular multinuclear Thuja acid or other molecules.Carrier can optionally include that mediation carrier keeps and realize the component/element (example of its desired use Such as, required sequence is replicated, the gene of medicine or antibiotic resistance, MCS is given, and/or can makes to be cloned gene Promoter/the enhancer element of the operation connection of expression).Carrier can come from, for example, plasmid, bacteriophage, or plant or animal Virus." cloning vector ", " shuttle vector " or " subcloning vector " generally comprises the element of operation connection, to facilitate clone and Asia Cloning process (for example, containing the MCS in multiple restriction endonuclease sites).

As it is used herein, term " expression vector " refers to include can promote coded sequence in specific host organism The carrier of the polynucleotide sequence of the operation connection of interior expression.For example, bacterial expression vector can promote coded sequence in bacterium Middle expression.Plant expression vector can promote coded sequence to express in plant cell.It is many that promotion is expressed in prokaryotes Nucleotide sequence can include, for example but be not limited only to, promoter；Operator；And ribosome bind site.Carrier for expression of eukaryon (for example, plant expression vector) includes promoter, enhancer, termination and polyadenylation signal (and other sequences), and they one As from it is different used in prokaryotic expression carrier.

SNP：As it is used herein, term " SNP " (SNP) can refer to works as genome There is single nucleotide acid between the member of species or between intraindividual pairing chromosome in (or other total sequences) Mutant dna sequence during difference.In colony, minimum gene frequency (minor allele can be assigned to SNP Frequency), it is the minimum gene frequency at certain locus observed in special group.For monokaryon glycosides For sour polymorphism, here it is in two gene frequencies it is less that.Different groups are expected to show at least somewhat not Same gene frequency.Special group may show dramatically different gene frequency.In some instances, with RN resistances Chain mark is SNP marks.The high flux gene for newly entering can be used in accurately and quickly methods of genotyping Typing method, such asWithDetermine (Illumina, San Diego, CA), SNP is multiple Change, each sample carries out 384 weights extremely>100,000 resurvey determines.Although SNP marks are highly useful, however, it was found that SNP marks Need the DNA sequence dna information for obtaining high-quality.

SNP may be fallen in the noncoding region of the coded sequence of gene, gene, or the spacer region between gene.Due to losing The degeneracy of password is passed, the SNP in coded sequence can not necessarily change the amino acid sequence of produced protein.If SNP's Two kinds of forms cause identical peptide sequence, such SNP to be referred to as " synonymous " (sometimes referred to as silent mutation).If can produce Different peptide sequences are given birth to, then they are referred to as " non-synonymous." non-synonymous change is probably missense or nonsense, wherein wrong Justice changes can produce different amino acid, and nonsense change can produce Premature stop codon.It is not located in protein coding region SNP gene splicing, transcription factor may be combined, or the sequence of non-coding RNA can also produce impact.SNP is typically double etc. Position gene, thus it is easily determined in plant and animal.Sachidanandam(2001)Nature 409:928-33.

Plant：As it is used herein, term " plant " can refer to whole plant, from the cell or tissue culture of plant Thing, and/or the aforementioned any part of any one.Therefore, term " plant " includes, for example but is not limited only to, whole plant；Plant Part and/or organ (for example, leaf, stem and root)；Plant tissue；Seed；And plant cell.Plant cell can be, for example but Be not limited only to, in plant and/or plant cell, from the detached cell of plant, and by culture from the detached cell of plant The cell of acquisition.Therefore, term " vegetable lamb " can refer to, for example but be not limited only to, whole cotton plants；Multiple cottons are planted Strain；Cotton plants cell；Cotton plants protoplast；Cotton tissue culture (for example, can therefrom regenerate cotton plants)； Cotton plants callus；Cotton plants part (for example, cottonseed, cotton, cotton seed leaf, cotton leaf, cotton stem, cotton bud (cottonbud), cotton root and the cotton tip of a root)；With cotton plants cell, it has been in a part for cotton plants or cotton plants Whole.

" genetically modified plants " are in its at least one intracellular plant containing exogenous polynucleotide.In embodiment, outward Source polynucleotides are stably incorporated in cellular genome so that the polynucleotides can the heredity in successive generations.One In a little examples, exogenous polynucleotide can be incorporated in genome as a part for recombinant expression cassettes.Term " transgenosis () " (transgenic) is used to herein referring to the cell that any genotype is changed due to the presence of exogenous nucleic acid, thin Born of the same parents system, callus, tissue, plant part or plant.Therefore, this term is covered and be changed at first so as to include external source multinuclear The genetically modified organism and cell of thuja acid, and the life produced by the hybridization or vegetative propagation of genetically modified organism at first or cell Thing and cell.As it is used herein, term " transgenosis () " do not include it is (for example, only non-by conventional plant breeding method The hybridization of transgenic organism) or (for example, random to intersect insemination, non-recombinant virus infection is non-by the event of natural generation Recombinant bacteria conversion, non-recombinant swivel base and spontaneous mutation) and the genome (chromosome or dyeing are external) of introducing changes.

Plant " strain ", " kind " or " strain " is made up of the plant individual with identical parent (parentage) Colony.The plant for belonging to a strain is typically to a certain extent inbreeding, and general on most of genetic locis It is homozygosis and homologous." subbreed " can refer to the inbreeding subset of the offspring from common ancestor, these offsprings in heredity with come Can distinguish from offspring's subset of germanus other similar inbreeding.In some embodiments, can be by F₃-F₅Dai Jin The seed from single vegetable lamb is handed over, until remaining isolated genes seat is homozygosis on great majority or full gene seat Producing " subbreed ".

What industry cotton variety was typically so produced：It is miscellaneous from this by crossing controlled between the different parent of 2 heredity The F of friendship₃To F₅Single plant self-pollination, by produce offspring aggregation (aggregating) (" collecting (bulking) ") and .Although the usual seemingly homogeneous of such kind, it is final (for example, from the self-pollination kind of selected plant To F₈On behalf of only) can become the mixture of homozygote plant as described below, it is any to select initial in these homozygote plants F₃-F₅May all there is variation for the phenotype at the locus of heterozygosis in plant.In the embodiment described herein, by coming Come from selected F₃To F₅The individual seed sample of the self-pollination progenies of plant carries out Genotyping and produces multiple based on mark The subbreed of thing, these subbreed based on the quality mark thing polymorphism at one or more specific gene seats on DNA level each other It is different.Such seed sample can be as seed, or as the direct receptor gene parting of plant tissue from seed growth. In some instances, collecting in has the seed of homologous genes type at one or more special sign thing locus, so as to produce The homogeneous subbreed of heredity at one or more locus chain with proterties interested (such as RN resistances).

" breeding strain " or " breeding strain " is referred to be carried out seed selection to excellent agronomy performance and selects (generally by many pollings Select) obtained from strain excellent on agronomy.Breeding cotton strain has a lot, and is well known by persons skilled in the art. Breeding colony is a collection of breeding strain or the individuality from breeding strain, can be used to represent this area in given crop species (example Such as, cotton) available agronomy fine breed gene type in terms of state-of-art.Similarly, breeding germplasm or breeding germplasm strain It is the excellent germplasm of agronomy.Breeding germplasm can be obtained from the plant with excellent agronomy performance, and can be used in producing Plant with excellent agronomy performance, such as cotton of existing or newly developed breeding strain.

Relative with breeding strain, " external strain " or " external strain " (or " Exotic Germplasm ") refers to available good from being not belonging to Strain or germplasm that the cotton of the strain that grows cotton or germplasm strain obtains.In the linguistic context that two vegetable lambs or germplasm hybridize, outward The breeding germplasm for carrying out germplasm and being hybrid with it is not by birth closely near edge.Most commonly, select Exotic Germplasm be in order to During new genetic elements (for example, allelic form interested) are incorporated into into the procedure of breeding.

" ancestors' strain " refers to as or already functions as the source of genetic stocks, for example, develop the hereditary material of breeding strain The parent system in material source." ancestor group " refers to such a group ancestors, for developing the main body of the hereditary variation of breeding strain It is to be contributed by them." offspring " is the breeding that may be separated by many generations between the offspring of ancestors, and offspring and its ancestors.Example Such as, breeding strain is the offspring of its ancestors.Pedigree can be used to describe the relation between offspring and each of which ancestors.Pedigree can be across More a generation or many generations, therefore pedigree can describe offspring with it at a distance of 1,2,3,4, or the relation of the ancestors in more generations.

Proterties or phenotype：Term " proterties " and " phenotype " are used interchangeably herein, and indicate measurable or observable Heritable feature.In some instances, phenotype can be controlled directly (that is, monogenic character) by individual gene or genetic loci. In other examples, phenotype is probably the result (complex character) of multiple gene interactions.Therefore, QTL can be by single base Act on because of mechanism or by polygenes mechanisms play.In some instances, proterties or phenotype can be assigned " phenotypic number ", and its is right The quantitative value that the Ying Yuwei phenotypic characters are measured.

Term " molecular phenotype " can refer to the phenotype detected in the level of the colony of (one or more) molecule. In some examples, molecular phenotype can be detected in molecular level.The detectable molecule of phenotype can be nucleic acid (for example, Genomic DNA or RNA)；Protein；And/or metabolin.For example, molecular phenotype can be the table of one or more gene outcomes Up to spectrum (for example, in the moment of development of plants, or response environment condition or stress).

Proterties or phenotype：Term " proterties " and " phenotype " are used interchangeably herein.For the purpose of the disclosure, especially sense The proterties of interest includes the important proterties of agronomy, the proterties that for example can be expressed in crop plants.

Reniform nematode (RN) resistance：For the purpose of the disclosure, proterties of special interest is " reniform nematode resistance ".It is a certain Whether vegetable lamb or germplasm can be determined comprising RN resistances by any one of various methods known in the art, example Such as, one or more plants are measured or the RN in germplasm plant reappears the determination techniques of (reproduction).

IV. in cotton reniform nematode resistance mark

Embodiment of the present invention includes resisting sex-linked mark with reniform nematode, such as by tetraploid sea island cotton Resist with reniform nematode in the cotton that genotype print plus cotton (Inca Cotton) GB713 are produced with the hybridization of upland cotton sex-linked Mark.These marks can be used for, and for example but be not limited only to, and identification has the bigger vegetable lamb that may include RN resistant phenotypes And germplasm；Select these vegetable lambs and germplasm (for example, in mark assisted Selection program)；With identification and selection without more Vegetable lamb and the germplasm of RN resistant phenotypes may be included greatly.Worked as using one or more marks as herein described and this area Front available composition is compared with method, can be educated for plant in terms of the time involved by cotton breeding, cost and labour Kind person provides advantage.For example, compared with currently available mark for this purpose, one or more marks as herein described Thing can provide more excellent result in the mark assistant breeding of cotton RN resistances.

Disclosed herein is specific mark, they are accredited as being located at the RN resistance masters of the chromosome of cotton gene group the 21st Within secondary QTL regions on QTL regions or the 18th chromosome or near, they are polymorphism in parent genotype.These There is the special sign thing allele chain with cotton RN resistant phenotypes in QTL marks.In some embodiments, with cotton Select in the mark subset that the chain QTL marks of middle RN resistant phenotypes are provided from table 2-4.For example but it is not limited only to, with The chain main QTL marks of RN resistant phenotypes can be selected from the group in cotton：DASCTP_4812_64；DASCTP_60046_ 46；DASCTP_60046_472；DCTE1_261396_78；DCTE1_278814_262；DCTE1_214911_694；DCTE1_ 233925_865；DCTE1_232591_126；DCTE1_365870_69；DASCTP_39375_356；DCTE1_240643_ 347；DCTE1_208529_965；DCTE1_271930_223；DASCTP_59246_191；DASCTP_28910_164； BNL3279；DASCTP_1656_527；DASCTP_51689_504；BNL4011；DCTE1_214869_124；DASCTP_ 8602_496；DASCTP_8602_418；GH132；And DASCTP_10515_556.In some embodiments, the QTL marks Locus that will thing can be selected from table 2-4 and with the mark of any locus linkage listed in table 2-4, except mark Thing DASCTP_4812_64；DASCTP_60046_472；DCTE1_261396_78；DASCTP_60046_46；DCTE1_ 278814_262；DCTE1_214911_694；DCTE1_233925_865；DCTE1_232591_126；DCTE1_365870_ 69；DASCTP_39375_356；DCTE1_240643_347；DCTE1_208529_965；DCTE1_271930_223； DASCTP_59246_191；DASCTP_28910_164；DASCTP_1656_527；DASCTP_51689_504；DCTE1_ 214869_124；DASCTP_8602_496；DASCTP_8602_418；DASCTP_10515_556；BNL3279；BNL4011； Outside GH132.

Target group can be used to determine and RN resists sex-linked mark.In some embodiments, such positioning group Body can come from the hybridization of GB 713x RN sensitiveness Upland Cottons, but it is also possible to/other colonies can also be used.Can make Determine chain marker gene seat with any suitable software platform.For example but it is not limited only to, can makes in instantiation WithQTL drawing appliances,With In some embodiments, such as when the software used in linkage analysis, reflect that the data of the allelic information for detecting can With during use or before use by electric transmission or Electronic saving, such as in computer-readable medium.

In some embodiments, reflected by detecting multiple mark allele in the first vegetable lamb or germplasm Surely the first vegetable lamb or the germplasm of RN resistant phenotypes may be included.For example but it is not limited only to, specific embodiment includes using In identification may include RN resistant phenotypes plant or germplasm method, wherein with RN resist sex-linked mark allele from Detect in following molecular marker：DASCTP_4812_64；DASCTP_60046_46；DASCTP_60046_472；DCTE1_ 261396_78；DCTE1_278814_262；DCTE1_214911_694；DCTE1_233925_865；DCTE1_232591_ 126；DCTE1_365870_69；DASCTP_39375_356；DCTE1_240643_347；DCTE1_208529_965；DCTE1_ 271930_223；DASCTP_59246_191；DASCTP_28910_164；BNL3279；DASCTP_1656_527；DASCTP_ 51689_504；BNL4011；DCTE1_214869_124；DASCTP_8602_496；DASCTP_8602_418；GH132； DASCTP_10515_556；And its equivalent.In some embodiments, sex-linked mark allele is resisted with RN under Detect in the person of stating：The marker gene seat listed in table 2-4 and chain with any mark for listing in table 2-4 Mark, except mark DASCTP_4812_64；DASCTP_60046_472；DCTE1_261396_78；DASCTP_ 60046_46；DCTE1_278814_262；DCTE1_214911_694；DCTE1_233925_865；DCTE1_232591_126； DCTE1_365870_69；DASCTP_39375_356；DCTE1_240643_347；DCTE1_208529_965；DCTE1_ 271930_223；DASCTP_59246_191；DASCTP_28910_164；DASCTP_1656_527；DASCTP_51689_ 504；DCTE1_214869_124；DASCTP_8602_496；DASCTP_8602_418；DASCTP_10515_556； BNL3279；BNL4011；Outside GH132.

According to some embodiments, the plant that may include RN resistant phenotypes for identification or the method for germplasm are included from this Detection in a little molecular markers resists sex-linked mark allele more than a kind of with low RN.Specific embodiment includes using The method that the plant or germplasm of RN resistant phenotypes may be included in identification, wherein detecting mark equipotential base from molecular marker Cause, above-mentioned molecular marker resists sex-linked mark chain with least one selected from these marks and RN.

In some embodiments, detected allele is and the positively related allelic form of RN resistances.Or, Detected allele is the allelic form negatively correlated with RN resistances, and in this case, allele can be anti- Select.It is that each mark selects an allele in the case where detection is selected more than a mark allele；Cause This detection 2 or multiple allele.In some instances, mark can comprise more than favourable (for example a, positive correlation ) allelic form；In such example, any one of these favorable allels forms can be selected.

Therefore, multiple mark allele can be simultaneously detected in single plant, germplasm or plant population.So Method example in, can select containing the positive correlation allele for resisting sex-linked mark with RN from more than one Plant or germplasm.In specific example, resist the positive correlation allele of sex-linked mark can with RN from more than one In to be infiltrated target (for example, acceptor) cotton planting.It will be understood by those within the art that in same plant or germplasm from More than (and/or infiltration) is selected in a RN Resistance QTL mark simultaneously, positive correlation allele can be in plant or germplasm Produce additional (for example, collaboration) phenotype.

Although specific mark allele can be isolated with RN resistant phenotypes, these marker gene seats are not necessarily It is a part for the QTL locus for facilitating (for example, be responsible for) RNA resistances.For example, do not it is required that the mark for isolating is included in rush Into or give RN resistances gene in (for example, as a part for gene open reading frame).Special sign thing allele with Association between RN resistant phenotypes is likely due to isolate between mark allele and RN Resistance QTL allele at this In ancestors' cotton strain that RN resistance alleles are originated caused by original " coupling " chain phase.

Censuring the relation between two genetic elements (for example, facilitating the genetic elements and neighbouring mark of RN resistances) When, " coupling " mutually chain to refer to following situation, wherein on the positive correlation allele at RN Resistance QTLs and same chromosome chain Corresponding chain marker gene seat positive correlation allele physical interconnection.In " coupling phase ", two allele by after The offspring for having held the chromosome chain inherits together.Positive correlation allele in " repulsion " is mutually chain, at gene of interest seat Generally negatively correlated allele physical linkage at (for example, the QTL of RN resistances) and neighbouring marker gene seat, therefore two logical Often positively related allele will not cause hereditary (that is, two locus are each other " not common phase ").

As described herein, the mark allele of " positive correlation " be in target group as herein described with expectation table The mark allele that type (for example, RN resistances) is isolated.However, in view of aforementioned, it will be appreciated that mutually chain due to repelling Possibility, in other embodiments of different groups are related to, can equivalently using the mark of other allelic forms.

Similarly, in some embodiments can further/instead using not isolating with RN resistances Chain mark allelic form, because such allelic form can be used to identify can not possibly include RN resistant phenotypes Plant.For example, such allele can be used to exclude purpose (for example, counter-selection) during breeding, be resisted with RN with identifying Property negatively correlated allele, and/or RN susceptible plants or germplasm are removed from the breeding of subsequent passes.

QTL marks at least have a positive correlation allele, although in some instances, QTL marks can have There are two or more positive correlation allele found in colony.Any positive correlation allele of such mark can use In, for example, identification or build RN resistant cotton strains.In some instances, identification (or penetrating into plant) and RN in plant Resist 1,2,3 or more positive correlation allele of sex-linked unlike signal thing, and the whole of these positive correlation marks Or its subset can forward or backwards be selected during MAS.In some embodiments, plant as identification at least one Or germplasm, it has at least one such and positively related allele of RN resistant phenotypes.

Marker gene seat itself is proterties, therefore can be analyzed by standard linkage analysis, such as by dividing The marker gene seat is tracked during.Therefore, in some embodiments, determine it is chain between mark, for example, 1cM is equal to the first mark allele has 1% chance in the single generation by the way that (it can be any with the second locus Other proterties, (for example, the second marker gene seat), or another is comprising QTL or the character gene being contained in QTL Seat) exchange and separate.

The genetic marker chain with QTL marks (such as the QTL marks provided in table 2-4 and its equivalent), when Their (for example, enough close linkages) close enough with given QTL marks are so that the genetic marker and QTL marks It is useful especially when showing low recombination frequency.In some embodiments, chain mark and QTL marks show about 10% or lower recombination frequency (that is, given mark is within the about 10cM of QTL).According to definition, these linked gene seats Can isolate in the case of at least 90%.Really, mark and QTL marks are closer to the mark is used as anticipant character Index efficiency and advantage it is bigger.However, with QTL at a distance of exceed e.g., from about the mark of 10cM be also it is useful, particularly When combining with other chain marks.

Therefore, in some embodiments, chain locus such as QTL marker genes seat and the second marker gene Recombination frequency between the locus of seat display about 10% or less；For example but it is not limited only to, about 9% or less, about 8% or less, About 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less.One In a little examples, related gene seat (such as marker gene seat and target gene seat, such as QTL) shows about 1% or less restructuring frequency Rate；For example but it is not limited only to, about 0.75% or less, about 0.5% or less, and about 0.25% or less.Therefore, specific In embodiment, each locus can be at a distance of about 10cM；About 9cM；About 8cM；About 7cM；About 6cM；About 5cM；About 4cM；About 3cM； About 2cM；About 1cM；About 0.75cM；About 0.5cM；About 0.25cM；Or it is less.In some instances, specific chain mark can Determined with the genetic map by observation cotton gene group.

In certain aspects, it is chain to be expressed as the recombination frequency limit, or it is expressed as heredity or physical distance scope. For example, in some embodiments, two chain locus are at a distance of two locus less than 50cM map units.At some In example, chain locus is at a distance of two locus less than 40cM.In some instances, chain locus is apart Two locus less than 30cM.In some instances, chain locus is at a distance of two locus less than 25cM.One In a little examples, chain locus is at a distance of two locus less than 20cM.In some instances, chain locus is phase Away from two locus less than 15cM.In some instances, it is chain to be expressed as the scope with upper and lower bound；For example but It is not limited only to, about 10 to 20cM；About 10 to 30cM；About 10 to 40cM；About 0.5 to about 10cM；About 0.1 to about 9cM；About 0.1 to About 8cM；About 0.1 to about 7cM；About 0.1 to about 6cM；About 0.1 to about 5cM；About 0.1 to about 4cM；About 0.1 to about 3cM；About 0.1 To about 2cM；About 0.1 to about 1cM；About 0.1 to about 0.5cM.

In some embodiments, mark as herein described (those marks for for example, illustrating in table 2-4, and with extremely The chain mark of a few foregoing) and RN resistance positive correlations.Therefore, these marks may be with RN Resistance QTLs and/or property Shape is close enough, so that one or more in these marks can be used as prediction of RN resistance traits.Such prediction Ability is exceedingly useful in MAS, there is more detail discussion to this herein.

The use of the symbol thing chain with RN resistant phenotypes and/or QTL marks as herein described is not necessarily only limited In any specific cotton genetic map or methodology.It is noted that because of various factors, the list of chain mark is in figure May be different between figure and methodology and methodology.For example, the mark being placed on any two figure may not be Identical, and a figure may have the mark density bigger than another figure.In addition, the positioning for building genetic map Colony, methodology and algorithm may be different.It will be understood by those skilled in the art that genetic map not necessarily than another The degree of accuracy is greater or lesser, and technical staff will also be recognized that any cotton genetic map is used equally to determine and symbol The chain mark of thing.For example, specific chain mark can determine (for example, real from any genetic map known in the art Test figure or integration map), it is possible to determine from the location data group of any new drafting.

Embodiment of the present invention be not limited only to any specific Cotton Population or using any particular methodology (for example, Any specific software or any specific software parameters group) identifying or determine that symbol thing is chain with RN resistant phenotypes.Mirror In the disclosure, one of ordinary skill in the art can be by the feature deduction of mark described herein to any cotton interested Gene pool or colony, and using any specific software and software parameters when do so.

V. in cotton reniform nematode resistance markers detection

For detecting that (identification) carries the vegetable lamb of the specific allele of RN resistance markers locus or germplasm Method is the feature of some embodiments.In some embodiments, available multiple markers detection scheme in this area Any one is used equally to detect mark allele that this depends on being detected the type of mark.In instances, for indicating The appropriate method of analyte detection can include by, for example, but be not limited only to that PCR, LCR and the amplification method based on transcription are (for example, ASH, SSR detection, rflp analysis and many other methods) amplification and the amplification mark obtained by identifying.

Usually, the detection of genetic marker relies on one or more attribute of nucleic acid.For example, some detection genetic markers The technology of thing using probe nucleic acid with corresponding to the genetic marker nucleic acid (for example, using cotton genomic dna molecule as Template produce amplification of nucleic acid) hybridization.In particular embodiments, crossing pattern, including for example but being not limited only to, solution Phase, solid phase, mixed phase and in situ hybridization are determined, and can be used for allele detection.Extensive guidance with regard to nucleic acid hybridization can be with Find in such as following documents, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid ProbesElsevier,NY.With colony into The corresponding mark of genetic polymorphism can be detected that such method is for example by any one of many methods between member But it is not limited only to, the method based on nucleic acid amplification；With the nucleotide sequencing in polymorphic marker area.In some instances, it is many Detection method (including based on amplification and based on sequencing method) can easily be changed and be suitable for high throughput analysis, for example, lead to Cross and use available high-flux sequence method, such as sequencing by hybridization.

Amplimer for expanding SSR type marker gene seats is included in the specific embodiment of some embodiments. Table 3-4 provides the concrete primer and primer pair for expanding symbol thing described herein.However, those skilled in the art Can immediately recognize that, other sequences on given primer either side can also be used for replacing given primer, as long as the primer can Nucleotide sequence of the amplification comprising allele to be detected.And, the definite probe for allele detection may not Together.For example, can identify any probe of mark amplification subregion to be detected can replace the exemplary probe listed herein. And, the construction (configuration) of amplimer and detection probe can also be different.Therefore, embodiment is not limited only to The primer and probe of concrete citation herein.Although there is provided herein many specific primer examples (being shown in Table 3-4), for the present invention Appropriate primer for example but can be not limited only to any suitable software program design,

Can be include but not limited to by the molecular marker of the available detection of method for building up in this area：ASH or its It is used for the method for detecting SNP；AFLP is detected；Amplification variable sequence detection；RAPD is detected；RFLP is detected；Autonomously replicating sequence System detectio；SSR is detected；SSCP is detected；With isodynamic enzyme marker detection.Although the exemplary mark thing provided in table 2-4 is SNP and SSR marks, but any aforementioned markers type can be used in particular embodiments identifying comprising to cotton The chromosome sections of the contributive genetic elements of RN resistant phenotypes.

For example, the mark comprising RFLP can be by, for example, and (it is typically subfragrnent to make the probe of nucleic acid to be detected Or corresponding to the synthetic oligonucleotide of subfragrnent) with the genomic DNA hybridization of Jing restrictive diges-tions being detected.Selectional restriction Enzyme is providing in Different Individual or colony the restricted fragment of at least two selectable (or polymorphism) length.It is miscellaneous for each Hand over and determine that the restriction enzyme of one or more generation informedness fragment is simple program, there is provided can be with after target DNA sequence Easily realized by those skilled in the art.According to length in suitable matrix (for example, agarose or polyacrylamide) After being separated and being transferred to film (for example, celluloid or nylon), tape label probe can be allowed to cause probe and target Balance hybridizes under conditions of combining, and subsequently removes unnecessary probe by cleaning, and detects the marker probes.

In some embodiments, amplification step is used as a part for the detection/Genotyping of marker gene seat.So And, it is all in all cases necessary to marker detection that amplification step is not.For example, by entering to genome DNA sample Row Southern traces can simply detect nonamplifie genomic DNA.Can also omit in amplification/detection method individually Detection probe, for example but be not limited only to by following manner：Real-time amplification reaction is carried out, when mixing product by amplimer Labeled nucleotide mixes and by monitoring that the molecule of amplicon rotates property relative to not expanding in the modification, the amplicon that are subject to Increase the change (for example, by fluorescence polarization) of precursor to detect the formation of product.

PCR, RT-PCR, real-time PCR and LCR are particularly widely used as to expand and detect nucleic acid (for example, including mark The nucleic acid of locus) amplification and amplification detection method.May refer to regard to the details for using of these and other amplification method Any one of many standard textbooks, including for example：Sambrook is edited, Molecular Cloning-A Laboratory Manual (2000) the 3rd edition, the 1-3 volume, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY；Current Protocols in Molecular Biology (augmenting for 2002) F.M.Ausubel et al., editor, Current Protocols,a joint venture between Greene Publishing Associates, Inc.and John Wiley&Sons,Inc.；With PCR Protocols A Guide to Methods and Applications (1990) Innis et al. are edited) Academic Press Inc., San Diego, CA.With regard to plant Other details of amplifying nucleic acid detection can also see, e.g. Plant Molecular Biology (1993) Croy (editor) BIOS Scientific Publishers,Inc。

The technology of specific amplification in vitro and detection method, including polymerase chain are completed with regard to be enough to guidance technology personnel The technology of reaction (PCR), ligase chain reaction (LCR), Q β duplication enzymatic amplifications and other RNA polymerases mediations is (for example NASBA), additional detail and the example can also see, for example, United States Patent (USP) 4,683,202；Arnheim and Levinson (1991)J.NIH Res.3:81-94；Kwoh et al.(1989)Proc.Natl.Acad.Sci.USA86:1173； Guatelli et al. (1990), ibid；Lomell et al.(1989)J.Clin.Chem.35:1826；Landegren et al.(1988)Science241:1077-80；Van Brunt(1990)Biotechnology8:291-4；Wu and Wallace(1989)Gene4:560；Barringer et al.(1990)Gene89:117；With Sooknanan and Malek (1995)Biotechnology13:563-4.The modification method of large nucleic acids is expanded by PCR, it may be the one of map based cloning It is useful in a little applications, in Cheng et al. (1994) Nature369:684 and its bibliography quoted in have more detailed Description, wherein the PCR amplicons up to 40kb can be produced.

Many available biological textbooks have widely discussion to PCR and related amplification method.Technical staff would recognize that Arrive, substantially any RNA can be transformed into using reverse transcriptase and polymerase (such as by RT-PCR) and be suitable for restricted disappearing Change, PCR amplifications and the double-stranded DNA being sequenced.

In some embodiments, can be with nucleic acid probe nucleic acid of the detection comprising mark allelic nucleotide sequence. Such probe can be with for example, for separation and the chain nucleotide sequence of mark allelic sequences in map based cloning.Can Nucleic acid probe for specific embodiment is not restricted by any concrete size limitation.In some embodiments, nucleic acid Probe can be, for example but be not limited only to, and at least 20 nucleotides are long；At least 50 nucleotides are long；At least 100 nucleotides It is long；At least 200 nucleotides are long.The nucleic acid probe of marker gene seat can be cloned and/or synthesized.

Any suitable label can be used together in instantiation with probe.It is suitable to be used together with nucleic acid probe Detectable include any to pass through spectrum, radio isotope, photochemistry, biochemistry, immunochemistry, electricity The composition of, optics or chemical means detection.Therefore, hybridization probe can using for example, autoradiograph, fluorescence imaging, Or other similar detection techniques are detected, this depends on concrete label to be detected.Useful label includes biology Element (being dyeed with the Streptavidin conjugate of mark), magnetic bead, fluorescent dye, radioactively labelled substance, enzyme and colorimetric marker. Other labels include being incorporated into the part with fluorogen, the antibody of chemiluminescence agent and enzyme mark or specific binding target. Probe can also include radiolabeled PCR primer, for producing radiolabeled amplicon.With regard to the mark of labeling nucleic acid The more information of the tactful and corresponding inspection policies of note may refer to, for example, Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals, the 6th edition, Molecular Probes, Inc., Eugene OR；With Haugland (2001) Handbook of Fluorescent Probes and Research Chemicals, the 8th Version, Molecular Probes, Inc., Eugene, OR (have CD-ROM can use).In specific example, using double labelling Fluorogenic oligonucleotide probe, for exampleProbe (Applied Biosystems) is implemented PCR and is detected and quantitative.

In some embodiments, primer is not labeled, and can be for example, to mark PCR amplicons size being carried out After parsing is (for example, by agarose gel electrophoresis), by the visualization of mark PCR amplicons.In specific example, by inciting somebody to action PCR amplicons are dyeed after size parsing with Ethidum Eremide, can be by corresponding to the difference of unlike signal thing allele The amplicon visualization of size.

Primer used in embodiment is not limited only to the primer of the amplicon that can produce any particular size.For example, Primer for expanding special sign thing locus and allele is not limited only to expand the whole region person of related gene seat.Draw Thing can produce the amplicon of any appropriate length of the length provided in longer or shorter than allele definition.In instances, Amplicon can be, for example but be not limited only to, and at least 20 nucleotides are long；At least 50 nucleotides are long；At least 100 nucleotides It is long；At least 200 nucleotides are long.

For manufacture oligonucleotides and useful composition comprising oligonucleotides (for example, probe, primer, molecular beacon, PNA and LNA) synthetic method it is usually well known to the skilled person.For example, oligonucleotides can be according in example Such as Beaucage and Caruthers (1981) Tetrahedron Letts.22 (20):Solid phase described in 1859-62 is sub- The chemical synthesis in addition of phosphamide ester process.These methods can adopt the synthesizer of automation, for example but be not limited only to, Needham-VanDevanter et al.(1984)Nucleic Acids Res.12:Described in 6159-68.Oligonucleotides (including the oligonucleotides through modification) for example but can be not limited only to, The Midland with ordering from many commercial sources Certified Reagent companies；The Great American Gene Company；ExpressGen Co., Ltds；With Operon Technologies Co., Ltds.Similarly, PNA can be ordered from any one in many sources, for example but not only It is limited to, PeptidoGenic；HTI Bio-Products Co., Ltds；BMA Biomedicals Ltd(U.K.)；With Bio.Synthesis Co., Ltds.

In some embodiments, it is possible to use computer implemented method detects mark allele.For example, can be with Nucleotide sequence comprising marker sequence is stored in a computer.Can be using suitable nucleotide searches algorithm (for example but not It is only limitted in BLAST^TMMiddle offer), or or even simpler word processor (word processor) identifying expectation Marker gene seat sequence (or its homologue).

In some embodiments, mark allele detected with the detection method of PCR-based, wherein comprising this The size of the PCR amplicons of mark and sequence indicate the presence or absence of special sign thing allele.In some instances, PCR primer hybridizes with the conserved region of polymorphic marker area both sides.The primer for being so used for amplifier molecule mark is sometimes referred to as " PCR marks " or referred to as " mark ".

A mainspring of molecular marker is to improve to plant by mark assisted Selection (MAS) in exploitation crop species The possibility of thing breeding efficiency.Can utilize with proterties interested or the genetic marker of gene linkage to identify such plant Thing：They include desired mark allele at one or more locus, it is therefore contemplated that can be by desired mark Allele passes to their offspring together with proterties interested or gene.Genetic marker can be used for identification in a base Contain the plant of specific gene type because of seat or at multiple non-chain or chain locus (for example, haplotype) places.It is similar Ground, can by mark allele as herein described penetrate into any desired cotton genetic background, germplasm, plant, strain, In kind etc., as one of the overall MAS procedures of breeding for being designed for raising output of cotton or general improvement cotton variety Point.

According to some embodiments, mark as herein described provides a kind of identification and includes RN resistances (or RN neurological susceptibilities) Vegetable lamb and germplasm means：Identification is at certain locus (locus as illustrated in table 2-4) place comprising specific Allele plant and germplasm, and the marker gene seat chain with aforesaid at least one.By identification lack with The plant of the mark allele that RN resistances are isolated, can identify RN neurological susceptibilities plant and germplasm (or to RN infection and/ Or plant of the injury with relatively low resistance), for for example it to be excluded from subsequent hybridization and breeding.

According to aforementioned, embodiment of the present invention include with significant probability with contribute to or give RN resistant phenotypes The molecular marker that QTL is isolated.These QTL marks can be in the mark assisted Selection of anticipant character (such as RN resistances) Middle application, and with other purposes.Embodiment of the present invention be not limited only to it is any be specifically checked or analyzed these mark The method of thing.

Using data mining and bioinformatics tools, comparison dna sequence information is same with available in each species gene type Source sequence typically facilitates discovery SNP marks.The sequence that is usually directed between homologous sequence of SNP marks exploitation compare or In reference gene group position, Multiple sequence alignments, for SNP find data mining, it is assumed that SNP positions at allele The estimation of frequency, and candidate SNP is selected for verifying.This process is phase for the low diploid species of genetic complexity To simple.However, the false positive rate of SNP detections is with the increasing of genome complexity, Genome Size and Ke get sequence informations Plus and increase.

For example, multiple plant species (such as cotton) are provided with multiple lists times in long-term evolution in its Matrix attachment region The chromosome of series, therefore facilitate the generation of polyploid plant species.These polyploid species can be further classified Into autopolyploid (that is, with similar multiple haploid genome series persons (such as " AAAA ")) and allopolyploid is (i.e., With multiple different two sub-gene group series persons (such as " AADD ")).Cotton is a kind of allotetraploid, with A and D sub-genes Group.Therefore, SNP has found the challenge with height in such tetraploid crop species, because it is polyploid, genome Complexity is high, and genome size is big.With 52 chromosomes, the genome of 2500MB, they are averaged distribution to tetraploid cotton Into 13 pairs of A and D genomes.Hendrix&Stewart(2005)Ann.Bot.95(5):789-97.

SNP detects program, such as AutoSNP (Batley et al. (2003) Plant Physiol.132:84-91), Gene frequency is usually used as the tolerance of identification candidate SNP.However, only with such method possibly for polyploid (such as cotton) is simultaneously unreliable, because they have a large amount of paralog sequences in its genome, and then causes high SNP to examine Survey false positive rate.This needs exploitation to can be used in big and high complexity genome and lower new of vacation SNP verification and measurement ratio risks Strength SNP detects pipeline.

In view of it is aforementioned, detect the efficiency of SNP from homologous sequence and reduce due to homologous to increase in cotton and mustard High false positive rate caused by genome, some embodiments use " HAPSNP " pipeline.In particular embodiments, can with The SNP of presumption is searched in the contig nucleotide sequence that high stringency is generated, thereby in different cotton genotypes (such as from difference Genotype inside cotton species or species) in generate haplotype and gene frequency, by paralog/homeologous SNP makes a distinction with homologous SNP.Haplotype cluster pipeline can be using assembly algorithm (assembly algorithm) (for example, CAP3；Huang&Madan(1999)Genome Res.9(9):868-77), SNP mining algorithms (for example, QualitySNP； Tang et al.(2006)BMC Bioinformatics 7(1):438), as SNP calling module (calling Module), SNP filtering modules, haplotype calling module (calling module) and SNP Format Series Lines modules The part of (formatting module).The sequence produced from upland cotton and sea island cotton genotype has been used to demonstrate this Pipeline.

In haplotype cluster pipeline, SNP can be classified into the true SNP of (1) from individual gene seat；(2) one SNP homologous in heterologous but other genotype in individual genotype；(3) equal paralog/homeologous in each genotype SNP.Real SNP marks and homologous in heterologous but other genotype SNP in a genotype are alternatively used for base Because of parting and positioning purpose.Contig information can be used to produce the SNP marks with flanking sequence information, for being directly used in The design of high flux Genotyping platform assay, for example but is not limited only to,With (Illumina) determine.In some instances, HAPSNP pipelines can realize higher checking rate (validation rate), This aforementioned three kinds of different types of SNP partially due to it expeditiously can classify on computers.

VI. reniform nematode resistance markers are penetrated in cotton

As it was previously stated, the identification of vegetable lamb or germplasm, the vegetable lamb or germplasm are comprising chain with RN resistant phenotypes One or more mark allele, be implement cotton mark assisted Selection provide the foundation.In some embodiment party In case, at least one vegetable lamb, the plant is selected to include at least one with the positively related mark allele of RN resistances；And Can be with vegetable lamb of the counter-selection comprising the mark allele negatively correlated with RN resistances.

Expectation mark allele positively related with RN resistances can penetrate into (such as good with specific genetic background Kind) cotton in, so as to produce the vegetable lamb or germplasm that are infiltrated with RN resistances.In some embodiments, multiple RN resistances Mark can established sequentially or simultaneously be selected and/or penetrated in cotton.The RN resistance marks that can be selected in single plant or germplasm The concrete combination of will thing is not limited, and can be included：Those illustrated in the combination of mark, such as table 2-4；Show in table 2-4 The subset of the mark for going out；Any mark chain with the mark illustrated in table 2-4；Or positioned at dye cotton Autosome 18 and chromosome 21 on the QTL for limiting herein it is interval in any mark.

In embodiments, identify with the ability of the positively related QTL marks allele of vegetable lamb RN resistances and be also The plant with favourable marker gene seat is selected to provide method.For example, can select any identified comprising expectation mark The plant of thing allele (for example, with the positively related mark allele of RN resistances), while this etc. can be lacked with counter-selection The plant of position gene (or comprising allele negatively correlated with RN resistances).Therefore, in particular embodiments, plant first In thing or germplasm after appraisal mark thing allele, penetrating into method includes selecting first vegetable lamb or germplasm, or selects First plant or the offspring of germplasm.In some instances, the vegetable lamb as a result selected or germplasm can plant with the second cotton Thing or germplasm (such as breeding cotton) hybridize, so as to produce the expectation comprising the mark allele and the second plant or germplasm The offspring of feature and/or allele.

In some embodiments, penetrating into the method for RN Resistance QTLs may include, for example, there is provided at least one and RN resistances Chain mark (mark for for example, isolating with RN resistances)；In the first vegetable lamb comprising RN Resistance QTLs or germplasm Middle determination mark allele；With the mark allele is penetrated in the second vegetable lamb or germplasm, so as to produce Vegetable lamb or germplasm that raw Jing penetrates into.In particular embodiments, the second vegetable lamb or germplasm are planted with first cotton It can be susceptible that thing or germplasm mutually compare RN infection and/or injury, and vegetable lamb that Jing penetrates into or germplasm and second cotton Flowering plant or germplasm are compared comprising RN resistances.As hereinafter will be discussed in greater detail, by these and other embodiment The vegetable lamb or germplasm that the Jing of generation penetrates into is also included within embodiment of the present invention.

In some embodiments, when by provided herein is any one method produce vegetable lamb or the germplasm that Jing penetrates into When, the vegetable lamb or germplasm that Jing penetrates into the ability of reniform nematode infection and/or duplication can be supported with the plant or germplasm come Characterize.For example but it is not limited only to, after with～2,000 adults and young reniform nematode inoculation plant about 6 weeks, what Jing penetrated into Plant or germplasm can include 138 to 24,806 nematodes.Resistance level is categorized into using the RN countings of gained resistance (<250RN), resistance (>251 Hes<1500RN), weak resistance (moderately resistant) (>1501 Hes<9200RN), and Neurological susceptibility (>9200RN).(for example educated by standard in desired genetic background except selected mark allele is penetrated into Kind of method) so as to RN Resistance QTLs are penetrated in background outside, in some embodiments, it is possible to use transgenic method is produced Raw RN resistant cottons plant and/or germplasm.In some embodiments, with cotton at least one mark as herein described During chain exogenous nucleic acid (for example, gene or ORFs) be directed into target plant or germplasm.For example, with this paper institutes The chain nucleic acid coding sequence of at least one mark stated can be cloned (for example, by figure position gram from cotton genomic dna It is grand) and be incorporated in target plant or germplasm.

Therefore, specific embodiment includes the method for producing vegetable lamb or germplasm comprising RN resistant phenotypes, wherein During the method includes for exogenous nucleic acid being incorporated into target vegetable lamb or its offspring, the wherein exogenous nucleic acid and nucleosides as described below Acid sequence is substantially the same：At least one positive correlation mark on the nucleotide sequence and one or more marker gene seats Allele linkage, the positive correlation mark allele is chain with RN resistances.In some instances, marker gene seat can To be selected from：Those (those for for example, illustrating in table 3-4) illustrated in table 2-4, and with aforesaid at least one chain (example Such as, show recombination frequency less than mark 10%).In some embodiments, it is possible to use multiple chain mark structures Build genetically modified plants.Which mark as herein described used in this multiple chain mark practitioner tailoring scope it It is interior.

Any one in many methods is used equally to provide exogenous nucleic acid in vegetable lamb or germplasm.In some embodiment party In case, nucleotide sequence by map based cloning separate, and by with it is chain with the positively related mark allele of RN resistances Identified.For example, nucleotide sequence can correspond to following ORFs (ORF), its coding table in vegetable lamb Up to when can cause or facilitate cotton that there is the polypeptide of RN resistances.The nucleotide sequence can subsequently be incorporated into exogenous nucleic acid molecule In.The definite composition of exogenous nucleic acid can be with difference.For example, exogenous nucleic acid can include expression vector, and it is introducing the external source The expression of the nucleotide sequence is provided in the plant of nucleic acid.

Resist sex-linked mark that the method comprising mark assisted Selection can be utilized to penetrate into RN (for example, thereby to ooze Enter RN resistant phenotypes) in vegetable lamb or germplasm.In embodiments, MAS using be accredited as with notable likelihood with The polymorphic marker that RN resistance traits are isolated is carrying out.These marks (those for for example, illustrating in table 2-4) are deduced For be positioned at facilitate plant RN resistances (with comprising wild type gene plant compared with) gene internal or near.These marks May be considered the indicant of the proterties, it is possible to referred to as QTL marks.In embodiments, test plants or germplasm be extremely The presence of positive correlation allele in a few QTL mark.

In embodiments, determine which polymorphic marker's allele shows using linkage analysis aobvious with statistics The likelihood of work is isolated with RN resistant phenotypes.Identify with after the positively related mark allele of RN resistant phenotypes, The mark can be used to carry out plant lines fast and accurately RN resistance alleles screening, without making plant growth Jing Cross whole life cycle to wait phenotypic assessment.And, the identification of mark allows to carry out specific RN resistance alleles Heredity is selected, even if in the case where the molecular identity of actual RN Resistance QTLs is unknown.Can be from the offspring cotton produced by hybridization Flowering plant obtains small tissue samples (for example, from the first piece leaf of plant), and is screened with suitable molecular marker.Mat This, can quickly determine whether the offspring should be advanced in further breeding.Chain mark can also be eliminated may shadow The impact of the environmental factor of phenotypic expression is rung, so as to allow to select RN resistant cottons in the way of environment neutrality.Therefore, although respectively Plant and see and may obscure with the use of mark described herein on contribution surface of the environmental factor to plant to the neurological susceptibility of nematode, But a special advantage of actually these marks is exactly that their application is independent of environment.

In some embodiments comprising MAS, polymorphism QTL marker gene seat can be used to select containing a kind of or many Plant the plant with the positively related mark allele of RN resistant phenotypes.For example, can be in the biology from the plant to be selected Nucleic acid corresponding with marker nucleic acid allele is detected in sample.Such detection can take probe nucleic acid and mark etc. Position gene or its amplicon hybridization form (for example, using allele specific hybridization, Southern analyze, Northern analyses, in situ hybridization, and primer hybridization subsequently carry out PCR amplifications to indicating object area).Demonstrating biological sample After middle presence (or not existing) symbol thing allele, the plant is selected, and in some instances, can be by the plant For producing progeny plants by selection breeding.

During vegetable lamb breeding, RN resistance markers locus can be with other anticipant characters (for example, high yield) Mark/gene merge, with the cotton variety of development and improvement.By non-molecular method, (for example, the proterties in vegetable lamb is commented Estimate) a large amount of samples of screening are usually costly, time-consuming, and unreliable.The as herein described and chain polymorphism of RN Resistance QTLs The application of mark provides a kind of effective method to select desired kind in the procedure of breeding.Relative to land for growing field crops assessment, The advantage of the mark assisted Selection of RN resistances includes that for example, any moment that MAS can be in 1 year is carried out, with Growing season It is unrelated.And, as previously described, ambient influnence is substantially unrelated with mark assisted Selection.

(for example, resist with RN sex-linked multiple with one or more linkages of characters marker gene seats when multiple in colony Mark) separate when, MAS is relative to the in hgher efficiency of phenotypic screen, because all of marker gene seat can be in laboratory In it is evaluated together from single DNA sample.In specific embodiments of the present invention, the multiple marks illustrated in table 2-4, with And the mark chain with aforesaid at least one, can be from single sample, or from multiple parallel samples simultaneously or sequentially Ground is determined.

Another kind applications of the MAS in plant breeding is to aid in recovering recurrent parent genotype by back cross breeding.Carry out The purpose of backcrossing is typically by one or a few mark or QTL locus from donor parents (for example, comprising desired RN The parent of resistance markers locus) penetrate into from backcross parent (for example, the cotton strain of script high yield), in its other party In the preferable genetic background in face.The backcrossing round for completing is more, and recurrent parent is bigger to the Genetic Contributions for eventually penetrating kind. In some examples, many wheel backcrossings can be carried out, for example, because RN resistance plants are because of such as low yield, low setting percentage Etc. (fecundity) it is undesirable.By contrast, the strain that the intensive procedure of breeding is produced may have the yield of breeding, educate Property etc., also RN can be infected compared with recurrent parent and/or be injured more resistant.From donor source (its may or not Can be that breeding kind as samsara strain contributes excellent genetic background) special sign thing mark auxiliary backcrossing in, from Industry personnel can select the backcross progeny with the donor mark, then utilize as many as possible with the backcrossing repeatedly of samsara strain Ground rebuilds the genome of samsara strain.

According to above, the mark that mark as herein described and method can be used for the cotton variety for instructing as described below is auxiliary Help selection or breeding：It has the complement (series) of the allelic form of desired chromosome sections, the chromosome segment Section and excellent agronomy performance (for example, RN resistances, and any other obtained mark for yield, disease resistance etc.) phase Close.Any described mark allele can be by penetrating into (for example, by conventional breeding, by conversion, or both) In being incorporated into cotton strain, to produce the vegetable lamb with excellent agronomy performance.If the nucleic acid from plant is to desired Genetic marker allele is positive, then in some embodiments the plant can inseminate to produce with same base certainly Because of the pure breeding system of type, or can by it with comprising identical mark allele or other expect marks and/or feature Plant hybridization, to create the hybridization generation of sexual hybridization.

In some embodiments, the method for the present invention is applied to the vegetable lamb of at least one relationship, for example, come autonomous The ancestors of topic vegetable lamb pedigree or the relationship vegetable lamb of Progeny Lines Derived, so as to follow the trail of desired RN resistance alleles Heredity.Can be to limit for example but not only according to the algebraically being separated by between the vegetable lamb that the method for these embodiments is applied to In 1-20 generations；1-5 generations；With 1,2 or 3 generations.For example, this method can apply to the direct offspring or parent of vegetable lamb (i.e., It was separated by for 1 generation).

Genetic diversity is important in the procedure of breeding.In the case where diversity is limited, when all favourable equipotentials When gene has been fixed in breeding colony, the genetic gain realized in the procedure of breeding eventually arrives at platform.Therefore, plant educates The purpose planted is that diversity is included in breeding pond without losing the genetic gain having been carried out, and using as far as possible Few investment.MAS can point out which genome area and which is selected from the favorable allels of original ancestry And retain with the time, so as to help people to merge favorable variation from Exotic Germplasm source (parent unrelated with fine breed gene pond), with Phase finds the effort of the currently non-existent favorable allels in fine breed gene pond.Therefore, in some embodiments, herein Described mark can be used for the MAS of the hybridization for being related to (breeding x is external) cotton strain, and to separating offspring MAS is implemented, to keep Main yield allele and specific RN resistance markers allele.

As it was previously stated, in some embodiments can be with molecular marker locus as herein described and allele (those for for example, illustrating in table 2-4, and the mark chain with aforesaid at least one) identify RN Resistance QTLs, then can be with RN Resistance QTLs are cloned by familiar program.These RN resistance clones can first by itself and genetic linkage as herein described It is identified.For example, " figure position gene cloning " is limited containing RN Resistance QTL genes using the physical proximity of RN resistance markers Separation chromosome segment.The detached chromosome segment can be produced by these well-known methods, for example but not only It is limited to, with one or more restriction enzymic digestion chromosomal DNA, using PCR and any suitable replacement amplified reaction amplification dyeing Body area.Subsequently the fragment that Jing digests or expands can be connected in the carrier for being suitable for replicating and/or express institute's Insert Fragment. ORF related to phenotypic character neighbouring mark can be special with DNA clone (for example, from the clone of genome dna library) Property hybridization identifying the clone for having the ORF (or fragment of ORF) above.If mark is farther with RN Resistance QTL genes, can To identify the fragment containing the ORF by the screening and separation of many wheels continuous to clone, the clone collectively constitutes (comprise) a continuous DNA sequence dna.This process so-called " chromosome walking ", and can be used to producing " contig " or " contig figure ".

The scheme that be enough to the guidance technology personnel separation clone related to chain mark may refer to, for example, Sambrook et al. (editor) Molecular Cloning:A Laboratory Manual, second edition, the 1-3 volume, Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,1989；With Ausubel et al., compile Volume, Current Protocols in Molecular Biology, the 2nd chapter, Greene Publishing and Wiley- Interscience,NY,1995。

VII. the plant of reniform nematode resistance markers is included

Some embodiments are included for manufacturing the method for vegetable lamb, and further include these vegetable lambs itself. In particular embodiments, such method can include making the first parent vegetable lamb hybridize to produce with the second vegetable lamb Cotton wool plant generations, the first parent vegetable lamb includes at least one with the positively related mark equipotential base of RN resistances Cause.These vegetable lamb offsprings can be determined and resist sex-linked mark allele with RN, it is possible to select desired offspring. These progeny plants or its seed can be used for various uses, including for example but being not limited only to, can be used to produce cotton with commercial distribution Flower；Process to obtain desired cotton production (for example, cotton fiber)；And/or it is further used for the breeding of subsequent round.According to one The vegetable lamb of a little embodiments includes following progeny plants, and it includes at least one allele of mark described herein Form, so that further offspring can inherit and resist sex-linked mark allele with RN.

Some embodiments are included for vegetable lamb of the generation comprising RN resistances (for example, compared with parent vegetable lamb With higher RN resistances) method.In particular embodiments, these methods can include being educated by conventional plant Kind or by the way that the foreign DNA (such as transgenosis) comprising RN Resistance QTLs is incorporated into into cotton variety or plant in producing this The plant of sample.

In particular embodiments, the method for producing the vegetable lamb comprising RN resistances can be included RN resistances QTL is incorporated into comprising root-knot nematode (RKN from sea island cotton；Meloidogyne incognita) resistance upland cotton in.At some In example, the presence of RKN resistances in the vegetable lamb comprising RN Resistance QTLs can cooperate with the RN resistances for increasing the vegetable lamb； The RN resistant phenotypes observed i.e. in such plant can resist than the RN in the similar vegetable lamb not comprising RKN resistances Property is bigger.

In some embodiments, compared with non-resistance control vegetable lamb, the vegetable lamb comprising RN resistances can be right Reniform nematode infects and/or injures and shows relative (comparative) resistance.Control vegetable lamb possibly removes what is discussed The external heredity of disease resistance allele is upper, and similar (for example, it may have as the vegetable lamb comprising RN resistances Parental breed).The relevant antagonism of this kind of plant can by under similar conditions equally or approximately equal ground be exposed to Reniform nematode infects to determine.

Therefore, some embodiments include the host cell with nuclear transformation corresponding with RN Resistance QTLs or organism, it Be to resist sex-linked mark to identify with RN using as herein described at least one.In some instances, these nucleic acid can With including coding to the chromosome interval (for example, genomic fragment) of the contributive expression product of RN resistant phenotypes of cotton, ORF, and/or cDNA.

Host cell can (for example, cloning vector, shuttle be carried with the carrier comprising the ORF chain with RN resistance markers Body or expression vector) carry out genetically engineered (for example, transduction, transfection, conversion etc.).Carrier includes, for example but is not limited only to, matter Grain, phasmid, agrobacterium, virus, naked polynucleotides (linear or ring-type)；With the polynucleotides being coupled.Many carriers can be by In being incorporated into bacterium, especially for breeding and the purpose for expanding.

Carrier can be incorporated into the plant of plant tissue, culture by any one in multiple standards method known in the art In thing cell and plant protoplast, including for example but being not limited only to：Electroporation (From et al. (1985) Proc.Natl.Acad.Sci.USA82:5824)；With viral vector such as cauliflower mosaic virus (CaMV) infection (see for example beautiful State's patent 4,407,956)；With little particulate Ballistic penetration (ballistic penetration) (the Klein et comprising nucleic acid al.(1987)Nature327:70)；Using pollen as carrier (pct international patent discloses No.WO 85/01856)；With use Carry Agrobacterium tumefaciens or rhizobiaceae (the Fraley et al. for wherein cloning the T-DNA plasmids for having DNA fragmentation (1983)Proc.Natl.Acad.Sci.USA80:4803).Can adopt in certain embodiments of the present invention it is any can be with height Appropriate method in effect nucleic acid being incorporated into into cell or protoplast, includes but are not limited to the certain party clearly identified herein Method.

The host cell of engineering can be in conventional nutrient medium or the culture in the culture medium of modification, with for example Activation promoter selects transformant.In some embodiments, host plant cell can be trained genetically modified plants.In example As there is the description from the protoplast regeneration plant cultivated in following documents, for example：Evans et al.(1983) " Protoplast Isolation and Culture, " is embodied in Handbook of Plant Cell Cultures 1, MacMillan Publishing Co.,NY,pp.124-176；Davey(1983)“Recent Developments in the Culture and Regeneration of Plant Protoplasts, " Protoplasts, Birkhauser are embodied in, Basel, the 12-29 page；Dale(1983)“Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops, " it is embodied in Protoplasts, ibid, the 31-41 page；With Binding (1985) " Regeneration of Plants, " is embodied in Plant Protoplasts, CRC Press, Boca 21-73 page of Raton, FL, the.Other resources of the useful details with regard to culture plant cell and regeneration can be provided includes Payne et al.(1992)Plant Cell and Tissue Culture in Liquid Systems,John Wiley&Sons, Inc.,NY；Gamborg and Phillips (editor) (1995) Plant Cell, Tissue and Organ Culture； Fundamental Methods,Springer Lab Manual,Springer-Verlag(Berlin Heidelberg NY)；With R.R.D.Croy (editor) Plant Molecular Biology (1993) Bios Scientific Publishers,Oxford,UK(ISBN 0 12 198370 6)。

The conversion plant cell produced using any one in above-mentioned transformation technology can cultivate regeneration and have what is converted Genotype therefore with the full plants of desired phenotype.These regeneration techniqueses are commonly relied on to tissue culture growth culture The operation of certain plants hormone in base, depends particularly on and intracellular killing livestock is introduced together into desired nucleotide sequence Agent and/or herbicide marker.Regeneration and growth course for producing full plants is generally comprised the steps：Select to turn Change body cell and bud；Transformant sprout is set to take root；With seedling is grown in soil.

The Plant Transformation of the nucleic acid nucleic acid of mark described herein (for example, including) that RN resistances are made contribute be able to can be used In species of the conversion in addition to cotton.For example it is contemplated that, when from QTL contributive to cotton RN resistant phenotypes expression produce When thing is converted and expressed in the important plant species of other agronomy and gardening, it is also possible to cause higher nematode resistance.This A little species include dicotyledon, for example but are not limited only to, following section：Pulse family (including pea, beans (beans), French beans, Peanut, Chinese yam beans, cowpea, suede beans, soybean, clover, alfalfa, lupin, nest beans, lotus, daghestan sweet clover, Chinese wistaria and sweet Beans) and composite family.Other can comprising the plant to the contributive nucleic acid of cotton RN resistances (for example, comprising mark as herein described) Being the plant from such as subordinate：Allium, apium, Arachis, Btassica, Capsicum, olecranon Macroptilium, Cucumis, Cucurbita (Curcubita), Daucus, Fagopyrum, Glycine, Helianthus, Lactuca, Lens culinaris category (Lens), tomato genus, clover category, Pisum, mung bean category, potato category, Clover, Vigna etc..

VIII. it is used for the system for detecting and/or associating reniform nematode resistance markers

Also system, including automated system are included in some embodiments, the system is used for identification and includes at least one The plant of the mark chain with cotton RN resistant phenotypes, and/or for by the presence of specific chain mark allele with RN resistances are associated.Example system can include can be used to detect the spy of the allele at marker gene seat described herein Pin；For the detector of the label in detection probe；Suitable fluid operative components and temperature controller, such as mixed probe With template and/or amplification template；And/or be associated mark analyte detection with the presence of symbol thing locus or allele System explanation.

In particular embodiments, there is provided the system for identifying vegetable lamb, the vegetable lamb is predicted It is the vegetable lamb with RN resistances.Such system includes, for example but is not limited only to：One group mark thing primer and/or probe, They be configured to detection at least one with RN resist sex-linked mark (mark for for example, illustrating in table 2-4, and with it is aforementioned At least one chain mark) at least one allele；Detector, it is configured to from the group mark physical prospecting pin or draws Thing or its amplicon detect one or more signal outputs, thereby identify the presence or absence of the allele；And with regard to should The system explanation that the presence or absence of allele is associated with RN resistances.

Performing the system of marker detection and/or association can include detector, and it is configured to detection from one group of mark One or more signal outputs of note probe or primer or its amplicon.The accurate configuration of detector is likely to be dependent on for detecting The type of the label of mark allele.Specific example can include photodetector and/or radioactivity detector.Example Such as, light transmitting or other properties of label probe are detected, may be indicated whether to exist to interact with the probe and (for example, be led to Cross specific hybrid) mark allele.The detector optionally monitors one or more signals from amplified reaction. For example, detector can monitor the optical signal corresponding to " real-time " amplification assay result.

There is diversified signal detector available, including for example but being not limited only to, photomultiplier；Spectrophotometric Meter；Ccd array；Array and Array Scanner；Scan detector；Photoelectric tube and photodiode；Microscope stage；Galvanometer is swept Retouch；With microfluid nucleic acid amplification detection means.In addition to for the mark type for detecting mark allele, detector Accurate configuration may rely partially on the most readily available instrument of user.In some instances can be using detection fluorescence, phosphorus The detector of light, radioactivity, pH, electric charge, absorbance, luminous, temperature or magnetic.

The part class of the system can be depended on according to the exact form of the instruction provided in the system of some embodiments As change.For example, instruction can exist as the systems soft ware in one or more integrated units of system, or they can In to be present in one or more computers or computer-readable medium for being connected with detector operation.In some instances, System command includes at least one reference table, and reference table includes in plant or germplasm the presence of symbol thing allele or not Presence is associated with the qualitative or quantitative resistance level for infecting reniform nematode and/or injuring of prediction.Instruction can also be instructed User interface is set up using the system, for example, to allow user to check sample analysis result and to system |input paramete.

In particular embodiments, system can be included for storing or sending, for example in automation (for example entirely certainly It is dynamic) storage or the part of mechanized data is transmitted in system, the mechanized data is represented or specifies what is be detected Mark allele.For example, computer-readable medium can be provided, it includes caching, hosts and stored memory, and/or its It is used to store electronic data memory unit (for example, hard disk drive, floppy disk and the storage drive of computer code Device).The data of allele that representative is detected by the inventive method can also by network, such as Intranet or internet or Its combination, is transmitted as the computer data signal in transmitting medium by electronics, optics or magnetics.Or/also, system can By wireless, infrared or other available replacement transmission means transmission datas.

In the course of the work, system generally includes sample to be analyzed, such as plant tissue, or from the detached material of tissue Material, such as genomic DNA, the genomic DNA of amplification, cDNA, the cDNA of amplification, RNA, RNA of amplification etc..

In some embodiments, system can be made up of detached element, or can be integrated into single unit, with Facilitate the detection of mark allele, and associate optionally for the extra mark-phenotype that performs.In specific embodiment In, system can also include sample, for example but be not limited only to, from cotton or the gene from selected Cotton tissue Group DNA, the genomic DNA of amplification, cDNA, the cDNA of amplification, RNA, RNA of amplification etc..

The automated system for providing in some embodiments arbitrarily includes the part for sample operation, such as machine People's equipment.For example, automated system can include robotic liquid's control cage (liquid control armature), be used for Solution (for example, plant cell extract) is transferred to into destination (such as from microtiter plate to array base palte), mesh from source Ground can with digital computer operation be connected (for example, in integrated computer system).One feature of automated system Can also be input equipment, it is used to enter data in digital computer, to control the high pass of robotic liquid's control cage Quantity of fluid transfer (and, optionally, control transfer of the control cage to solid support).Many automatic machinery people fluid processing systems System is commercially available.For example, can obtain various automatic from Caliper Technologies Corp. (Hopkinton, MA) Change system, these automated systems utilize various ZYMATE^TMSystem, and generally include robot and fluid treatment module.Class As, it is conventionalRobot, it is used for kinds of experiments chamber system (such as micro titre trays operation), also has It is commercially available, for example can be from Beckman Coulter, InC (Fullerton CA) purchases.As the replacement of Conventional robotic, now Can obtain varied from Caliper Technologies and Agilent technologies (Palo Alto, CA) For perform fluid process and detect microfluid system.

In particular embodiments, the system for molecular marker analysis can include, for example but be not limited only to, and wrap Include the digital computer of high pass quantity of fluid control software；Including the image analysis software for analysis mark substance markers thing data Digital computer；Including the digital computer of data translation software；For solution to be transferred to into the robot of destination from source Liquid control cage (armature)；For (such as controlling high pass to pass through robotic liquid's control cage to system input data Quantity of fluid shift) input equipment (for example, computer keyboard)；With for will from the label signal of marker probes numeral The image analyzer of change.

The light checked and/or record by camera or other equipment (for example, photodiode and data storage device) Image (for example, hybridizing pattern) is learned, can be further processed in any embodiment of this paper.For example but it is not limited only to, These images can be acted upon by being stored by image digitazation and/or on computers to image and being analyzed.Have Various commercially available ancillary equipment and software be available for the digitlization of digitized video or Digital Optical image, storage and point Analysis, for example, by using various computers and programming platform.

Some embodiments also include that can be used for the identification in cotton includes at least one with the chain mark of RN resistant phenotypes The plant of will thing, and/or the kit that the presence of specific chain mark allele is associated with RN resistances.In some realities In example, such kit can include suitable primer or probe, and it is used to detect that at least one resists sex-linked mark with RN Will thing and specific mark allele；And instruction, illustrate how to use these primers or probe in detecting at least one mark Will thing and by the mark allele with prediction to reniform nematode infect and/or injure the qualitative or quantitative level of resistance It is associated.In some instances, kit can be included for packing probe, primer and/or the packaging material of instruction；And control (for example compareing amplified reaction, it includes probe, primer or the template nucleic acid for amplification, and molecular size mark).

In some embodiments, for the identification in cotton comprising at least one and the chain mark of RN resistant phenotypes Plant and/or the kit or system that are associated the presence of specific chain mark allele with RN resistances can include Detect the nucleic acid of concrete SSR and/or SNP QTL marks as herein described.For example, can include can be for system or kit Cotton is nucleic acid-templated to be above polymerized to produce the amplimer pair of cotton mark amplicon by archaeal dna polymerase starting DNA, wherein The mark amplicon is corresponding to the mark being selected from the group：The cotton mark that illustrates in table 2-4 or with aforesaid at least one Individual chain mark.For example, the primer pair that can be illustrated from table 4 is selected special to SSR marks in its equivalent Primer pair.

It is used to illustrate some specific features and/or embodiment there is provided the following examples.It is not considered that these realities Apply the disclosure is limited to example these illustrations specific features or embodiment.

Embodiment

Here exemplified with some embodiments, including：From RN sensitiveness Upland Cottons x prints plus cotton (Inca Cotton) the F of GB 713₂Reniform nematode Resistance QTL is identified in colony；Identification and the mark of the RN resistance trait close linkages Thing；And verify and confirm the chain mark for the mark assisted Selection RN resistance plant in cotton.In these enforcements In example, using passing through(Illumina) is determined from 181 F₂The genotype information obtained in individuality is generated Linkage map with 4062 SNP and 5 SSR marks throughout 26 chromosome.By these F₂Planting is with the kidney shape In the soil of nematode infections, the data related to resistance are collected, be expressed as reduction of the nematode breeding relative to susceptible standard variety. Using corresponding F₂RN resistant phenotype information (that is, the nematodal accounting data from surrounding soil to root) is detecting two RN resistances QTL。

The mark DASCTP_28910_164 (22.43cM) and DASCTP_1656_527 (23.55cM) on chromosome 21 Between observed a main QTL, across 0-32.62cM, maximum LOD must be divided into 13.84.According to 1000 kinds of permutation and combination, It must must be 5.2 to calculate under 4.4, p=0.01 that LOD threshold values are calculated under p=0.05.This QTL explains 29.8% phenotypic variation.

Also between SNP marker DCTE1_214884_1077 (53.99cM) and DASCTP J7253J37 (55.94cM) A secondary QTL is observed on chromosome 18, across 39.17-66.09cM.This QTL explains 7.7% extra phenotype and becomes It is different, it was observed that between.

Therefore, the two QTL contribute to 37.5% of RN nematodes always variation in soil.

KASPar is further transformed into mark^TMIn measure, planted with the new RN resistances of mark assisted Selection with verifying Thing.

Embodiment 1:Material and method

Vegetable material. by print plus a kind of cotton (Inca Cotton) GB713 (short-day sea island cotton) reniform nematode (RN) resistance Genotype produces F with the hybridization of RN susceptible genotypes₁Seed.In next winter, by F₁Plant selfing obtains F₂Generation.

Extracting genome DNA and amplification. using CTAB DNA extraction schemes from individual F₂Plant and the leaf tissue of parent Extract genomic DNA.Kohel et al.(2001)Euphytica 121:163-72.DNA mass is carried out by gel electrophoresis Assessment, uses Quant-iT^TM Quantification kit (Invitrogen, Carlsbad, CA) making according to manufacturer With explanation estimation DNA concentration.DNA concentration is normalized into into 50ng/ μ L, so as to using iSelect (Illumina) determining carries out Genotyping.

Phenotype analytical. in greenhouse research, in the terrine containing pasteurize fine sand loam of 10cm diameters 214 are sprouted Individual F₂, 10 BC₁F₁And parental seed, ratio is 1 seedling of every terrine.Experimental design is the completely random area with 5 repetitions Group.On the sowing same day, 2000 vermiform RN are inoculated with these tanks, containing the young and adult nematode.All basins are as needed Watered and applied fertilizer.After being inoculated with 6 weeks, by calculating vermiform RN to F₂Plant carries out RN resistant phenotype measure.Referring to Robbins et al.(1994)J.Nematology 26:659。

Genotyping is carried out with molecular marker. two groups of iSelect of design(Illumina) determine, make Indicated with the SNP that 454 sequencing exploitations are carried out by the simplified library (PstI and EcoRI) to upland cotton and sea island cotton genotype Data are subsequently analyzed by thing by HAPSNP pipelines.Finally in the first and second iSelectOn chip 5023 and 8040 measure are manufactured altogether respectively.

Using from 181 F₂Genotype and the DNA sample of parent, according to the operation instruction of manufacturer gene point is carried out Type.The marking of genotype cluster is usedCustomization polyploid in v2011.1 software kits (Illumina) SNP scoring methods (custom polyploid SNP scoring method) are completed.Polymorphic marker in target group Genotype interpretation (calls) is subsequently converted at first with four allele with ternary fashion (ternary fashion) marking A (P06x.4433 allele), B (GB713 allele) and H (Heterozygous alleles).Indefinite genotype record is turned Missing data (being represented with dash (-)) is changed into avoid separating pseudomorphism (segregation artifact).Genotype data Be formatted into seat file (locus file) (.loc) so as toIt 4.0 is analyzed.See Van Ooijen (2006)“JoinMap 4,Software for the calculation of genetic linkage maps in experimental populations,”Kyazma B.V.,Wageningen,NL。

Quantitative trait locus (QTL) is positioned. uses6.0(Van Ooijen(2009) 6,Software for the mapping of quantitative trait loci in experimental Populations of diploid species, " Kyazma B.V., Wageningen, NL) QTL is positioned.6.0 require three input files, including locus gene type file, map file and quantitative data file.Gene The seat genotype codes of (.loc files) containing segregating population full gene seat genotype file.Map file by Generate, the estimation figure position containing full gene seat.Quantitative data file (.qua files) is containing the nematode from target group Count and resistance interpretation (call).In .qua files, resistance interpretation is converted into numeric scores from character data type：HR= 1；R=2；MR=3；S=4.Analyzed using the interval between two marks, and with probability logarithm (LOD) score Form calculus QTL be located at the interval in likelihood.When LOD scores exceed remarkable threshold predetermined in linkage group, that is, examine Go out one to separate the position with maximum LOD on QTL, and linkage group is QTL estimated locations on the diagram.

Embodiment 2：Phenotype reniform nematode resistant determination

Although print plus cotton (Inca Cotton) GB 713, a kind of sea island cotton genotype, with photoperiod property, with to root Upland cotton genotype (upland cotton) of the tie lines worm (Meloidogyne incognita) with high-level resistance generates different flower and awards Powder.F is generated by traditional hybridization practice₂Target group.By using～2,000 adults and young line in this colony Collect nematodal accounting data to carry out the phenotypic screen of reniform nematode resistance, nematode meter from soil after worm/basin inoculation is individual 6 weeks Number data represent RN breeding degrees.The number and resistance that phenotypic data contains nematode in each basin is categorized as resistance (HR< 250RN), resistance (251<R<1500RN), common resistance (1501<MR<9200RN) with susceptible (S>9200RN).

During phenotypic screen, the scope of nematode count is 138-24,806.In phenotype test, numerical value is transformed into log₁₀(X+1), so as to by variance criteria.The log of each generation in this research is given in Fig. 1-2₁₀(X+1) frequency of numerical value Rate is distributed and the generation based on nematodal accounting-mean analysis (generation-mean analysis).According to generation-average point Analysis, F₁,F₂, and BC₁F₁There were significant differences for nematodal accounting data between instead of.In BC₁F₁Generation (with susceptible parent backcrossing), averagely log₁₀(X+1) value deflection neurological susceptibility parent.This shows that the heredity of the proterties is by partial dominance.

Two are had to the cotton customization of 13063 SNP marks and 6 simple sequence repeats (SSR) marksDetermine (cotton custom-builtAssays), firstly evaluate them and detect parent This polymorphism between being, is also used for them to generate genotyping information.A total of 4091 marks, including 4086 SNP and 5 SSR, is polymorphism between parent, and allele interpretation is recorded for positioning group with ternary fashion Body.For codominance and dominant mark, most of mark meets 1:2:1 typical F₂Segregation ratio.

With throughout all 26 chromosomes, total linkage distance across 4755.2cM 4062 SNP and 5 SSR mark structures Build genetic linkage map.Average SNP mark density is approximately 156 marks of every chromosome, average between adjacent markers Distance is 1 mark/1.17cM.Table 1.

The SNP marks of table 1.P06x.4433x GB713 genetic maps are covered and linkage distance information

Embodiment 3：QTL is positioned

According to deciding field result, RN resistances are navigated on a main QTL and the chromosome 18 on chromosome 21 Individual QTL.

Chromosome 21 has 182 marks altogether, and across 194.8cM, average mark coverage rate is 1.07cM.It is above-mentioned Main QTL areas are concentrated between 0 to the 32.62cM of the distal chromosome, and maximum LOD must be divided into 13.93, in DASCTP_28910_ Observe between 164 and DASCTP_1656_527.This main QTL explains in resistant phenotype 29.8% variation.Although in dye Two extra QTL LOD peaks are detected on colour solid 21：(i) QTL LOD peak DASCTP_39375_ on chromosome 21 Between 356 (19.33cM) and DCTE1_208529_965 (20.74cM)；(ii) a QTL LOD peaks region is on chromosome 21 Between DCTE1_214869_124 (27.49cM) and DASCTP_8602_418 (28.33cM), they are counted as a main QTL The different piece in area.

Mark, LOD scores and % in the QTL areas related to RN resistances on chromosome 18 and 21 of table 2. is explained.

Main QTL and secondary QTL on chromosome 18 on table 3. and chromosome 21 chain SNP marks and its resistance and easy Sense genotype

The mark chain with QTL on chromosome 21 of table 4.

Mark	Chromosome	Primer	SEQ ID NO.
				BNL3279	21	It is positive	SEQ ID NO：73
BNL3279	21	Reversely	SEQ ID NO：74
				BNL4011	21	It is positive	SEQ ID NO：75
BNL4011	21	Reversely	SEQ ID NO：76
				GH132	21	It is positive	SEQ ID NO：77
GH132	21	Reversely	SEQ ID NO：78

Embodiment 4：SNP for polyploid species detects pipeline

Test and verify that the SNP based on haplotype detects pipeline as pattern species using cotton：HAPSNP pipelines.Use Genome complexity reduces approach, with 454 sequencing technologies to two cotton varieties, a upland cotton species and a sea island cotton thing Kind, it is sequenced.The sequence information for so generating is processed by HAPSNP pipelines, and the pipeline includes following modules：(1) Assembling/positioning；(2) SNP interpretations；(3) SNP is filtered；(4) haplotype interpretation；(5) SNP Format Series Lineses.The figure table of pipeline Show the flow chart being summarized as such as Fig. 5.

Can be directly inputted in the assembling/plot module of HAPSNP pipelines from the sequencing data of cotton, such as Fig. 5 institutes Show.After assembling/positioning (for example, seeing the module 1 of Fig. 5), " output .ace " (or ACE) file can be input to SNP interpretation moulds In block (for example, shown in the module 2 of Fig. 5).Based on sequence relatively determining all possible SNP, sequence compares SNP reading modules It is to carry out in the reference sequences for compare consideration in all of list entries and optionally for sequence.Contig and mark Knowing (identifier) can be included in all SNP, as shown in Figure 5 as the output after SNP interpretations.Then, to these SNP/ contigs carry out SNP filtrations (for example, seeing the module 3 of Fig. 5).SNP filtering modules are it may also be determined that whether SNP is located at poly- In thing area (homopolymer region).After SNP is filtered, false-positive SNP is removed and is input to SNP Format Series Lineses In changing module, as shown in Figure 5.

On the other hand, all possible SNP is input to into haplotype reading module (for example, seeing the module 4 of Fig. 5).Dan Bei Type reading module optionally includes the haplotype filter element independently of SNP filtering modules.Haplotype information can be input into To in SNP Format Series Lines modules, to consider to be associated with genotype after merging with filtered SNP.

Finally, SNP Format Series Lineses module (for example, seeing the module 5 of Fig. 5) makes the SNP after filtration comply with (comply) side Wing sequence and haplotype information (optionally through filtering), so that it is determined that the contig containing high-quality SNP marks is used to survey Examination purpose.

Candidate or true SNP are dialleles, and each genotype supports an allele under the conditions of homologous, at least There are 2 sequences.Half SNP (Hemi-SNP) is triallelic, and a genotype wherein in haplotype cluster is double equipotentials Gene or heterozygosis, another genotype is monoallelic or in homozygous conditions.The SNP of paralog is in gene In type diallele or heterozygosis.True SNP and half SNP can be used for Genes location purpose.Paralog SNP is fixed in heredity Use in position is less.According to the position of candidate SNP locus, flanking sequence letter is obtained at SNP upstreams or downstream 100bp Breath, and be used to determine design with [allele 2 of the allele 1/] information formatization at SNP locus.

It is sequenced by 454 of the simplified library (PstI and EcoRI) to upland cotton and sea island cotton genotype, subsequently passes through HAPSNP pipelines are analyzed to data, and with the SNP marks of gained two groups of iSelect are devised (Illumina) determine.Finally in the first and second iSelect5023 He is altogether manufactured on chip respectively 8040 measure.This research is used for Genotyping purpose using these chips.

The HAPSNP pipelines contribute to identifying high-quality SNP with high checking rate in polyploid species such as cotton.Will be homologous SNP is distinguished with paralog and homeologous SNP and is more prone to than conventional method and more accurate.Such pipeline will provide essence The computer SNP candidates of choosing, for being verified in polyploid species, while reducing the appearance of false positive SNP.

Embodiment 5:For the checking and application of the SNP mark related to reniform nematode resistance of Molecular breeding in upland cotton

The F1 plants for being produced by the hybridization of RN neurological susceptibility Upland Cottons x prints plus cotton (Inca Cotton) GB713 at first Backcross population (BC1F1) is generated with RN neurological susceptibility mixing breeds, so as to RN resistances are penetrated into into Upland Cotton from GB713 In.Using this colony's proof mark thing-RN resistance trait correlation.

Carry out the phenotypic screen of reniform nematode resistance in this colony, collect from～2,000 adults and young line The nematodal accounting data obtained in soil after worm/basin inoculation is individual 6 weeks, the data represent RN breeding degrees.Phenotypic data contains There is the number of nematode in each basin.

Using 6 SNP marks in the main QTL areas of chromosome 21, i.e. DASCTP_39375_356 (19.33cM), DASCTP_ 59246_191(21.87cM),DASCTP_1656_527(23.55cM),DASCTP_51689_504(24.39cM),DASCTP_ 8602_496 (28.05cM), DASCTP_8602_418 (28.33cM) proof mark thing-RN resistance trait correlations.

Table 5. is used to test the SNP marks of mark-trait associations

Chromosome	Mark	Position (cM)
			21	DASCTP_39375_356	19.33
21	DASCTP_59246_191	21.87
			21	DASCTP_1656_527	23.55
21	DASCTP_51689_504	24.39
			21	DASCTP_8602_496	28.05
21	DASCTP_8602_418	28.33

KASPar measure is carried out to SNP marks, and for carrying out Genotyping to BC1F1 colonies.Believed using genotype Breath implements " single analysis of markers (Single Marker Analysis) " with the combination of phenotypic data, uses " Windows QTL Cartographer " programs are carried out.

Single analysis of markers is fitted data in simple linear regression model y=b0+b1x+e.Result below is every Individual mark gives b0, and the valuation of b1 and F are counted.Whether the analysis is frequently used for proof mark thing chain with QTL.So Inspection be by determine b1 it is whether dramatically different with 0.The H0 that F statistics it will be assumed:B1=0 and alternative H1:B1 is not equal to 0 and enters Row compares.Pr (F) is the tolerance that H0 has much supports.Pr (F) is less to represent less to the support of H0, therefore the support to H1 is got over Greatly.Conspicuousness under 5%, 1%, 0.1% and 0.01% level uses respectively *, * *, * * * and * * * * to indicate.Note, likelihood Ratio test statisticses be compare two nestings (nested) it is assumed that and be the negative natural logrithm of likelihood ratio twice.For example, It is assumed that assume that H0 is nested in H1, and they have respectively likelihood L0 and L1.So, " likelihood ratio test statisticses " It is -2ln (L0/L1).- t 1 is the number of analyzed proterties.

Table 6. is used for single analysis of markers of mark-RN resistance traits association

Mark DASCTP_39375_356 (19.33cM), DASCTP_1656_527 (23.55cM), DASCTP_ 51689_504 (24.39cM), DASCTP_8602_496 (28.05cM), DASCTP_8602_418 (28.33cM) is found in In 5% level significantly, and in pr (F) row indicated with *.This confirms that RN resistance traits with the main QTL in chromosome 21 The SNP marks in area are related, and select RN resistance plants by MAS.

Because these marks are identified related to RN resistances by statistical test, we from two hybridization to educating The genotype information of kind of propulsion generation is assessed, to predict phenotype and be pushed further into resistance base in the procedure of breeding Because of the strain of seat.

Using RN resistance markers information, we can be based on resistance markers locus and provide selection for propulsion, So as to help the breeding of RN resistant cottons.Mark information between these candidate gene seats of total figure can be used to develop The new SNP mark related to RN resistances.

Although in order to the purpose being aware and understand has been described in detail to foregoing embodiments, the technology of this area Personnel can clearly realize that by reading the disclosure, can be on the premise of without departing substantially from true scope of the present invention in form and thin Various changes are carried out on section.For example, above-mentioned whole technologies and device can be used according to various combinations.

Sequence table

<110>R Bu Yalapu

R appoints

MG McPphersons

SP khoums Pa Tela

It is sub- together that C-SA sand receives Barcelona vara

JW Leon Spinks

Subphylum is special in K handkerchiefs

<120>Resist sex-linked mark with reniform nematode

<130> 2971-P11485.2 (72022)

<150> US 61/799,059

<151> 2013-03-15

<160> 78

<170> PatentIn version 3.5

<210> 1

<211> 1002

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (501)..(501)

<223>SNP mark DCTE1_217844_513

<400> 1

agatgacgat tccgttcagt caacagaaac agaagggccc attgattctc acgcaacaac 60

gaatgttcaa agttccttac aattggagac caggccaaga cgtcaaactc gaccatcatc 120

tcgtttgaga gattttgagg tgatacaaga tgatgttgtt gatgatgaag gagacatgat 180

tcagtttgca atgtttgcaa atgttgaccc atcatcgtat gaagaagcag cagaagaaga 240

agtgtggtgt aatgcaatga aagaagaaat gttggctatt gaagaaatgg tacatgggac 300

ttggtcgact tacctgctgg aaaagaagca atcggtttga agtgggtttt caaaacaaag 360

taccaagcag atggaaaaat acaaaaatat aaggcacggc ttgtagcgaa agggtaccgc 420

caacaacaag gaattgatta tgaagaaacg ttttctcctg tggtacgttt tgaaatagtc 480

agaattgctc tagcattggc ycgctcaatt gaaatggcct gtttttcaat ttgatgtaaa 540

gtcagcgttt ttaaacggtg agttgaagga agaggttttt gtgtcacagc catatgggtt 600

tgtaatcaaa ggcaaggaga gtaaggtata caagctgaaa aaggctcttt acggcttgaa 660

gcaagcgccg cgagcctggt acagtaagat tgatgcttat tttcaaaatt caggttttgt 720

tagaagtgaa aatgaaccta ctctttatct taaaaacaag cgaatgattt gttactagtt 780

tgtctctatg tagattatat gatttacatg gggtctatct tcttcacttg tttttgattt 840

ttaaagaaaa tatgatgaag tcttttgaga tgacagattt tgggaaagct tcattttttt 900

tctggtgtgg agatttattc aaacaggaga tggggatttt tatatcccaa aagaagtatg 960

cagctgatct cttcgaaggt ttaacatgct aaattgcaac tc 1002

<210> 2

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (101)..(101)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (121)..(121)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_66915_767

<220>

<221> misc_feature

<222> (288)..(288)

<223>N is a, c, g, or t

<400> 2

agctaagtca gcatatatca taatgaaagg gattcgctcc attcttattg ctttaggcct 60

ctttgctttt gcttgctcat cggcctcagc atatgacccc nagtcctctc caggactttt 120

ngtgtagcta tcaaggacat caagaatggc rgtacgcatt caagactttg tgttaatgtt 180

catcctcatg atgttaataa agcatgagct gaacaacatt acgtttcaat gttctaatat 240

aactttttaa catttcttca ttgcagtgtt tgtaaatggt aaattttngc aaagacccaa 300

a 301

<210> 3

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (35)..(35)

<223>SNP mark DCHA20258_34

<400> 3

cagcaactgc aaatacatct ggtgctggtg cttcyatctt ggactcctat attgcggatc 60

catattatgg ccgttattct ggtga 85

<210> 4

<211> 742

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (501)..(501)

<223>SNP mark DCTE1_217106_782

<400> 4

caagaaatat actggaaact gattaaaaac ttgaaaagtt cctaccaatt tcaagaagca 60

ctttgttggc aagacgtaac cggtactttc atcctcagca cgggcaataa tgtctcgcca 120

ttcacaaagc gcctataagc catttaagtg agacataagt caaagtaatg gataccatct 180

atccatctaa ttctgcacat gaaatgtgta ccaaaattta aattggacat ttcttgtggt 240

ttatcaacca ttcgattgtt attttaaaag taaaagggtc aatcagatat gaaaacaact 300

agaacttaca gcaacaattg ctagctgctc agcattgaaa ccaccgccct gcaacctata 360

tcaaatatta caacaaatgc aaagatttag tttgaatata catcagtgat ggaatcagac 420

cggccagtta tactggtgcc tgtctggtcc agtacatacc caaaaaccag ggaaccaatg 480

aacccaaccg gagaaaacta rcttttaact tttcttttgt tatttagtaa aattattttt 540

gtaagtgatg gcatttgaac ttttaaccac ttagtgatat actaaatacc ataccattaa 600

gccaaataca tacattttat aacaaataac tacatttaaa actatttaat atcaaaatca 660

catatttaac atttaattta cattttaatt gaataatcaa gttcaaccag tgaatctgga 720

ggtcacacag gttagacctc ca 742

<210> 5

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (16)..(16)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_42527_351

<400> 5

ggacatggta aaaaanccaa acaagattgt cttttgagcc agacattatg ttgatgtccg 60

caagagaaag agaaagtatt accatctcag ggctaagtgg agaagcaaat gcttggccag 120

ttatttcacc ttcagtaaag attgtaagtt rttgatcgtt tcttaggtgc atggggatct 180

gtcttttgat gagtagcagc caacgaaact atttgttcct gtaggctact ggagcaaccg 240

ttccggttct aaagaaacca agccatggct ttgggtcact tctggggaat gctgcctcaa 300

a 301

<210> 6

<211> 643

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (479)..(479)

<223>SNP mark DCTE1_391589_479

<400> 6

ttctgtgctg atacttagat atgtcccacc agacgaggtg ccacttccgt tatcacttca 60

ggagtcaatt gattcttaca agctgagaca taatagtgat gctcagatga ttaatggaga 120

aaacccagcg aagagtcctg gagatccttg tctgcctttg cttggagaaa agaatgttgc 180

agttgattgt actgttgtta gaaaaaatag aagctctatc cagttgtaag ttccttgcct 240

tatgtctact tttgatatag aaatctattt gctccttcaa tttcccgaaa gcatccttgc 300

ccgaccctca tatgcaatgc atatttggac atggtggggc gtatgattct ccaaatatat 360

gaaaaaattg gagaatatga gaatacttgt gttgggcaca tacatgtatc cgacattagc 420

ctttgagtcc aagtgacaga tacagatctt ataaaatcat tttcattagc agtatacart 480

gtcttggttt gtcacagact ttagagatgt gcttgaaggt ttttccttgc ttttatatgt 540

gcaaactgat gataagatgt aatgtaattg aatattgtga aacttcttat caaggtttca 600

atatgaaatg cttcacagtt atgaacatta ttaccatgga att 643

<210> 7

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (16)..(16)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (115)..(115)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_18966_467

<220>

<221> misc_feature

<222> (275)..(275)

<223>N is a, c, g, or t

<400> 7

aaggatacaa ctagantcac tggaagtttc acgacctgaa gaacctccta ccatggaagc 60

tactgaatcg acattggaaa atatcatact gtttgaagat aatttgaaca aaaanggaaa 120

ccaataatgg taacagtagc ccagaagatg raactatgtc cctatcagaa ttatctacaa 180

gcttccaaca atgcttgcag tccatcaatg atcaaaacag aaacatggtt aaagtcgaga 240

aaccaaaaga agccattgat gttccgcaaa tgaangccat ttgattatga agcagccatg 300

a 301

<210> 8

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (62)..(62)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_2912_350

<400> 8

tacgatgagt cgtcggggat ttgggaagac atatggggtg accatatgca ccatggatat 60

tnacgagccg ggttccgata tttcgggttc agatcatcgt gccgctcaga ttcgaatggt 120

cgaagaatcg ctccgttttg ctggaatatc rggttagttt tctttacata tttttcaata 180

atccacgtgt tagtttctgt tggaagatta gattatgtcg ttgaatttat cgttagcgag 240

agtagtgcca agtccaaaca tataggtgct ctttccaact gttcatgaac tggaatatct 300

g 301

<210> 9

<211> 681

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (501)..(501)

<223>SNP mark DCTE1_214884_1077

<400> 9

tgtatgtatg agaatgagat atgagtgaga gaaagaagag ggaaaagcaa agaagtaggc 60

accactatat tggtttgtta tttatttttc ccttgctttg cgagtgtctt tcttcctgta 120

gaaattagaa accaagtttt ctgagtgtta ggaccctttt tttggcacaa agtcaattgc 180

atctaggaaa agggttgtaa atattgtgtg attctgatgc cagacttgtt tgatattttc 240

ttttaaggtt ggttgcatga gccatatgaa ctaagttctg ggttcatagt ctttttgttt 300

gttttttggt ctttttaagt aacgcattac aggagctata cctatggcat tggcatccat 360

gaaaatttaa tggagagaca attacgtttt tgttgaaaaa atataaatat ttaaagaaaa 420

taatgtagca aaaaagatat aggcaaatgc aactcagcaa agaatgctca aattctagaa 480

aatgaaaaag aaaaatattc rattttcagc agacatgttc tgagagagag aaaagttagg 540

agggaacaga gaaagaaaag aaacaaaaag aaaaagagct aaaataaaga aaaaagagag 600

gaaatgattt ggtggtaatt tgacagaatc gatgtctaac tttgactagt gatattttct 660

ttttgtataa acttcggaat t 681

<210> 10

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (8)..(8)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (31)..(31)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (111)..(111)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (142)..(142)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_58378_518

<220>

<221> misc_feature

<222> (212)..(212)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (227)..(227)

<223>N is a, c, g, or t

<400> 10

gcatgagnca agcgattctc aattctttat naatgggaaa tggctcaaga acattgataa 60

cggtagcagc aacaacctca ttgctaatgc caaccgccct tcctcttcat nctgctgccg 120

cttacatgcg tgacaagttt tngctgtctc ycaagagaag acatgatccg tttcctcatc 180

ggttgccttg gtgcactagc tcctcttccc cntgtcctct atttccntca ctcggagttg 240

ttaacctaaa ctatagctca attgaagcct cccttccagc tgtagaagca actcaaaaac 300

a 301

<210> 11

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (44)..(44)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (52)..(52)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (59)..(59)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_34272_162

<400> 11

ctgatgacgc attgtttccc ttggcaattg ctattgttga cgtnggaaag tngatgaana 60

attggatgtg gttcatgtca gagttgagga agcttcttgg tgtgaacact gaaaacatgc 120

ctagacttac tatactgtct gaaagacagc raggcatggt agatgcagta gaaacccatt 180

ttcctagtgc gtttcatggt ttttgtctac gttatgtcag tgaaaatttc cgtgatacat 240

ttaagaacac aaagttggtt aatatcttct ggaacgctgt ttatgctctc acgactgtag 300

a 301

<210> 12

<211> 407

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (123)..(123)

<223>SNP mark DCTE1_317552_123

<400> 12

aattcattat tcatctatcc atcatattca tcagtttatt caatccatct tccacttttt 60

attttatcgg cttttcactt ttaagaaaac aaacgagaat aaaatatgga gaaaaattac 120

ccyagtcgta caaacctcga atcctctcaa aacaaaatcc ctctctcaaa acaacattct 180

ctctttttat tctttttggt cacacctatt agggttataa tggcctttta aataggctaa 240

gattagagtt ctaatatgac tggaatatct ctggaataat caaagtttga ctgggaaaag 300

taaccctatt aaaagtgata aatgactaaa agtaacacta ttttagcctc attcttaatg 360

ctatttttgg atgtattatt cgatagtaaa atggtagtaa ttttatg 407

<210> 13

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (23)..(23)

<223>SNP mark DCHA31095_22

<400> 13

cagccccggt taaaaagaga atytctcctg ttccagtctc tcctacagac aggcagttac 60

aaagaaagtt accaaagaaa gtaag 85

<210> 14

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (22)..(22)

<223>SNP mark DCHA5179_21

<400> 14

cagcaagacc atgcaatttg grtcatgagg ttgatagttt gacctagaaa aagaggtgtt 60

gaaagatgga catgtctgct acatt 85

<210> 15

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (29)..(29)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (54)..(54)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (71)..(71)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (95)..(95)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_17253_337

<220>

<221> misc_feature

<222> (170)..(170)

<223>N is a, c, g, or t

<400> 15

atatcctttt gctcgtgtac tttcaaaant gttatttgca atggagccac tacnaccaga 60

tgcaaaaatt ngagatcata atggcagaaa attantactg aaataatgca ataagggcat 120

cactaagcaa agagagagac agagaaagac rcacgcgaac tggaaccccn aaagggccaa 180

gagcatcagt gaaatcagag cttggaattg gagtacccca cttaggtatc tttctgcaaa 240

gcattagaaa tagaggttgc atgttacaag acttagctag agttttcaac ttcctggcaa 300

g 301

<210> 16

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (2)..(2)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (12)..(12)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (20)..(20)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (26)..(26)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (38)..(38)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (66)..(67)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_36168_373

<220>

<221> misc_feature

<222> (217)..(217)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (250)..(250)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (268)..(268)

<223>N is a, c, g, or t

<400> 16

gnatttttca antgggctgn acttgnattt tagattantg ttttaattta gtttccattt 60

tggcgnnagg ggggtggttc tgggctgcaa attggacggc aacagtgtgg caggaattgc 120

ttaacctata ggatttttgt tataccatta ytatataggt taaactcaat ccttctctta 180

tgattgaaac atttgggata agtgaaggaa atctaantgg actcaaattg agatgaatag 240

tattttttcn agtaagtgat attggggnta cactacacaa ctatagagat actttcaatt 300

t 301

<210> 17

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (35)..(35)

<223>SNP mark DCHA17412_34

<400> 17

ctgcgatccc aggatctccc ttcgcaaaga ccatytcgac taaatcgatt gtcaccctaa 60

agaaaggcca ttcattgtac atctc 85

<210> 18

<211> 446

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (230)..(230)

<223>SNP mark DCTE1_293065_230

<400> 18

aattcgacat aagaatcaac acgaaaccga cttaccaccg atgacaaacc acctctaacg 60

cgtattaaac cgttgaagtg taattcccta ctccacaatc tactcctaaa cacgaattat 120

cactaatcaa tcactacata atgttatcgc taaaatccca aaccaaaagt taacgacaat 180

tgagttctac ttaccaaacc aaaaggagaa cccaacaatt ccaacagcay gatcaaacaa 240

tggattcacg agctgaatca aaagagaaat aaaatagatg gttttcgaaa aagagaaaag 300

aaatgccaag aaggggaaaa acaaaaaaaa ttagacgtca tattttattt gaaggagaga 360

gaaattttta gaaaaataat aaaaataaaa gatatcccac atatccctta ttttactctt 420

agtaacccac taactcataa gtccct 446

<210> 19

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (20)..(20)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_156_330

<220>

<221> misc_feature

<222> (162)..(162)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (181)..(181)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (187)..(187)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (205)..(205)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (239)..(239)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (291)..(291)

<223>N is a, c, g, or t

<400> 19

tcattcctga acctggctan gtcgatcgag caatttgcaa acaagagctt cgcatgatcc 60

aggctttgta cgctccatat gtacgaggga atttgtaact acacgcataa ctcgcccttc 120

tgaatcatca gcaatgacct acatttcgtg mgttgacaaa antattaaga agtgaacaaa 180

ntataantgc aaagttacct aaatngtcaa gtggtgcaca cctctttaag ggaagggant 240

aagcaacatg gatagtggta tacatgatga tgtgatcttt gcattttctt ncatcatcac 300

t 301

<210> 20

<211> 888

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (388)..(388)

<223>SNP mark DCTE1_388919_388

<400> 20

agccaagtct aatggcatca ctgttagggc cttggttgta ataaatccag gcaacccaac 60

aagacaggta tcaatgaaat gctttcagaa aagtatatta taaatgaaaa aggttcctgc 120

aatttcattt ttctcatttt gattgcaggt tcttgctgag gaaaaccaga aggcaatcgt 180

ggagttctgc aaggaagaag gtcttgtttt gctagtagat gaggtaggtt gatttgcata 240

tgttcttgct ttaccattcg ttaacaatga cttcgctttc ccaaggacaa gtttgcactg 300

accttgataa cgaccttggt ttcacaggtt taccaggaga atgtttatgt tcctgaaaag 360

aaattccact cttttaagaa ggttgccyga tctatgggtt acggcgagaa ggatatacac 420

ttggtatctt ttcagtcggt ctcgaaaggt aacattggtt tatcttattt ctattatttc 480

atttatggaa tgtgaatgag cttatacaaa gtttggaatt ttgacacaga atataaactc 540

ttttactgtc tccagggtat tatggagagt gtggaaaaaa gggaaggagg ttacatggag 600

gttactgggt ttggtgctga tgtgaaggag catatataca aattagcatc tgtgaatgtg 660

tgttctaaca tcactggtca aattcttgct agtcttgtaa tgagtccacc taaggttata 720

tcttttgtca tttaaagcca gttatctgtt tgtattcaaa tgacattgga tgtatgacat 780

aggactaaat agattttgag ctttgacaag ctgcttataa acattgtttc ttatagaaga 840

aacacattat tggagaaagc ttttgaatct cccaactatt ggatttgg 888

<210> 21

<211> 230

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (6)..(6)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (38)..(38)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_36697_528

<400> 21

ttcttngtcg gatcaagtgg ctattactcc tttccagnca gcgttgggta gctaaaatgc 60

tcggctacaa ttttgaagtt tcttaccgaa aagggattaa caacaaagtt gcggacgctc 120

tctctcgaca accacaactt gaacatagtc rtactatcag atttcagtaa gctcagttat 180

ctcggacctc ttggtacagg tgcagcaatc ttatactcaa gacaattgac 230

<210> 22

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (66)..(66)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_36697_380

<220>

<221> misc_feature

<222> (154)..(154)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (186)..(186)

<223>N is a, c, g, or t

<400> 22

ttctttagcg aagctcttag ggtgtgacat caagcattat ccatttatga aaaagaaatg 60

ctggcnagtt ttactagcag ttcgaaagtg gcatgcttac ttggttggtt gccattttaa 120

aattagtaca gatcatcaaa gtttgcgttt yttngtcgga tcaagtggct attactcctt 180

tccagncagc gttgggtagc taaaatgctc ggctacaatt ttgaagtttc ttaccgaaaa 240

gggattaaca acaaagttgc ggacgctctc tctcgacaac cacaacttga acatagtcag 300

t 301

<210> 23

<211> 578

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (114)..(114)

<223>SNP mark DCTE1_245250_114

<400> 23

aaaaagaagc agtagaccta agcgactacc atggaaggag ataatcgatg gtgttggttt 60

ccatctacgg taatggtttt gggtatggtg ttgtttgttt ggctgcttgg aaayatgaga 120

aattgaacat cgataatagc aatgtataac aagaaaagaa aaaaatagaa cacacagaat 180

tttacgtgga aatcctttcg aaaaaaaatc acgggcagag gaaaagaaat tcactatgtc 240

gaatttgaat gattacaacc agaaagacga ttacatctat ttataggttt aaaaaactta 300

ttctagtcaa attcaaataa aagtaatgta gtaagtttaa aatactttat tctaatcaac 360

atcaaataga tgaagtaaac ttctataggg attttgcttg tgcagccagt atcgtactac 420

gagggtcgaa ggcctccggt tgtaacccca gtctagtatt ttgcttatta gggatctacg 480

atgggctgcg gccttcgccc tatcttgcca cttgtatttt atttttaaca ggaattttgg 540

tcacacaact tctaacaatc tccaccttga cacgaatt 578

<210> 24

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_48698_177

<400> 24

cgcatcaagg agcttgaaac tgagtgctgg tcctcaaaga agaaacatga gcacttcttg 60

agaaaagtta gtgaggaaag agctgcgtgg cgaagcagag agcatgagaa aatacgtgca 120

tttgtcaatg atgtcaaagc tgatttgaat ygggaaaaga aaaaccggca gagacttgag 180

attgtcagtt ccaaattggt gaaagagctg gctgctgcca agttatcagc aaagcaatat 240

atgcaggact atgaaaagga aagaaaggac cgagaactaa ttgaagaagt atgcgatgag 300

c 301

<210> 25

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (104)..(104)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_48698_527

<400> 25

agattccatg aagcttcgag acgaagtcga tgaggaaaga aagatgttgc agatggctga 60

ggtctggcgt gaagaacgtg tacagatgaa gctcattgat gcanaaagta gcacttgaag 120

aaaggtattc acagatgaac aagcttgttg yagatttaga tacttttcta aggtcaagga 180

ccggaactct ggatgtgaaa gatatgagag aagcagaatc acttcgacaa gctgctgctt 240

cagttaatgt ccaagaaatc aaggaatttg catatgagcc ggcaaaccct gatgacattt 300

t 301

<210> 26

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_48698_434

<220>

<221> misc_feature

<222> (197)..(197)

<223>N is a, c, g, or t

<400> 26

ggaaagaaag gaccgagaac taattgaaga agtatgcgat gagcttgcta aggaaattgg 60

agaagacaag actgaagtgg aagcattaaa gagagattcc atgaagcttc gagacgaagt 120

cgatgaggaa agaaagatgt tgcagatggc ygaggtctgg cgtgaagaac gtgtacagat 180

gaagctcatt gatgcanaaa gtagcacttg aagaaaggta ttcacagatg aacaagcttg 240

ttgcagattt agatactttt ctaaggtcaa ggaccggaac tctggatgtg aaagatatga 300

g 301

<210> 27

<211> 120

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (72)..(72)

<223>SNP mark DCMA76208_71

<400> 27

ctgccgaaag gaaataacaa attttattga gaaaaagaat aattttctct cacacacaac 60

atcattggac cytttttcat ctcttcatca gcagagatcg gaagagcggt tcagctcagc 120

<210> 28

<211> 590

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (377)..(377)

<223>SNP mark DCTE1_269021_377

<400> 28

accgttgctt tagaagccaa acctcatatt gaaccttagg aacccaccct aggtagaatt 60

actctctacc ttagtagaca cctcattagg agcttttact atttcttggt cgtattgtat 120

ttaattgata ctgatttttt gtgttttact ttgattgcat ggcatatcat tataaaggcg 180

ttggttcgtg ttcggttgct cgatagaaag tttatcatgg gaaatgggtt cttgatagaa 240

tggaggacaa tgcggctgtc caaacttggg ctgagagggc gcaacgcgaa aaaggtgata 300

gtttagccga cgggtatgta tcagaattat gagacttcac ccgtgtcatc gtagcccaga 360

acaacttgca agagttraag gaaatttggg atcaatggaa tattgaggtt agacagttat 420

ctattcaaat tataggaatt tacttatttg cttgatatga aggtagacaa gcgtctgctt 480

cgagctcttg cccaattttg gaatccagcc tatagtgctt cacgttcggg aaggttgatt 540

tggtacctac aatagaagaa aacatggcct tacttcggtg ttcgagaatt 590

<210> 29

<211> 423

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (256)..(256)

<223>SNP mark DCTE1_291290_256

<400> 29

ctcctctgac aatggatatg gcttaccgtg aagtctaaac catgtcatgt aatatgaaga 60

ggacgataaa ttcggttgga caattgattc gcgcataagt ataaaatcgt acctatattt 120

ccagatgtag atatatttgg catgaaattt aggccaatct ttattggttc tctctgcaag 180

tcgatcttgt gaagttcatc gagctcttgg ggtgacgtcg gaatacttta cataaaccta 240

aactgacgca tcactyagtc raatttgtgc atctcaatca ttgtaaaaac tatcaatgac 300

actttgacgt gccatatatt tcaattcgcc aaaaattcaa tcatgatgta ttcttgaatt 360

atcggatcaa agtgtagcat tcactcaaac tatagtaaca aagttataaa ttttattacg 420

tac 423

<210> 30

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (30)..(30)

<223>SNP mark DCHA11045_29

<400> 30

ctgcctctgt gctgttatgc tctccaacay tgctaaaacg tccggaatca agccagaaat 60

agccataaca atccctaaac gatgc 85

<210> 31

<211> 548

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (250)..(250)

<223>SNP mark DCTE1_221931_250

<400> 31

tgagctacac atcttattgt caaatttgtt ctttttttaa attaaaatca tgataaaaaa 60

atagtattgt ttcaaggaag gcactaaacg ttcgaaccaa gagcaaatga ttacaatgag 120

agaacctgtt tcaaacttgt tgtaatgtaa aaggcaaggt ttctagctta atagttgatg 180

gtgagagttg tactaatatt gcaagtaaaa ttatggtgga acagctccat tttcctacaa 240

caaaagaccy tcaaatgtac aacttgcaat ggttgaatga gaatggtaaa atcaaagtca 300

ccaaacaagt gatggtctct ttctcacttg gtaagtacaa agatgaagta ctttgtgaca 360

ttgtcttgat gcaagcctgc catatctttc ttaggcgtct atggcaattc gatcgacgag 420

ttcattatga tagccatgct aatcagtaca cttttgagtg tacaaaataa gaccacctca 480

catcattgac ttcggatcaa attcgtgaaa accaagctaa gatgatgaaa ttgagagtaa 540

ttataaga 548

<210> 32

<211> 349

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (78)..(78)

<223>SNP mark DCTE1_261396_78

<400> 32

gaatataatt ttatatataa aattaacact acattttggc gaaatcctag gaggaagcaa 60

agaagttcat gacctctycc agaaaaattc agtaccacat tggggggtat gcgagccgcg 120

ttaggtttga agttcgggat gcaagtgtca caacgcaata acttaacggg ggtgtggcag 180

caacggtttt atttgatgtt ggtggacatt ctgctatcaa tgaaatttta tatcgcaaag 240

atgttagagt tttgtacttc tcattgtcgt catctttaaa atacgaaaaa gggatatggt 300

ataatatata tacacagtaa gagttctttt aggcatgaat ttaattatt 349

<210> 33

<211> 196

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (46)..(46)

<223>SNP mark DASCTP_60046_46

<400> 33

gacgctcccc attgtgggtc atccaatgtg ttgggtgggc ctgttmctgt tgaaggaaat 60

gctggcaact acagtgtcaa tggaagtaac tcgggtagta accacgcaag caatgggcca 120

catggaagta gcaccctagc cgatactgta gggacaaaca tagaaagcga taatggaata 180

gctgggaaaa gtggaa 196

<210> 34

<211> 214

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (64)..(64)

<223>SNP mark DASCTP_4812_64

<220>

<221> misc_feature

<222> (153)..(153)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (203)..(203)

<223>N is a, c, g, or t

<400> 34

ggtataatat ggcatcgaat gcaaataagt tttcctttgt caacaccgga agctcatctg 60

cacrtgatag taagatagaa ctgacaagaa aaggatcagt atgcgatgtt caatctcctt 120

tggttaatga cctttccaat cagtactcaa atngttggta gcaataacat caatatggcc 180

tctacaactg ataatgcttt tgnccaagcc agct 214

<210> 35

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (11)..(11)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (103)..(103)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (130)..(130)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_60046_472

<220>

<221> misc_feature

<222> (216)..(216)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (230)..(230)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (288)..(288)

<223>N is a, c, g, or t

<400> 35

atcgatgctt nccggaaaaa ggtatccaat gtcgacctgt tagcataatt tctgagattc 60

agagtaaatt cttctcaaca ttttgccttt ctttttccag gtnccgatat caaagtagga 120

aaaggttagn cacaacaacg acctcgtatc ygagggcaat tcgtgtgaca aacggtgaac 180

accaatgacc cctcatctga aggcaattca tccgancaaa tagcaaagan ggaaaacact 240

agaagagtcc agctgataag gtaacaagat ttgatccact cctctganta aaataccttg 300

t 301

<210> 36

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (32)..(32)

<223>SNP mark DCHA212383_31

<400> 36

cagctcctga atttatacat aggatagaag aratttactt tcatatatag agaacaaaag 60

cgaacttgca tcacatgatt aacac 85

<210> 37

<211> 601

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (301)..(301)

<223>SNP mark DC7_59671475

<400> 37

ttctaaaagg cttgtctgat gacggtgctt ggtctttgtt caaagaaata gcatttgagc 60

aaagatatgc agactcaacg aattcagcct ttgtggaaat aggaaaacag attttagaaa 120

ggtgtagtgg tgttccctta gtcataagga cgatagcaag tacattatct tacaaagaaa 180

ctgaaaagga gtggcattct tttagagata atgaacttgt tagaatatct caaaacgaag 240

gtaaaattct acctacactt tagtttagct acgatcatct cccatcccat ttgaagcatt 300

sctttgctta ttgccgactt tatccaaaag atcttgtgat tgatgtgcaa acacttgttg 360

agttttggat tgcacaaggt ttcgtaaagc aattgaatca aagtcaatct cttgaagaga 420

tcgggtttgg atattttaaa gatttagtag aacgaagctt tagctttagc tcttttgcct 480

ttctgttttg ttttttattt tttttctcag tcaattgatt acaagaaaag gcgttgggat 540

cacccaacct ataccagttc aggcaaggaa tcaaaagcag ccaaactata ccaaaacaga 600

g 601

<210> 38

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (23)..(23)

<223>SNP mark DCHA165008_22

<400> 38

cagcctctcc aatgacagac atkgtgaaat tatgagaagg aaggagaaga gagaaatgaa 60

gaagccaaaa cttgaaattc aggga 85

<210> 39

<211> 389

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (262)..(262)

<223>SNP mark DCTE1_278814_262

<400> 39

aactacatag gccctggctg gtgctcttgc ctctgactgt tgtgtaacta tgttactact 60

tctaagaaca ccttcacctc tggataccga actacctcta cctaaccctc tgcctctagt 120

agtaggcatt gatatctgag atgatgtagg tatagcacat tcactttttg gacaatctca 180

gatgaaatga tctgtggacc cgcattgaaa acaccgtcga gttatcttta aacattcgcc 240

aaagtgcttc tttccatagt gytcacagtc ggggatatca atatttcgta aaggtccctt 300

tacactacct gtagaaacag tagtctgttt accccgattt ttatcaccca gatccgatct 360

gaaacttgac ctccaattgc ctctgaatt 389

<210> 40

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (24)..(24)

<223>SNP mark DCHA280949_23

<400> 40

ctgccgtatt gtagctctaa caayctgagt caacattgca ttggctaacc tccgagttcc 60

cctaactcca cccatcaaaa tcgcc 85

<210> 41

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (75)..(75)

<223>SNP mark DCHA58490_74

<400> 41

cagcattatc aacttcatca ccaattattg cttctctatc ttcgtctctt gaaaatgtag 60

agataaaagt agtcmtgttc gaaca 85

<210> 42

<211> 843

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (501)..(501)

<223>SNP mark DCTE1_214911_694

<400> 42

gtatgtatag ttttagtaaa taatactaag ggtttgaact tgattttgat tttctgggtt 60

tcttgattgg attttgtttg attgttttta caagaattgg tgccacatgt tagagagatt 120

ggaagcttgt attgcttgta atgggagatt gttgatctcc tttgaatgac tgagttgaat 180

tgttcaataa aatcaagatt gacgagttat gttgtgtcaa cttttgaatt aatttgtctt 240

gtcaagacag aaccatcaat gctcatgcat aaaatccaat ggttctaggc tgatgtttct 300

agcttgtact cagcgttgtt tgtttcctta gagtctaaga aaaactgata agcaaagaga 360

atattagata tgcaatggtt gataccgtgc atgaaaagag aagatatttt gtgctgcaac 420

atgtaaccaa aggtatctcc ttagcaacct tcattatttt ggttccatca actcggtatc 480

aggtgcgctt cccaacaatc ycagcagggt gcactctatc ttcaagcagg gcaccatgga 540

caccagtgag tgctcgggtg cgaggaggcc gaaatgcaga tccttttttg ggtggcctca 600

atatcctcct gttggcaagc actacaacag ccggcaaaag catgcatgaa ccaatacaac 660

ataaattaag tctaccagcc aagtaactcc tggctgcagc aatgagaggt ttggtttata 720

aaaaggcaac gccttcgcca cgacgagagg tttggttgag gaaattagga tgaactcgta 780

ttgaaagaag caatggtggt gtggttgcct ttccaaagcc gaaatggttt cttatacaga 840

gct 843

<210> 43

<211> 531

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (501)..(501)

<223>SNP mark DCTE1_233925_865

<400> 43

ttcattttca tttcattttt ttttataaaa attcgattca aaaaattagt ttcgacaacc 60

tattaatcca atttagacta ataactattg taagttctct ttaaacaaaa aaattcaata 120

aattaataaa taacaaaaat aattaagtaa aagtgaaaaa atggcaaaaa agtcattcaa 180

tctctcgctt tttgtctttc aattagaccg ttgcagagtc tcaaatcact tttttaatat 240

caaacttaaa aacacaatgt attccagagt gaaccttcaa gtttcattta aaaatgggag 300

agacaagtga acctctgcgg caaacccctg aaccaaactc tgcttctttg gagtctctgt 360

tccatgttct cgaccctatt tctctcattc ataattcaag ccccggtaac ccaattcctt 420

taaggcttac aacagagagt tctataatgg aaaggggtcc cagatatgga gcttatgcag 480

agctaagaga aacaaagctg rgaatgaaga gtgggatgat gcaacaagaa a 531

<210> 44

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (57)..(57)

<223>SNP mark DCHA162845_56

<400> 44

cagcattggc tctacaaatt gattgggttc gacttcgaag tattatacag accaggraag 60

ttaaatacag tggccgacgc cttat 85

<210> 45

<211> 120

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (25)..(25)

<223>SNP mark DCMA55989_24

<400> 45

cagcaatggc acccaaaaat gccaraaacg tccaaggaca cctaaaataa gtatcataac 60

ctttagctga gatcggaaga gcggttcagc aggaatgccg agaccgatct cgtatcgtat 120

<210> 46

<211> 567

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (126)..(126)

<223>SNP mark DCTE1_232591_126

<220>

<221> misc_feature

<222> (524)..(524)

<223>N is a, c, g, or t

<400> 46

aaaaaagttc aggccccaat ttgcgaacaa ttgccaagtt caatgactaa cttagacaaa 60

aaaaaatttc aagcaccaat gtgggaacaa ttattaagtt caggccccaa atagtgcatt 120

aaccamaaaa aaaggtgaac caaaatagat atgcagaaag ccaaacctat atgtgtgcat 180

gaatgcactt gtgaacttgg acatgtgtct tatggtatat ttccctaata aggctaaagg 240

tagtttcatt ttcatggatg ggtaagagac ctctagattt gtaaatcact gaagggaaag 300

aaattaccca tgagaatatg tcatcttctg ctgagcttga cgcaggactc atcgggctct 360

ggctgtgagc tgtccatcgt tgttctatag gagcaggagc aggtaactca ttcgtctcaa 420

tgagtttctt tagctcttca atgtactcaa ggtccttcat actaggcttg gaaagagttc 480

cagcaggaaa acaggtttct gatacccatt gttggccccc caanaatcaa agcccaagat 540

atcatcactc catccaactc gatatcc 567

<210> 47

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (20)..(20)

<223>SNP mark DCHA153168_19

<400> 47

cagcaccgcc gtttgcaccr ccaccgccgg acccttgcat tctcttcaaa tagaggcgat 60

atttctgtaa atgactagcg acgtt 85

<210> 48

<211> 120

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (80)..(80)

<223>SNP mark DCMA93983_79

<400> 48

cagcttcaaa ttgaaggatg gaggggtgat gccaagtttc cgagttggct ttctttgctc 60

acaaatctcg tcagaatttr cataagtggt agtaatttca aacatctccc gtcctgtcct 120

<210> 49

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (34)..(34)

<223>SNP mark DCHA224779_33

<400> 49

ctgcaaggtt tcttggaagg tacgctgtct gctycgtctc ggtttattgt gtctccagaa 60

ggtgctctta ttccgaatcc agaag 85

<210> 50

<211> 374

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (69)..(69)

<223>SNP mark DCTE1_365870_69

<400> 50

aaattaggcc acttgcagag cttaatgtga tgagcatctt ctgtcccttt acaagtaaac 60

aaagctcgrt gcttcttaaa caagataacc tttgaaagtt gaataattgg aacccaatgt 120

gactactgga ttccgggagt tccttggacc aaaagtggaa aaacctcagc agctgtacaa 180

agtccccagc ttgaagactg gatagattag ataggtgtca tttataggct gtactgacac 240

cataaacaat attttgataa aacctaaacc aaaatatcct atacacaatg ccaaattata 300

cagaaatgaa agcaaatata gagctatact ttgttcatgg aaacaaatca ccttaattct 360

ttgttctttt tttc 374

<210> 51

<211> 357

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (223)..(223)

<223>SNP mark DCTE1_271930_223

<400> 51

aattctcctc cattctattt gctcttcttt gctcttgtgc atagagaaca tttatcagct 60

ttgtcaaggg tatagctgat aggtccctag agtcctccaa agaagagatt tttgactcat 120

acttctctgg aagagttgta atgacttttt caactatcct tttgtcactg aactaatcct 180

caagaagcct tatgtttttt actgtagcca taatcctgtc agmatactag ttgatggttt 240

ctgactcttt catcttcaga ttctcgaagt cccttctcag gtttatcaac tgctactgcc 300

tggtcttatc tgacccttga actcttcctt gagtctgtcc caagcctact ttggagt 357

<210> 52

<211> 990

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (501)..(501)

<223>SNP mark DCTE1_208529_965

<400> 52

agcgttgcct gccttccaag gatccctaaa attaggcctt aactttctct ccacaagcat 60

ccttggtccg tagactggtg actcctctgg tctatcatct tccaccatca ccgccatatc 120

cgccatagag tgtttcgcat aagccctgga agcccccttg ttcttgttgg agttggaatc 180

ctcaattgta aaagtcgctg tatcaaatat ttcgaccatt aatcaaacaa taacagaaac 240

ttaactaaat aggagaaaaa atcaatttgt acgaaagggc tagtgaagta gtttacacaa 300

ttattgcttc tttcctgatc ggcccgttat aaaattcaaa aagttttgaa tccttttagg 360

tgcggccctt cttcctctgt ttccacgtcg atatcacgtt ttcgttttac aagggaagtg 420

ctgcctgcgg atccatcgtt ttcagtagag tgatgatgga tatgttccaa gtttggagaa 480

gattgaatgg tatggcactg yagctcttyt atttcttcca aatctttctc atacattatt 540

ctaacaccac acttcttcac cttaacagaa ggataccttt ttggagaaga ttggaaagac 600

atctcaagcg ataaatcctt ggcttcacat ttatccttta atgataatgg cgagaaatat 660

cgaagaaaaa ggtggtcctt ctttatgcgt cgtccacaaa aattaaggaa actggaatcg 720

accattcgac tattttaact tgaaaaacag atccattact actagattgt ccagaatatc 780

tagaatggat ataagttgga ccaccgataa cctttatctc ggaaccatca tcatcaccga 840

aaatgcagca gaaagcaact ccaatacatt gactgtcatt ctgaatattg agaggcaaag 900

gtatttcgat tacagagccg cctctctgtt ggctgaacca ttctgggatt tcatttccag 960

gtataacagc atcaaatctt ttcttgaatt 990

<210> 53

<211> 85

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (53)..(53)

<223>SNP mark DCHA6086_52

<400> 53

cagcaaccga cgtgattttt gcccactaca acatatatta gttttaaggg caytttattt 60

ttaaattttt caaaaaatgc tgggc 85

<210> 54

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_39375_356

<400> 54

aggatacctc taccactcaa tattcagaat gatagtcaat ggattggagt agcttcctgt 60

tgcatttttg tcaatgatga tgcttcccgg gataagttta tcaactgcag agctgttatc 120

cattgtagaa attctggaca aggcggtcga ratggatctg tcttccgaga tacagatctt 180

cgacgtgtcg atgcaagtag ttggttgttt gggaaacgtt ttaaccagcc cataacgaag 240

gatcacctat ttcttagata ttggtcgcgt gataaattat atccattttc cttagaggat 300

a 301

<210> 55

<211> 458

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (347)..(347)

<223>SNP mark DCTE1_240643_347

<400> 55

attacctgat ctacacgcaa cacagtgcag gcagcatctg cagcatattt gagagcaaaa 60

aacctgacaa aaaaagtcaa caattgtaaa ttataaaaga atcagaggtt aattttcgat 120

gcaaaagaga acagaactga gccagaaata attgtaaata gcgacttact tagtgacata 180

aaggtcccaa acattcacgg ttgagacatc cttgcaaaca caatcctcag agtcatctcc 240

cctcaagtcg atgcccactt tagcattccc agatgcatgc ttttcataca agttagaaat 300

aatatccatt ggtttgagtc cagcattctc tgccagggtt ctcggtrcca attcaaaact 360

ttcagcaaac ttagcaatgg catactgatc caatctgcaa aatatcccgt agttaatatt 420

aatagcttct tctagtactc atctaatcta ctataaat 458

<210> 56

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (140)..(140)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_59246_191

<220>

<221> misc_feature

<222> (292)..(292)

<223>N is a, c, g, or t

<400> 56

aggggttgag ttaatgattt taccgcaaat gataaccgtg ttagttgatt ttacgagtgc 60

agaagacttc agctgtgaca tcgagtctga tggaaaagtg ctaatcaaag ggataacaac 120

aaccggtgag aaagttgtan gtcaagaact ytcaagtctt tcatatgctc acacaaaatt 180

tatgcccatc tggacacttc actgtttcat tcgagctacc gggccctgtc gatcccgaaa 240

aagttactag ttgtttagct aatggattgc ttgaagccgt tgtgaagaaa anggtaactg 300

a 301

<210> 57

<211> 601

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (301)..(301)

<223>SNP mark DC7_56573714

<400> 57

gaaattaacc agttttaaaa ttccacaaat tagttatata ttttttaaat aatttatttt 60

tcacttacat gttcctgtca tagcatatta acccattgaa taataacacg accttttttt 120

attaactttt gattaaagta tagacaactc tttataattt gtcaaaaaca ttttgtttga 180

ttcaaatata ggattttttt tatcgaatcc atacatatta ccgcatcacc ttcgacttca 240

atgtcactac caccatcaga taccacccca acatagaaag acgacaatgt tgcctgcaaa 300

macaataaat gaaggattca agcttgactt atggaagaac caactcaacc aagtagctgc 360

ttttgcttgc ttacgctgta gctaagacag tgagggttgt agttgcagtt gtagccataa 420

gggaagggtt tgggtgtagt gaagtccatg ttgaagcagt gatcgttgac catcagagct 480

tgggtggtgt tgctttagac aggggacgac tctattactt ccagagttgt tctgctcttg 540

gtcttgggtt tccattttcg gatcctcatg cttgtaatct agatccaaac acagatcctc 600

a 601

<210> 58

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (57)..(57)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (73)..(73)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_28910_164

<400> 58

agcgttacct tcttgttttc ggtgggagtt cacatgctac tttcttcaat gatttgncat 60

gtccttgatt tgncaaactg taagataatt acacttttca gttttctcat ttgcttgtct 120

gaggaattaa tttctttatc tttccttctt ytttttttct ttcttttttt tggtatttgg 180

atttgtagat ggaatggtca aaacctgcac aactaggtga gttaccaact cctagagctg 240

gtcatgcagg agtgactatt ggggaaaact ggtttattgc tggaggtgga gacaacaaaa 300

g 301

<210> 59

<211> 601

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (301)..(301)

<223>SNP mark DC7_56523319

<400> 59

aattcacttt acttccttca ctgctacttc cattatttgc aacatcagtt ggaatgcctt 60

ttgtagtgca gtttttagtg agaggaggtc cgcaaagatg attgcccacg taagacaagt 120

ttgcaaagct ttgaagttga gtacttgttg ggatttgtcc tgtcaaattg ttgtacgaca 180

cattgaagtg attcaagaaa ttcaatttgg agaaacttgg agggatttca ccatttagtc 240

gattcatggc caaatcaaga gattccaaaa acttcatgtt gccaatgttg tctggtatat 300

wtcctgttag gtgattccct gagaaattta aagacaatag tccaacgaga ctaccaattt 360

ctttggggat ctctcctgtg aaactgttaa cagaaaggtc caagttggta acaagtccta 420

gtgtgtgacc atattcatcc tctcgccctt tcaacaccaa taaggcactc aaataaaatg 480

ggtaattaat gaaatacagc gtagaaagtt catttttggt tttgtttgtt gtggccattg 540

cacttaaatt attgaagcat tttggaataa ctcctgaaat gttgttgtgg gcaaggtcca 600

a 601

<210> 60

<211> 597

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (97)..(97)

<223>SNP mark DCTE1_240981_97

<400> 60

aaattctgag cctgaggaga cccttcccca gaccctcctg gagacatttg ggcaaccgag 60

atggcatcgg atgagacctg cttgatgaat ccagagrcag tacccaacag ttcagaaggc 120

tgagcctgag gagaccctcc ctcaaaccct cctggagttt gggcagccga gatggcatcg 180

gaagagatct gcttgatgag tccagaggca gtgcccgaca gtccagaatg ctgaacctga 240

ggaggccctc ccccagctgc tcctggagac atttgtgcag ccgagatagc ttcagatgag 300

acatcctaga caattggaag gacaaaacga tcaatgtcag ttacagattc actaaccggc 360

aattataaga agaagatgag gaagaagaat tgttgtatct cctttcattt gctagacatc 420

atcaaatttt tggatactct ttattaatca aaccttcgaa ctaaaaggtt ttgtaatttc 480

agtttaaatc cacagttttc caactcaacc acaaacttcc ccgaagattt aactgttcca 540

aaaggctttg aaatcacaaa ttttcaatgg tatattaata tcatagtacc aacaata 597

<210> 61

<211> 339

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (63)..(63)

<223>SNP mark DCTE1_317966_63

<400> 61

aattcaggaa cttctttaaa accttttgat ctaacggtgg agaaaaacag cttcttttgc 60

rwrgtttggt ggataaaaag taggtctagg tgttgggagg gcaaatgcgg ttgattttct 120

ctatctagtc tgcttttttt cttgtttggc taccgagaaa aattttgttc ttgtttctgt 180

ttctgggtct tttggtttta gtgaaggaga agtagaggga ataaaaaaaa acaaaacaga 240

gaagagatct ttttggtggt ttcttaattt gggttgaaga aaagatgaca acaaaatcag 300

ttgaagtaag gtttaatttt ctttttttct tttagggtt 339

<210> 62

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_1656_527

<220>

<221> misc_feature

<222> (192)..(192)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (200)..(200)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (212)..(212)

<223>N is a, c, g, or t

<220>

<221> misc_feature

<222> (245)..(245)

<223>N is a, c, g, or t

<400> 62

gtgccggaat ggcacccgcc attcccgaaa cttctgcatg aaaatcgtaa aactatgaaa 60

aattacatta aaaaaggctt aaatatcaag aaagaagagc caaaatatta cttaattcat 120

ttggaatttg tcccttcctc cccatttccg kacataattg ttctacggga actttcgctg 180

ctaatttagg tngatttagn agattttggg tncctaagga atgcaccgaa cttctagctt 240

ttgtngcatt ccttcacggc tgtcatccca gaacagtcta attctactga ttcgccacta 300

t 301

<210> 63

<211> 293

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (82)..(82)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_51689_504

<220>

<221> misc_feature

<222> (220)..(220)

<223>N is a, c, g, or t

<400> 63

aatatggaat caaagttcat acagattgct ttatatgcaa ggtattgtaa cacaaagtaa 60

aaacaggaag ggaaaacaaa anggagtcaa ttttggccat ctatctactc aataagctta 120

aaaaactcac ctagtcctgg aactgccaag rgtgagttct aggtcatccg atccgcaatc 180

ttcatgaatc ctctctcctt cccacggttt cacgagtccn tgttgcattg cttccaaatg 240

cgaactcgtc tgatataact tcagacattg gaacatcagc cgtatgatct gat 293

<210> 64

<211> 585

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (286)..(286)

<223>SNP mark DCTE1_237093_286

<400> 64

aaatttatca taataatttt tttccgtgtt ttaaagaaaa atcggtttca acgttgtgat 60

cgaaataatt aacaatatta actatttaga ctaactaatg atattataaa gtagagggac 120

caaattatat aaaaaaacat taaagtatat agattaaccc tcgagtttca gcataacata 180

ggaaccaaaa ctaaaatttg gccattctct tttaagttaa aaattaaaac atagagatta 240

aatcgtaaat taagacataa taaaaaggcc aaaactaaaa tttggycatg aatctataat 300

aaaagtaaca ccacagcatg aggtgccatg tgccaacaga gggaaggtcc tttacaactt 360

ctatataatt aattagtaaa tggaaacttt tggggtaggt ttattttttt taatgggatt 420

aggtttatta ccatttaatc atgtttgcca taattgaaaa aaaaggatta ataattatat 480

tatgttgaaa ttttaatttt agttttaatg atcaaatttt aatttacaat tattttaagt 540

agtaaaaaaa ttctaaataa aatttaaaga ctcacttata gaatt 585

<210> 65

<211> 120

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (29)..(29)

<223>SNP mark DCMA26817_28

<400> 65

cagcaattga ccacaatatt cattacacyc aagcttcagt tgagttaaat tactaaatac 60

atgcaaacct gaagtgttta caccgcagag atcggaagag cggttcagca ggaatggaat 120

<210> 66

<211> 120

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (60)..(60)

<223>SNP mark DCMA156739_59

<400> 66

ctgcggtgta aacacttcag gtttgcatgt atttagtaat ttaactcaac tgaagcttgr 60

gtgtaatgaa tattgtggtc aattgctgag atcggaagag cggttcagca ggaatggaat 120

<210> 67

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (101)..(101)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_8602_418

<400> 67

gcttaccaag ctgacaaact tcacaaacag tatcattgac ctcaaccttg gacatgtcct 60

caaccaagtt cagcctgtgc aacagatcaa gtgacctaaa nttggcgcgg cctaatctcc 120

tatgccaaag accaatgcta tcagcaagac kagtataagc ctttctctca atttgactca 180

catcaagcat aaaacaccta ttaatcatag ttactataac tagctcctaa ccaaatgagt 240

ctttaacaat gcacgaacca tctctaaaga ccaaagtgta tcattttaca ctaactgact 300

a 301

<210> 68

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (23)..(23)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_8602_496

<220>

<221> misc_feature

<222> (267)..(267)

<223>N is a, c, g, or t

<400> 68

gcaacagatc aagtgaccta aanttggcgc ggcctaatct cctatgccaa agaccaatgc 60

tatcagcaag acgagtataa gcctttctct caatttgact cacatcaagc ataaaacacc 120

tattaatcat agttactata actagctcct raccaaatga gtctttaaca atgcacgaac 180

catctctaaa gaccaaagtg tatcatttta cactaactga ctaacactaa gtaagttctg 240

gtctatgtca ggcacaaaaa gcacatncag aaattacttt gttacctgaa ctagagttga 300

t 301

<210> 69

<211> 624

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (124)..(124)

<223>SNP mark DCTE1_214869_124

<400> 69

tgtcatacat aagtcatgca cctaatgtac taacttttgg cttcgatatc aatggagcat 60

taactttcaa tacatcgaag cacatgagtt aggtactagg ggcgagtatt caatcgagtt 120

gagkcaaatc aagtaaaaaa aatttcgagt ttgttgagtt gatgaatcct attttagcaa 180

ctgaactcaa tttaaatttt tcgaatcgaa tcgaatctag tcaaaaaatt tcgagtcaag 240

tcgagtcgag ttaacgaatc ctattattga tactcaatgt tgcgtttaca tggatcgatt 300

atttaactag tagacgaagt acgagattat ttaactacat aaacaataca attgttttgt 360

cttttaattt aatgggtcaa cgtttatcaa aataacgtag ttttgccttt caacttaatg 420

gttttgactt ttaactttta aaaaagtaaa catttatcaa aacgacatag ttttggcttt 480

tcttattcgg attttcggat aactcgaatt gtgtaattca tattcaagtt aaaccgaaaa 540

acttaatttt ttattcgagt tgatctgaat aacttgatta actcaaataa ctcgaactat 600

ttaattcaaa attaaatttt ttat 624

<210> 70

<211> 219

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (61)..(61)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_10515_611

<400> 70

ttaatccact agcaagggca gttgcaaaag aatgcggcgg tttgccatta gctctcaaaa 60

ncggttggga agtcaatgag gaacaaaaga aggattgagt tgtggaaaca cgcacttcac 120

catttgcagc attcagaccc tcacgtcaaa macatcgagg acgaagtcta tcgtcgttta 180

aagttgagtt acgactcatt gccaagtaag atcctgcag 219

<210> 71

<211> 274

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221> misc_feature

<222> (116)..(116)

<223>N is a, c, g, or t

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_10515_556

<400> 71

gctgcttgga atttgtttgc tcaaaatgct ggtgatgtgg ttgaagtacc aagcattaat 60

ccactagcaa gggcagttgc aaaagaatgc ggcggtttgc cattagctct caaaancggt 120

tgggaagtca atgaggaaca aaagaaggat ygagttgtgg aaacacgcac ttcaccattt 180

gcagcattca gaccctcacg tcaaacacat cgaggacgaa gtctatcgtc gtttaaagtt 240

gagttacgac tcattgccaa gtaagatcct gcag 274

<210> 72

<211> 301

<212> DNA

<213>Upland cotton (Gossypium sp.)

<220>

<221>Mutation

<222> (151)..(151)

<223>SNP mark DASCTP_10515_498

<220>

<221> misc_feature

<222> (174)..(174)

<223>N is a, c, g, or t

<400> 72

tgtctgccga gcaatgatga cagatgaaga gatcaagtta gatgttctaa aacaagaagc 60

tgcttggaat ttgtttgctc aaaatgctgg tgatgtggtt gaagtaccaa gcattaatcc 120

actagcaagg gcagttgcaa aagaatgcgg yggtttgcca ttagctctca aaancggttg 180

ggaagtcaat gaggaacaaa agaaggattg agttgtggaa acacgcactt caccatttgc 240

agcattcaga ccctcacgtc aaacacatcg aggacgaagt ctatcgtcgt ttaaagttga 300

g 301

<210> 73

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Oligonucleolide primers

<400> 73

catgtccaat ggatgtgtca 20

<210> 74

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Oligonucleolide primers

<400> 74

gggccactta aaggcattct 20

<210> 75

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Oligonucleolide primers

<400> 75

acatttccac ccaagtccaa 20

<210> 76

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Oligonucleolide primers

<400> 76

aatcgttgac agcactgcac 20

<210> 77

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223>Oligonucleolide primers

<400> 77

tcatggaaca ccaaagttgg a 21

<210> 78

<211> 25

<212> DNA

<213>Artificial sequence

<220>

<223>Oligonucleolide primers

<400> 78

acatgataga ttattcagca atgca 25

Claims (32)

1. it is a kind of for first upland cotton (Gossypium hirsutum) plant of the identification containing reniform nematode (RN) resistance or The method of germplasm, the method includes：

The detection at least one in the first upland cotton plant or germplasm resists sex-linked mark with RN, wherein at least one mark Will thing is selected from the group：

And other marks chain with aforesaid at least one.

2. at least in method according to claim 1, wherein at least one mark and following persons

Individual close linkage：

3. method according to claim 2, wherein at least one of detected mark and following persons mark shows little In about 10% genetic recombination frequency：

4. method according to claim 1, wherein detected mark is not the mark being selected from the group：

5. method according to claim 1, wherein detected mark is the mark on chromosome 18.

6. method according to claim 1, wherein detected mark is the mark on chromosome 21.

7. method according to claim 1, the wherein germplasm is upland cotton strain or kind.

8. method according to claim 1, wherein the detection includes a part for the amplification mark or mark to produce The mark amplicon of raw amplification, and detect the mark amplicon of the amplification of gained.

9. method according to claim 8, wherein the amplification includes：

By amplimer or amplimer pair with from the first upland cotton plant or the detached nucleic acid blending of germplasm, wherein the primer or At least a portion complementation or partial complementarity of the primer pair with mark, and upland cotton nucleic acid can be used to cause as template DNA polymerizations caused by archaeal dna polymerase；With

Extend the primer or primer pair to produce at least one in the DNA polymerisations comprising archaeal dna polymerase and template nucleic acid Individual amplicon.

10. method according to claim 9, the wherein nucleic acid is DNA molecular or RNA molecule.

11. methods according to claim 8, the wherein amplification are included using PCR (PCR) or ligase chain type Reaction (LCR), from the first upland cotton plant or the detached nucleic acid of germplasm as template used in the PCR or LCR.

A kind of 12. methods for producing the upland cotton plant or germplasm of Jing gene transgressions, the method includes：

By at least one from the first Gossypium (Gossypium) plant or germplasm and the positively related mark of reniform nematode (RN) resistance Will thing allele gene transgression in host upland cotton plant or germplasm, to produce upland cotton plant or the kind of Jing gene transgressions Matter, wherein at least one mark is selected from the group：

And other marks chain with aforesaid at least one.

13. methods according to claim 12, wherein the host upland cotton plant or germplasm and first cotton or Germplasm mutually compares RN infection sensitivities, and wherein the upland cotton plant of the Jing gene transgressions or germplasm are planted with the host upland cotton Thing or germplasm are compared comprising increased RN resistances.

The upland cotton plant of the 14. Jing gene transgressions produced by method according to claim 12 or germplasm.

The upland cotton plant of the Jing gene transgressions of 15. claims 12 or germplasm, wherein being connect with about 2,000 reniform nematodes After about 6 weeks, the upland cotton plant of the Jing gene transgressions or germplasm include 138 to 24,806 to the plant of the kind plant or the germplasm Nematode.

16. methods according to claim 12, wherein the host upland cotton plant or germplasm are from breeding cotton variety or outer Come plant or the germplasm of cotton variety.

17. methods according to claim 12, wherein the host upland cotton plant or germplasm are upland cotton.

18. methods according to claim 12, wherein the host upland cotton plant or germplasm include root-knot nematode (RKN) resistance.

The upland cotton plant of 19. methods according to claim 18, wherein the Jing gene transgressions or germplasm with do not include RKN resistances Jing gene transgressions upland cotton plant or germplasm compare, with increase RN resistances.

20. methods according to claim 1, wherein the method include by represent detected mark data electric transmission or Electronic saving is in computer-readable medium.

21. methods according to claim 1, wherein the method include selecting the first upland cotton plant or germplasm.

22. methods according to claim 1, wherein the method include selecting the offspring of the first upland cotton plant or germplasm.

23. methods according to claim 21, including by the first selected upland cotton plant or germplasm and the second vegetable lamb or Germplasm hybridizes.

24. methods according to claim 23, wherein second vegetable lamb or germplasm are from breeding cotton variety or external The plant of cotton variety or germplasm.

A kind of 25. methods for producing the genetically modified plants with reniform nematode (RN) resistant phenotype, the method includes：

One or more exogenous nucleic acids are incorporated in target plant to produce genetically modified plants, wherein one or more external sources At least one of nucleic acid includes the endogenous nucleotide sequences from vegetable lamb, and the endogenous nucleotide sequences are planted in the cotton The mark being selected from the group with least one in thing is chain：

And other marks chain with least one of foregoing；Wherein the genetically modified plants of gained include RN resistances.

26. methods according to claim 25, wherein the target plant is vegetable lamb.

27. methods according to claim 25, wherein from the vegetable lamb the endogenous nucleotide sequences in the cotton It is chain with least one mark in plant so that the endogenous nucleotide sequences show the something lost with least one mark Pass recombination frequency and be no more than about 10%.

28. methods according to claim 25, wherein one or more exogenous nucleic acids are incorporated into into target plant including cotton Flowering plant hybridizes with target plant.

The system that 29. one kind are predicted to be the vegetable lamb with reniform nematode (RN) resistant phenotype for identification, the system bag Include：

One group mark physical prospecting pin or primer, it is configured to detection at least one and resists sex-linked mark with RN, wherein the mark Will thing is selected from the group：

And other marks chain with least one of foregoing；

Detector, it is configured to detect that one or more are defeated from the signal of the group mark physical prospecting pin or primer or its amplicon Go out, thereby identify the presence or absence of of the mark；And

By the presence or absence of system command being associated with RN resistant phenotypes of mark.

The system of 30. claims 29, the wherein detector detect one or more light transmittings, wherein the light transmitting Warning Mark Thing allele it is presence or absence of.

The system of 31. claims 29, wherein the instruction includes at least one reference table, the reference table includes at least one Presence or absence of the associating between low palmitic acid content phenotype of the mark allele of detected mark.

The system of 32. claims 29, the wherein system include the nucleic acid samples from plant or germplasm.