Diagnostic molecular markers for seed lot purity traits in soybeans

Abstract

The present invention is in the field of plant breeding. More specifically, the invention includes a method for breeding and selecting plants that uniform for one or more seed lot purity traits such as, such as distinct flower color, pubescence color, hilum color, and pod wall color. The invention further includes molecular markers associated with distinct flower color, pubescence color, hilum color, and pod wall color for uses in a breeding program.

Claims

1. A method of molecular marker assisted soybean breeding, the method comprising the steps of: a) genotyping two parental soybean plants to determine the allelic state of each parental soybean plant with regard to each of: (i) whether the parental soybean plant comprises the W1 Locus DD allelic form (W1 allele) represented by SEQ ID NO:1 or the W1 Locus II allelic form (w1 allele) represented by SEQ ID NO:2; (ii) whether the parental soybean plant comprises the T Locus CC or TT allelic form of the molecular marker represented by SEQ ID NO:8 (M0243191); (iii) whether the parental soybean plant comprises the Td Locus II allelic form represented by SEQ ID NO:13 or the Td Locus DD allelic form represented by SEQ ID NO:14; (iv) whether the parental soybean plant comprises the R locus AA or TT allelic form of the molecular marker represented by SEQ ID NO:19 (M0100925); and (v) for the haplotype defined by the molecular markers represented by SEQ ID NO:26 (M0202726), SEQ ID NO:33 (M0119618), and SEQ ID NO:40 (M0094170), whether the parental soybean plant comprises the L2 Locus AA or TT allelic form of the molecular marker represented by SEQ ID NO:26 (M0202726), whether the parental soybean plant comprises the L2 Locus AA or TT allelic form of the molecular marker represented by SEQ ID NO:33 (M0119618), and whether the parental soybean plant comprises the L2 Locus AA or GG allelic form of the molecular marker represented by SEQ ID NO:40 (M0094170); (b) crossing the two parental soybean plants genotyped in step (a) to generate an F.sub.1 population of soybean plants; (c) obtaining a DNA or RNA sample from a tissue of at least one F.sub.1 soybean plant of the F.sub.1 population generated by the cross in step (b); (d) determining by a nucleic acid based analyses, for at least the allelic forms in step (a) that differ between the two crossed parental plants, the allelic state of the F.sub.1 soybean plant; and (e) selecting based on the determination of allelic state in step d an F.sub.1 soybean plant for use in a soybean breeding program that is a true F.sub.1 soybean plant and not the result of self-pollination of one of the parental soybean plants.

2. The method of claim 1, further comprising: (f) self-crossing the true F.sub.1 soybean plant selected in step (e) to generate an F.sub.2 population of soybean plants; (g) obtaining a DNA or RNA sample from a tissue of at least one soybean plant of the F.sub.2 population generated by the cross in step (f) and: (i) determining whether the allelic state of the F.sub.2 soybean plant comprises the W1 Locus DD allelic form (W1 allele) represented by SEQ ID NO:1 or the W1 Locus II allelic form (w1 allele) represented by SEQ ID NO:2; (ii) determining whether the allelic state of the F.sub.2 soybean plant comprises the T Locus CC or TT allelic form of the molecular marker represented by SEQ ID NO:8 (M0243191); (iii) determining whether the allelic state of the F.sub.2 soybean plant comprises the Td Locus II allelic form represented by SEQ ID NO:13 or the Td Locus DD allelic form represented by SEQ ID NO:14; (iv) determining whether the allelic state of the F.sub.2 soybean plant comprises the R locus AA or TT allelic form of the molecular marker represented by SEQ ID NO:19 (M0100925); and (v) determining the allelic state for the haplotype defined by the molecular markers represented by SEQ ID NO:26 (M0202726), SEQ ID NO:33 (M0119618), and SEQ ID NO:40 (M0094170), whether the F.sub.2 soybean plant comprises the L2 Locus AA or TT allelic form of the molecular marker represented by SEQ ID NO:26 (M0202726), whether the F.sub.2 soybean plant comprises the L2 Locus AA or TT allelic form of the molecular marker represented by SEQ ID NO:33 (M0119618), and whether the F.sub.2 soybean plant comprises the L2 Locus AA or GG allelic form of the molecular marker represented by SEQ ID NO:40 (M0094170); and (g) selecting based on at least the determination of allelic state in (i), (ii), (iii), (iv), and (v) an F.sub.2 soybean plant that is homozygous and not segregating for the determined alleles.

3. The method of claim 2 further comprising self-crossing the selected F.sub.2 soybean plant to generate an F.sub.3 population of soybean plants.

4. The method of claim 3 further comprising determining the allelic state as determined in claim 2 of at least one generated F.sub.3 plant or any progeny or descendent thereof in a successive generation to determine whether the seed lot purity traits of flower, pubescence, hilum, and pod wall color have been fixed and/or to validate visual observations of flower color, pubescence color, pod wall color, and hilum color.

Description

I. DESCRIPTION OF THE INVENTION: DEFINITIONS

(1) The definitions and methods provided define the present invention and guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of common terms in molecular biology may also be found in Alberts et al., Molecular Biology of The Cell, 5.sup.th Edition, Garland Science Publishing, Inc.: New York, 2007; and Lewin, Genes IX, Oxford University Press: New York, 2007. The nomenclature for DNA bases as set forth at 37 CFR 1.822 is used.

(2) As used herein, an allele refers to one of at least two alternative forms of a genomic sequence at a given locus on a chromosome.

(3) As used herein, a homozygous allele is a locus on a chromosome having identical alleles for a signal trait.

(4) As used herein, a heterozygous allele is a locus on a chromosome having two different alleles for a signal trait.

(5) As used herein, a locus is a position on a genomic sequence that is usually found by a point of reference; e.g., a DNA sequence that is a gene, or part of a gene or intergenic region.

(6) As used herein, polymorphism means the presence of one or more variations of a nucleic acid sequence at one or more loci in a population of at least two members. The variation can comprise but is not limited to one or more nucleotide base substitutions, the insertion of one or more nucleotides, a nucleotide sequence inversion, and/or the deletion of one or more nucleotides. Exemplary examples of polymorphisms include single nucleotide polymorphisms (SNPs), insertions or deletions in DNA sequence (Indels), simple sequence repeats of DNA sequence (SSRs), a restriction fragment length polymorphism, and a tag SNP. A genetic marker, a gene, a DNA-derived sequence, a haplotype, a RNA-derived sequence, a promoter, a 5 untranslated region of a gene, a 3 untranslated region of a gene, microRNA, siRNA, a QTL, a satellite marker, a transgene, mRNA, ds mRNA, a transcriptional profile, and a methylation event may also comprise polymorphisms. In addition, the presence, absence, or variation in copy number of the preceding may comprise a polymorphism.

(7) As used herein, a marker is a detectable characteristic that can be used to discriminate between organisms. Examples of such characteristics may include genetic markers, biochemical markers, fermentation yield, fermentation efficiency, energy yield, secondary compounds, metabolites, morphological characteristics, and agronomic characteristics.

(8) As used herein, a marker assay is a method for detecting a polymorphism at a particular locus using a particular method. Exemplary examples of marker assays include measurement of at least one genotypic trait such as restriction fragment length polymorphism (RFLP), single base extension, electrophoresis, sequence alignment, allelic specific oligonucleotide hybridization (ASO), random amplified polymorphic DNA (RAPD), microarray-based polymorphism detection technologies, and nucleic acid sequencing technologies.

(9) As used herein, genotype means the genetic component of a phenotype that can be indirectly characterized using markers or directly characterized by nucleic acid sequencing.

(10) As used herein, genotyping refers to any method whereby the specific allelic form of a given genomic polymorphism is determined. For example, a single nucleotide polymorphism (SNP) can be genotyped by determining which nucleotide is present (i.e. an A, G, T, or C). Insertion/deletions (Indels) can be genotyped by determining if the Indel is present. Indels can be genotyped by a variety of assays including but not limited to marker assays.

(11) As used herein, the term adjacent, when used to describe a nucleic acid molecule that hybridizes to DNA containing a polymorphism, refers to a nucleic acid that hybridizes to DNA sequences that directly abut the polymorphic nucleotide base position. For example, a nucleic acid molecule that can be used in a single base extension assay is adjacent to the polymorphism.

(12) As used herein, interrogation position refers to a physical position on a solid support that can be queried to obtain genotyping data for one or more predetermined genomic polymorphisms.

(13) As used herein, a nucleic acid molecule is the complement of another nucleic acid molecule if they exhibit complete complementarity.

(14) As used herein, the term single nucleotide polymorphism, also referred to by the abbreviation SNP, constitutes a single base pair change, an insertion of one or more base pairs, or a deletion of one or more base pairs at a single site.

(15) As used herein, the term haplotype means a chromosomal region within a haplotype window defined by two or more polymorphic molecular markers.

(16) As used herein, the term haplotype window means a chromosomal region that is established by statistical analyses known to those of skill in the art and is in linkage disequilibrium. Thus, identity by state between two inbred individuals (or two gametes) at one or more molecular marker loci located within this region is taken as evidence of identity-by-descent of the entire region.

(17) As used herein, phenotype means the detectable characteristics of a cell or organism which can be influenced by genotype.

(18) As used herein, linkage refers to relative frequency at which types of gametes are produced in a cross. For example, if locus A has genes A or a and locus B has genes B or b and a cross between parent I with AABB and parent B with aabb will produce four possible gametes where the genes are segregated into AB, Ab, aB and ab. The null expectation is that there will be independent equal segregation into each of the four possible genotypes, i.e. with no linkage of the gametes will of each genotype. Segregation of gametes into genotypes differing from are attributed to linkage.

(19) As used herein, linkage disequilibrium is defined in the context of the relative frequency of gamete types in a population of many individuals in a single generation. If the frequency of allele A is p, a is p, B is q and b is q, then the expected frequency (with no linkage disequilibrium) of genotype AB is pq, Ab is pq, aB is pq and ab is pq. Any deviation from the expected frequency is called linkage disequilibrium. Two loci are considered genetically linked when they are in linkage disequilibrium.

(20) As used herein, quantitative trait locus (QTL) means a locus that controls to some degree numerically representable traits that are usually continuously distributed.

(21) As used herein, the term soybean means Glycine max and includes all plant varieties that can be bred with soybean, including wild soybean species.

(22) As used herein, the term elite line means any line that has resulted from breeding and selection for superior agronomic performance. Exemplary examples of elite soybean varieties that are commercially available to farmers or soybean breeders include AG00802, A0868, AG0902, A1923, AG2403, A2824, A3704, A4324, A5404, AG5903 and AG6202 (Asgrow Seeds, Des Moines, Iowa, USA); BPRO144RR, BPR 4077NRR and BPR 4390NRR (Bio Plant Research, Camp Point, Ill., USA); DKB17-51 and DKB37-51 (DeKalb Genetics, DeKalb, Ill., USA); and DP 4546 RR, and DP 7870 RR (Delta & Pine Land Company, Lubbock, Tex., USA); JG 03R501, JG 32R606C ADD and JG 55R503C (JGL Inc., Greencastle, Ind., USA); NKS13-K2 (NK Division of Syngenta Seeds, Golden Valley, Minn., USA); 90M01, 91M30, 92M33, 93M11, 94M30, 95M30 and 97B52 (Pioneer Hi-Bred International, Johnston, Iowa, USA); SG4771NRR and SG5161NRR/STS (Soygenetics, LLC, Lafayette, Ind., USA); S00-K5, S11-L2, S28-Y2, S43-B1, S53-A1, S76-L9 and S78-G6 (Syngenta Seeds, Henderson, Ky., USA). An elite plant is a representative plant from an elite variety.

II. DESCRIPTION OF THE INVENTION: OVERVIEW

(23) The present invention is an improvement over current methods of selecting soybean plants and seeds based on phenotypic characteristics because it provides methods to verify the accuracy of visual observations, such as field observations, that can be compromised by uncontrollable environmental conditions, human error, etc. In breeding operations, many resources may be wasted by investing in lines prior to discovering that the lines segregate for a desirable seed lot purity trait.

(24) The present invention is drawn to molecular markers to select for genetic loci associated seed lot purity traits. These genetic loci are: (i) the W1 locus containing the flavonoid 35 hydroxylase gene at linkage group F (chromosome 13); (ii) the T locus containing the flavonoid 3 hydroxylase gene at linkage group C2 (chromosome 6); (iii) the Td locus at linkage group N (chromosome 3); (iv) the R locus at linkage group K (chromosome 9); (v) the I locus at linkage group A2 (chromosome 8); and (vi) the L2 locus linkage group N (chromosome 3).

(25) TABLE-US-00001 TABLE 1 Markers spanning genomic regions associated with seed lot purity traits. Allelic form(s) Linkage Group Associated with (LG) SEQ ID Seed Lot Purity Locus Name (Chromosome) Marker Name Map Positions.sup.1 NO: Trait.sup.2 W1 Locus LG F (13) W1 allele 4552570-4557280 1 DD w1 allele 2 II T Locus LG C2 (6) M0243191.sup.3 18534618-18541507 8 CC or TT Td Locus LG N (3) 5644434-5647952 13 II 14 DD M006200746.sup.8 47152562-47152863 53 TT or GG M006200926.sup.9 47212539-47212840 54 TT or CC R Locus LG K (9) M0100925.sup.4 42903750-42905044 19 AA or TT M006934394.sup.19 42526481-42526782 58 AA or TT M006934399.sup.20 42533183-42533484 59 AA or GG M006934436.sup.21 42543758-42544059 60 AA or TT M006934505.sup.22 42563593-42563894 61 TT or CC M006934661.sup.23 42625198-42625499 62 AA or CC I Locus LG A2 (8) M006725263.sup.16 8356339-8356640 55 TT or CC M006725275.sup.17 8367092-8367393 56 TT or CC M006725283.sup.18 8372397-8372698 57 TT or CC L2 Locus LG N (3) M0202726.sup.5 583905-584201 26 AA or TT M0119618.sup.6 785516-786002 33 AA or TT M0094170.sup.7 950081-950475 40 AA or GG M006065284.sup.10 777977-778278 47 TT or AA M006065312.sup.11 789372-789673 48 AA or TT M006065346.sup.12 799426-799727 49 CC or AA M006065360.sup.13 816587-816888 50 TT or GG M006065367.sup.14 821682-821983 51 TT or CC M006065379.sup.15 829690-829991 52 GG or AA .sup.1The nucleotide positions of loci and markers is based on nucleotide positions of a physical map of soybean physical map of the linkage groups listed in column 2 of Table 1 (as described on the World Wide Web at soybase.org) and of Table 14 (Appendix to the Specification). Polymorphic nucleotide bases are designated in the sequence listing provided herewith according to the WIPO Standard ST.25 (1998), Table 1, as follows: r = g or a (purine); y = t/u or c (pyrimidine); m = a or c; (amino); k = g or t/u (keto); s = g or c (strong interactions 3 H-bonds); w = a or t/u (weak interactions 2H-bonds); b = g or c or t/u (not a); d = a or g or t/u (not c); h = a or c or t/u (not g); v = a or g or c (not t, not u); and n = a or g or c or t/u (unknown, or other; any.) .sup.2Both the maternal and paternal alleles of the single nucleotide polymorphisms that can be associated with a seed lot purity trait are shown. .sup.3The identified polymorphic allele of marker M0243191 is located at nucleotide 96 of SEQ ID NO: 8. .sup.4The identified polymorphic allele of marker M0100925 is located at nucleotide 137 of SEQ ID NO: 19. .sup.5The identified polymorphic allele of marker M0202726 is located at nucleotide 137 of SEQ ID NO: 26. .sup.6The identified polymorphic allele of marker M0119618 is located at nucleotide 48 of SEQ ID NO: 33. .sup.7The identified polymorphic allele of marker M0094170 is located at nucleotide 348 of SEQ ID NO: 40. .sup.8The identified polymorphic allele of marker M006200746 is located at nucleotide 201 of SEQ ID NO: 53. .sup.9The identified polymorphic allele of marker M006200926 is located at nucleotide 201 of SEQ ID NO: 54. .sup.10The identified polymorphic allele of marker M006065284 is located at nucleotide 201 of SEQ ID NO: 47. .sup.11The identified polymorphic allele of marker M006065312 is located at nucleotide 201 of SEQ ID NO: 48. .sup.12The identified polymorphic allele of marker M006065346 is located at nucleotide 201 of SEQ ID NO: 49. .sup.13The identified polymorphic allele of marker M006065360 is located at nucleotide 201 of SEQ ID NO: 50. .sup.14The identified polymorphic allele of marker M006065367 is located at nucleotide 201 of SEQ ID NO: 51. .sup.15The identified polymorphic allele of marker M006065379 is located at nucleotide 201 of SEQ ID NO: 52. .sup.16The identified polymorphic allele of marker M006725263 is located at nucleotide 201 of SEQ ID NO: 55. .sup.17The identified polymorphic allele of marker M006725275 is located at nucleotide 201 of SEQ ID NO: 56. .sup.18The identified polymorphic allele of marker M006725283 is located at nucleotide 201 of SEQ ID NO: 57. .sup.19The identified polymorphic allele of marker M006934394 is located at nucleotide 201 of SEQ ID NO: 58. .sup.20The identified polymorphic allele of marker M006934399 is located at nucleotide 201 of SEQ ID NO: 59. .sup.21The identified polymorphic allele of marker M006934436 is located at nucleotide 201 of SEQ ID NO: 60. .sup.22The identified polymorphic allele of marker M006934505 is located at nucleotide 201 of SEQ ID NO: 61. .sup.23The identified polymorphic allele of marker M006934661 is located at nucleotide 201 of SEQ ID NO: 62.

(26) SNP markers were discovered in or proximal to genes in soybean that determine flower color (W1 locus), pubescence color (T and Td loci), hilum color (R locus and I locus), and pod wall color (L2 locus). These molecular markers can be used in several stages of the breeding process to make breeding more efficient and more accurate. Illustrative examples of how such molecular markers can be used in the breeding process include distinguishing true hybridization events from self-pollinations and to separate plants that are fixed for the seed lot purity traits from those that are segregating. These molecular markers may be used in soybean breeding programs to increase the purity of seeds lots for commercialization.

III. GENOTYPES ASSOCIATED WITH PHENOTYPES

(27) (i): W1 LocusFlower color

(28) The gene flavonoid 35 hydroxylase controls flower pigmentation and is located within the W1 locus of linkage group F (chromosome 13). The two variant alleles of this gene were previously cloned and sequenced (Zabala & Vodkin, Crop Sci. 47(S2): S113-S124 (2007)). A sequence alignment of the two variant alleles illustrates a 53 base pair nucleotide insertion and a 10 base pair nucleotide substitution in the w1 allele relative to the W1 allele. The 53 nucleotide insertion occurs at nucleotide base position 4237 of the W1 allele followed by a substitution of 10 nucleotides (SEQ ID: 3) creating the w1 allele. For example, primers (SEQ ID: 4 and 5) and probes (SEQ ID: 6 and 7) were designed to distinguish the variant alleles (W1 and w1) of flavonoid 35 hydroxylase and thus could be used, for example, to identify, select, introgress, obtain, or produce lines differing in flower color phenotype. It is understood that other primers and probes may be developed to distinguish the variant alleles W1 and w1 and to determine the allelic state of a soybean plant with respect to a genotype associated with flower color phenotype. Detection of a deletion genotype, DD (W1 allele-SEQ ID: 1), corresponds to purple flower color and the detection of an insertion/substitution genotype, II (w1 allele-SEQ ID: 2), corresponds to white flower color. The data is presented in Table 2 and shows an exact correlation between the genotype and flower color phenotype.

(29) TABLE-US-00002 TABLE 2 Soybean lines Genotyped at the W1 Locus for Flower Color. Allelic Forms Associated with Flower Color Flower W1 (DD - SEQ ID NO: 1) Soybean Line Phenotype and w1 (II - SEQ IS NO: 2) AG0801 Purple DD AG0808 White II DKB10-52 White II AG1102 Purple DD AG2605 White II AG2606 Purple DD AG2909 White II AG3505 Purple DD DKB35-52 White II AG4403 Purple DD DKB46-51 White II AG5301 White II AG5501 Purple DD AG6702 Purple DD AG7201 White II
(ii) and (iii): T and Td LociPubescence Color

(30) Soybean pubescence color is controlled through the association of two loci, the T locus and the Td locus. All soybean plants have hair growing on the stem and leaves. This hair is referred to as pubescence, which expresses a definite color. Most soybean plants have either gray, tawny or light tawny colored pubescence.

(31) The T locus is located on linkage group C2 (chromosome 6) and contains the flavonoid 3 hydroxylase gene. Within the gene is a molecular marker, M0243191 (SEQ ID:8), which distinguishes tawny or light tawny pubescence color from a gray pubescence color. The identified polymorphic allele of marker M00243191 CC genotype can be associated with a tawny or light tawny pubescence color and a TT genotype can be associated with a gray pubescence color.

(32) The molecular marker associated with the T locusM0243191 (SEQ ID: 8)can be amplified, for example, using the primers indicated as SEQ ID NO: 9 and 10 and detected with probes indicated as SEQ ID NO: 11 and 12. It is understood that other primers and probes may be developed to determine the allelic state of this molecular marker and to, for example, identify, select, introgress, obtain, or produce a soybean plant with respect to a genotype associated with a certain pubescence color phenotype.

(33) The Td locus is located on linkage group is on linkage group N (chromosome 3) and is represented by SEQ ID: 13. The Td locus nucleotide sequence with the 12 base pair deletion is represented by SEQ ID: 14 and can distinguish a light tawny pubescence color (deletion genotype DD), from gray and tawny pubescence color (insertion genotype, II). The 12 base pair nucleotide deletion occurs at nucleotide base position 300 of SEQ ID: 13.

(34) In Table 3, 42 soybean lines were tested at the T and Td locus for pubescence color. In all tested soybean lines the T locus M0243191 marker, (SEQ ID: 8), distinguished tawny or light tawny pubescence color from a gray pubescence color. At the Td locus, the presence or absence of the 12 base pair deletion showed a near-perfect correlation between pubescence color genotypes and phenotypes. The presence or absence of the 12 base pair deletion can be amplified, for example, using the primers indicated as SEQ ID NO: 15 and SEQ ID NO: 16 and detected with probes indicated as SEQ ID NO: 17 and SEQ ID NO: 18. It is understood that other primers and probes may be developed to detect the presence or absence of this deletion and to determine the allelic variants of this marker to, for example, identify, select, introgress, obtain, or produce a soybean plant with respect to a genotype associated with a certain pubescence color phenotype.

(35) TABLE-US-00003 TABLE 3 Soybean Lines Tested for Pubescence Color at the T and Td Loci. Allelic Form of Allelic Form Associated Pubescence Marker with Td Loci Soybean Color Associated with T (SEQ ID: 13 II and Line Phenotype Loci (SEQ ID: 8) SEQ ID NO: 14 DD) 98820-33 Light tawny CC DD A3525 Gray TT II AG0801 Tawny CC II AG0808 Tawny CC II AG1102 Tawny CC II AG1702 Light Tawny CC DD AG2106 Light tawny CC DD AG2107 Gray TT II AG2110 Gray TT DD AG2204 Light tawny CC DD AG2406 Tawny CC II AG2605 Light tawny CC DD AG2606 Light tawny CC DD AG2802 Gray TT II AG2909 Gray TT DD AG3101 Gray TT II AG3205 Gray TT II AG3402 Tawny CC II AG3505 Gray TT II AG3705 Tawny CC II AG4005 Tawny CC II AG4303 Light tawny CC DD AG4403 Light tawny CC DD AG4801 Tawny CC II AG4903 Light tawny CC DD AG4907 Light tawny CC DD AG5301 Gray TT II AG5501 Gray TT II AG5606 Tawny CC II AG5803 Gray TT II AG6702 Tawny CC II AG7201 Tawny CC II AG7501 Gray TT II AG7502 Tawny CC II CST353 Light tawny CC DD CSTX365N Light tawny CC DD Dennison Light tawny CC DD DKB10-52 Light tawny CC DD DKB24-52 Light tawny CC DD DKB35-52 Light tawny CC DD DKB38-52 Gray TT II DKB46-51 Tawny CC II

(36) In another study, 772 soybean lines were evaluated at the T and Td locus for pubescence color. In all tested soybean lines in Table 15, the haplotype at the Td locus on linkage group is on linkage group N (chromosome 3) containing molecular markers M006200746 (SEQ ID NO: 53) and M006200926 (SEQ ID NO: 54), distinguished tawny or light tawny pubescence color from a gray pubescence color. At the Td locus, the presence or absence of the TT TT haplotype demonstrated a correlation between pubescence color genotypes and phenotypes for light tawny and tawny.

(37) TABLE-US-00004 TABLE 15 Soybean Lines Genotyped at the Td locus for Pubescence Color where the allelic state of the molecular marker represented by SEQ ID NO: 8 is CC. Haplotype Pubescence M006200746 M006200926 Number of Color (SEQ ID (SEQ ID Soybean Haplotype Phenotype NO: 53) NO: 54) Lines 1 Light TT TT 228 Tawny 2 Tawny GG TT 87 3 TT CC
(iv): R LocusHilum Color.

(38) The soybean seed hilum or eye is the point of attachment of the seed to the pod. Soybeans can be identified by the various hilum colors they express. Hilum colors include Black (B1), Brown (Br), Yellow (Y), Imperfect Black (Ib), Slate (Sl), Tan (Tn), Buff (Bf) and Gray (G). The R locus is located on linkage group K (chromosome 9). A molecular marker (M0100925-SEQ ID NO: 19) was identified that co-segregates with variability at the R locus The genotypic variation of the polymorphic molecular marker was tested among 177 soybean lines segregating for hilum color and the data is presented in Table 4.

(39) TABLE-US-00005 TABLE 4 Genotypic Variation Co-Segregates With Variation In Hilum Color. Genotype at M0100925 Genotype at M0100925 Hilum Color (SEQ ID NO: 19) = AA (SEQ ID NO: 19) = TT Brown 1 10 Black 83 1 Imperfect black 60 0 Yellow 0 1 Buff 18 3

(40) As shown in Table 4, the AA genotype is associated with black, imperfect black, and buff hila colors, and is linked to the R allele of the R locus. The TT genotype is associated with brown, yellow, and buff hila colors, and is linked to the r allele for the R locus, which has been demonstrated to influence these hila colors. Buff hila color can result from the presence or either the R or r allele, although more buff lines appear to possess the R allele.

(41) The molecular marker associated with the R locus (M0100925-SEQ ID NO: 19) can be amplified, for example, using the primers indicated as SEQ ID NO: 22 and 23 and detected with probes indicated as SEQ ID NO: 24 and 25. It is understood that other primers and probes may be developed to determine the allelic state of this molecular marker and to, for example, identify, select, introgress, obtain, or produce a soybean plant with respect to a genotype associated with a certain hilum color phenotype.

(42) In another marker-trait association study, genotypic variation among 211 soybean lines segregating for hilum color identified molecular markers at the R locus (linkage group K-chromosome 9) (SEQ ID NO: 55-57) and the I locus (linkage group A2-chromosome 8) (SEQ ID NO: 58-62). The preferred haplotypes for hilum color identification (BL, IB, BF, BR) are shown in Table 16. The results also show the interaction of other genes controlling hilum color in soybean seed: pubescence color (T, t), flower color, (W1, w1) (Fehr, W. R., 1978. Breeding. In: A. G. Norman (Ed.), Soybean, Physiology, Agronomy and Utilization, pp. 119-155. Academic Press, New York.).

(43) TABLE-US-00006 TABLE 16 The relationship of haplotypes for the molecular markers SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62 to hilum color. (The genotypes associated with pubescence color and flower color are described elsewhere herein). Haplotype Number of Hilum SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID Pubescence Flower Soybean Haplotype Color NO: 55 NO: 56 NO: 57 NO: 58 NO: 59 NO: 60 NO: 61 NO: 62 color Color Lines 1 BL CC TT TT AA GG AA TT AA Tawny White 68 2 CC TT TT AA GG AA TT AA Light Tawny Purple 3 IB CC TT TT AA GG AA TT AA Gray Purple 110 4 BF CC TT TT AA GG AA TT AA Gray White 19 5 CC TT TT TT AA TT CC CC Gray Purple 6 BR CC TT TT TT AA TT CC CC Light Tawny Purple 14 7 CC TT TT TT AA TT CC CC Tawny White
(vi): L2 LocusPod Wall Color.

(44) A marker-association study for the L2 locus for pod wall color consisted of a set of 2371 lines that had information for pod wall color and genotypes on linkage group N (chromosome 3). An analysis of variance indicated that three markers, M0202726 (SEQ ID NO: 26), M0119618 (SEQ ID: 33), and M0094170 (SEQ ID: 40), were significantly associated with pod wall color (P<0.0001). When the three molecular markers were combined into a haplotype, certain haplotypes were significantly associated with tan pod walls, and others were significantly associated with brown pod walls. This is illustrated in Table 5.

(45) TABLE-US-00007 TABLE5 MolecularmarkerhaplotypesM0202726(SEQID:26)/ M0119618(SEQID:33)/ M0094170(SEQID:40)forpodwallcolor. 2.Scores (Closerto1.0 indicatestan Haplotypedefinedby: 1.Num- podwallcolor, M0202726(SEQIDNO:26)/ berof closerto3.0 M0119618(SEQIDNO:33)/ Soybean indicatesbrown M0094170(SEQIDNO:40) Lines podwallcolor) AAGGAA 1324 1.2 AAGGGG 87 1.1 AAGTAG 37 1.6 AAGTGG 21 1.3 AATTAA 39 1.6 AATTGG 573 2.2 TTGGAA 25 2.6 TTTTAA 171 2.9 TTTTGG 94 2.7

(46) In Table 5, the first two and last two haplotypes are associated with tan and brown pod wall color. Several haplotypes are not clearly associated with one category. The haplotype (AA TT GG) has a score of 2.2, which indicates that it is slightly more predictive of brown pod walls, but the haplotype has almost as many soybean lines with tan pod walls.

(47) The molecular marker M0202726 (SEQ ID: 26) can be amplified, for example, using the primers indicated as SEQ ID NO: 29 and 30 and detected with probes indicated as SEQ ID NO: 31 and 32. The molecular marker M0119618 (SEQ ID: 33) can be amplified, for example, using the primers indicated as SEQ ID NO: 36 and 37 and detected with probes indicated as SEQ ID NO: 38 and 39. The molecular M0094170 (SEQ ID: 40) can be amplified, for example, using the primers indicated as SEQ ID NO: 43 and 44 and detected with probes indicated as SEQ ID NO: 45 and 46. It is understood that other primers and probes may be developed to determine the allelic state of the molecular markers comprising this haplotype and to, for example, identify, select, introgress, obtain, or produce a soybean plant with respect to a genotype associated with a certain pod wall color phenotype.

(48) A second marker-trait association study was conducted on the L2 locus for pod wall color on a set of 308 soybean lines. An analysis of variance indicated that six molecular markers, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, were highly significantly associated with pod wall color (P<0.001). When the three markers were combined into a haplotype, a distinct haplotype were associated with brown pod walls and another haplotype was associated with tan pod walls (Table 17).

(49) TABLE-US-00008 TABLE 17 The relationship of haplotypes for the molecular markers SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52 to pod wall color. Number of Pod Haplotype Soybean Wall SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID Lines Haplotype Color NO: 47 NO: 48 NO: 49 NO: 50 NO: 51 NO: 52 Evaluated 1 BR TT AA CC TT TT GG 190 2 TN AA TT AA GG CC AA 118 Haplotype 1 is with brown pod wall color. Haplotype 2 is associated with tan wall color.

IV

(50) The present invention further provides that a soybean plant is selected from the group consisting of members of the genus Glycine, more specifically from the group consisting of Glycine arenaria, Glycine argyrea, Glycine canescens, Glycine clandestine, Glycine curvata, Glycine cyrtoloba, Glycine falcate, Glycine latifolia, Glycine latrobeana, Glycine max, Glycine microphylla, Glycine pescadrensis, Glycine pindanica, Glycine rubiginosa, Glycine soja, Glycine sp., Glycine stenophita, Glycine tabacina and Glycine tomentella.

(51) It is further understood that a soybean plant of the present invention may exhibit the characteristics of any relative maturity group: 000, 00, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. An allele of a QTL can comprise multiple genes or other genetic factors even within a contiguous genomic region or linkage group, such as a haplotype. As used herein, an allele of a QTL can therefore encompass more than one gene or other genetic factor where each individual gene or genetic component is also capable of exhibiting allelic variation and where each gene or genetic factor is also capable of eliciting a phenotypic effect on the quantitative trait in question. In one aspect of the present invention the allele of a QTL comprises one or more genes or other genetic factors that are also capable of exhibiting allelic variation. The use of the term an allele of a QTL is thus not intended to exclude a QTL that comprises more than one gene or other genetic factor. Specifically, an allele of a QTL in the present invention can denote a haplotype within a haplotype window. A haplotype window is a contiguous genomic region that can be defined, and tracked, with a set of one or more polymorphic markers wherein the polymorphisms indicate identity by descent. A haplotype within that window can be defined by the unique fingerprint of alleles at each marker.

(52) The present invention also provides for parts of the plants of the present invention. Exemplary plant parts include seed, endosperm, ovule, and pollen. Plants or parts thereof of the present invention may be grown in culture and regenerated. Methods for the regeneration of Glycine max plants from various tissue types and methods for the tissue culture of Glycine max are known in the art (See, for example, Widholm et al., In Vitro Selection and Culture-induced Variation in Soybean, In Soybean: Genetics, Molecular Biology and Biotechnology, Eds. Verma and Shoemaker, CAB International, Wallingford, Oxon, England (1996). Regeneration techniques for plants such as Glycine max can use as the starting material a variety of tissue or cell types. With Glycine max in particular, regeneration processes have been developed that begin with certain differentiated tissue types such as meristems, Cartha et al., Can. J. Bot. 59:1671-1679 (1981), hypocotyl sections, Cameya et al., Plant Science Letters 21: 289-294 (1981), and stem node segments, Saka et al., Plant Science Letters, 19: 193-201 (1980); Cheng et al., Plant Science Letters, 19: 91-99 (1980). Regeneration of whole sexually mature Glycine max plants from somatic embryos generated from explants of immature Glycine max embryos has been reported (Ranch et al., In Vitro Cellular & Developmental Biology 21: 653-658 (1985). Regeneration of mature Glycine max plants from tissue culture by organogenesis and embryogenesis has also been reported (Barwale et al., Planta 167: 473-481 (1986); Wright et al., Plant Cell Reports 5: 150-154 (1986).

(53) In certain embodiments of the invention, a method of selecting for varietal purity in a soybean line, such as for a seed lot, comprises (A) crossing at least one first soybean plant comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO: 1, 2, 8, 13-14, 19, 26, 33, and 40 with at least one second soybean plant in order to form a population, (B) screening the population with one or more nucleic acid markers to determine if one or more soybean plants from the population contains the nucleic acid molecule, and (C) selecting from the population one or more soybean plants comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO: 1, 2, 8, 13-14, 19, 26, 33, and 40.

(54) The present invention also includes a method of introgressing an allele into a soybean plant comprising: (A) crossing at least two soybean plants in order to form a population; (B) screening the population with one or more nucleic acid markers to determine at least one allele at one or more of the loci W1, T, Td, R, I, or L2 of one or more soybean plants from the population and (C) bulk individuals from the population with similar alleles of at least one of the W1, T, Td, R, I, or L2 loci.

(55) The present invention includes isolated nucleic acid molecules. Such molecules include those nucleic acid molecules capable of detecting a polymorphism genetically or physically linked to the W1, T, Td, R, I, or L2 loci. Such nucleic acid molecules capable of detecting a polymorphism genetically or physically linked to the W1, T, Td, R, I, or L2 loci include SEQ ID NO: 1 through SEQ ID NO: 46, fragments thereof, complements thereof, and nucleic acid molecules capable of specifically hybridizing to one or more of these nucleic acid molecules.

(56) In certain embodiments of the invention, a nucleic acid molecule of the present invention includes those that will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1 through 46 or complements thereof or fragments of either under moderately stringent conditions, for example at about 2.0SSC and about 65 C. In certain embodiments of the invention, a nucleic acid of the present invention will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1 through 46 or complements or fragments of either under high stringency conditions. In certain embodiments of the invention, a marker nucleic acid molecule of the present invention comprises the nucleic acid sequence set forth in SEQ ID NO: 1 through 34 or complements thereof or fragments of either. In certain embodiments of the invention, a marker nucleic acid molecule of the present invention shares between 80% and 100% or 90% and 100% sequence identity with the nucleic acid sequences set forth in SEQ ID NO: 1 through 46 or complements thereof or fragments of either. In certain embodiments of the invention, a marker nucleic acid molecule of the present invention shares between 95% and 100% sequence identity with the sequences set forth in SEQ ID NO: 1 through 46 or complements thereof or fragments of either. In certain embodiments of the present invention, a marker nucleic acid molecule of the present invention shares between 98% and 100% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 1 through 46 or complement thereof or fragments of either.

V. MOLECULAR ASSISTED BREEDING TECHNIQUES

(57) Genetic markers that can be used in the practice of the instant invention include, but are not limited to, are Restriction Fragment Length Polymorphisms (RFLP), Amplified Fragment Length Polymorphisms (AFLP), Simple Sequence Repeats (SSR), Single Nucleotide Polymorphisms (SNP), Insertion/Deletion Polymorphisms (Indels), Variable Number Tandem Repeats (VNTR), and Random Amplified Polymorphic DNA (RAPD), and others known to those skilled in the art. Marker discovery and development in crops provides the initial framework for applications to marker-assisted breeding activities (US Patent Applications 2005/0204780, 2005/0216545, 2005/0218305, and 2006/00504538). The resulting genetic map is the representation of the relative position of characterized loci (DNA markers or any other locus for which alleles can be identified) along the chromosomes. The measure of distance on this map is relative to the frequency of crossover events between sister chromatids at meiosis.

(58) As a set, polymorphic markers serve as a useful tool for fingerprinting plants to inform the degree of identity of lines or varieties (U.S. Pat. No. 6,207,367). These markers form the basis for determining associations with phenotype and can be used to drive genetic gain. The implementation of marker-assisted selection is dependent on the ability to detect underlying genetic differences between individuals.

(59) Certain genetic markers for use in the present invention include dominant or codominant markers. Codominant markers reveal the presence of two or more alleles (two per diploid individual). Dominant markers reveal the presence of only a single allele. The presence of the dominant marker phenotype (e.g., a band of DNA) is an indication that one allele is present in either the homozygous or heterozygous condition. The absence of the dominant marker phenotype (e.g., absence of a DNA band) is merely evidence that some other undefined allele is present. In the case of populations where individuals are predominantly homozygous and loci are predominantly dimorphic, dominant and codominant markers can be equally valuable. As populations become more heterozygous and multiallelic, codominant markers often become more informative of the genotype than dominant markers.

(60) In another embodiment, markers that include. but are not limited, to single sequence repeat markers (SSR), AFLP markers, RFLP markers, RAPD markers, phenotypic markers, isozyme markers, single nucleotide polymorphisms (SNPs), insertions or deletions (Indels), single feature polymorphisms (SFPs, for example, as described in Borevitz et al. 2003 Gen. Res. 13:513-523), microarray transcription profiles, DNA-derived sequences, and RNA-derived sequences that are genetically linked to or correlated with seed purity, regions flanking seed purity loci, regions linked to seed purity, and/or regions that are unlinked to seed purity can be used in certain embodiments of the instant invention

(61) In one embodiment, nucleic acid-based analyses for determining the presence or absence of the genetic polymorphism (i.e. for genotyping) can be used for the selection of seeds in a breeding population. A wide variety of genetic markers for the analysis of genetic polymorphisms are available and known to those of skill in the art. The analysis may be used to select for genes, portions of genes, QTL, alleles, or genomic regions (Genotypes) that comprise or are linked to a genetic marker that is linked to or correlated with seed purity, regions flanking seed purity loci, regions linked to seed purity, and/or regions that are unlinked to seed purity can be used in certain embodiments of the instant invention.

(62) Herein, nucleic acid analysis methods include, but are not limited to, PCR-based detection methods (for example, TaqMan assays), microarray methods, mass spectrometry-based methods and/or nucleic acid sequencing methods. In one embodiment, the detection of polymorphic sites in a sample of DNA, RNA, or cDNA may be facilitated through the use of nucleic acid amplification methods. Such methods specifically increase the concentration of polynucleotides that span the polymorphic site, or include that site and sequences located either distal or proximal to it. Such amplified molecules can be readily detected by gel electrophoresis, fluorescence detection methods, or other means.

(63) A method of achieving such amplification employs the polymerase chain reaction (PCR) (Mullis et al. 1986 Cold Spring Harbor Symp. Quant. Biol. 51:263-273; European Patent 50,424; European Patent 84,796; European Patent 258,017; European Patent 237,362; European Patent 201,184; U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,582,788; and U.S. Pat. No. 4,683,194), using primer pairs that are capable of hybridizing to the proximal sequences that define a polymorphism in its double-stranded form.

(64) Methods for typing DNA based on mass spectrometry can also be used. Such methods are disclosed in U.S. Pat. Nos. 6,613,509 and 6,503,710, and references found therein. Polymorphisms in DNA sequences can be detected or typed by a variety of effective methods well known in the art including, but not limited to, those disclosed in U.S. Pat. Nos. 5,468,613, 5,217,863; 5,210,015; 5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876; 5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039; 7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of which are incorporated herein by reference in their entireties. However, the compositions and methods of the present invention can be used in conjunction with any polymorphism typing method to type polymorphisms in genomic DNA samples. These genomic DNA samples used include but are not limited to genomic DNA isolated directly from a plant, cloned genomic DNA, or amplified genomic DNA.

(65) For instance, polymorphisms in DNA sequences can be detected by hybridization to allele-specific oligonucleotide (ASO) probes as disclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No. 5,468,613 discloses allele specific oligonucleotide hybridizations where single or multiple nucleotide variations in nucleic acid sequence can be detected in nucleic acids by a process in which the sequence containing the nucleotide variation is amplified, spotted on a membrane and treated with a labeled sequence-specific oligonucleotide probe.

(66) Target nucleic acid sequence can also be detected by probe ligation methods as disclosed in U.S. Pat. No. 5,800,944 where sequence of interest is amplified and hybridized to probes followed by ligation to detect a labeled part of the probe.

(67) Microarrays can also be used for polymorphism detection, wherein oligonucleotide probe sets are assembled in an overlapping fashion to represent a single sequence such that a difference in the target sequence at one point would result in partial probe hybridization (Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al., Bioinformatics 21:3852-3858 (2005). On any one microarray, it is expected there will be a plurality of target sequences, which may represent genes and/or noncoding regions wherein each target sequence is represented by a series of overlapping oligonucleotides, rather than by a single probe. This platform provides for high throughput screening a plurality of polymorphisms. A single-feature polymorphism (SFP) is a polymorphism detected by a single probe in an oligonucleotide array, wherein a feature is a probe in the array. Typing of target sequences by microarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122; 6,913,879; and 6,996,476.

(68) Target nucleic acid sequence can also be detected by probe linking methods as disclosed in U.S. Pat. No. 5,616,464, employing at least one pair of probes having sequences homologous to adjacent portions of the target nucleic acid sequence and having side chains which non-covalently bind to form a stem upon base pairing of the probes to the target nucleic acid sequence. At least one of the side chains has a photoactivatable group which can form a covalent cross-link with the other side chain member of the stem.

(69) Other methods for detecting SNPs and Indels include single base extension (SBE) methods. Examples of SBE methods include, but are not limited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431; 5,595,890; 5,762,876; and 5,945,283. SBE methods are based on extension of a nucleotide primer that is adjacent to a polymorphism to incorporate a detectable nucleotide residue upon extension of the primer. In certain embodiments, the SBE method uses three synthetic oligonucleotides. Two of the oligonucleotides serve as PCR primers and are complementary to sequence of the locus of genomic DNA which flanks a region containing the polymorphism to be assayed. Following amplification of the region of the genome containing the polymorphism, the PCR product is mixed with the third oligonucleotide (called an extension primer) which is designed to hybridize to the amplified DNA adjacent to the polymorphism in the presence of DNA polymerase and two differentially labeled dideoxynucleosidetriphosphates.

(70) If the polymorphism is present on the template, one of the labeled dideoxynucleosidetriphosphates can be added to the primer in a single base chain extension. The allele present is then inferred by determining which of the two differential labels was added to the extension primer. Homozygous samples will result in only one of the two labeled bases being incorporated and thus only one of the two labels will be detected. Heterozygous samples have both alleles present, and will thus direct incorporation of both labels (into different molecules of the extension primer) and thus both labels will be detected.

(71) In another method for detecting polymorphisms, SNPs and Indels can be detected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930; and 6,030,787 in which an oligonucleotide probe having a 5 fluorescent reporter dye and a 3 quencher dye covalently linked to the 5 and 3 ends of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye results in the suppression of the reporter dye fluorescence, e.g. by Forster-type energy transfer. During PCR forward and reverse primers hybridize to a specific sequence of the target DNA flanking a polymorphism while the hybridization probe hybridizes to polymorphism-containing sequence within the amplified PCR product. In the subsequent PCR cycle DNA polymerase with 5.fwdarw.3 exonuclease activity cleaves the probe and separates the reporter dye from the quencher dye resulting in increased fluorescence of the reporter.

(72) In another embodiment, the locus or loci of interest can be directly sequenced using nucleic acid sequencing technologies. Methods for nucleic acid sequencing are known in the art and include technologies provided by 454 Life Sciences (Branford, Conn.), Agencourt Bioscience (Beverly, Mass.), Applied Biosystems (Foster City, Calif.), LI-COR Biosciences (Lincoln, Nebr.), NimbleGen Systems (Madison, Wis.), Illumina (San Diego, Calif.), and VisiGen Biotechnologies (Houston, Tex.). Such nucleic acid sequencing technologies comprise formats such as parallel bead arrays, sequencing by ligation, capillary electrophoresis, electronic microchips, biochips, microarrays, parallel microchips, and single-molecule arrays, as reviewed by R.F. Service Science 2006 311:1544-1546.

(73) The markers to be used in the methods of the present invention should preferably be diagnostic of origin in order for inferences to be made about subsequent populations. Experience to date suggests that SNP markers may be ideal for mapping because the likelihood that a particular SNP allele is derived from independent origins in the extant populations of a particular species is very low. As such, SNP markers appear to be useful for tracking and assisting introgression of QTLs, particularly in the case of Genotypes.

EXAMPLES

(74) The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1. Identification of Self-Pollinating Events in an F1 Generation

(75) Two lines are used as parents (Parent A and Parent B) of an F.sub.1 population. The parents differ in alleles at the W, T, Td, R, and L2 loci. Molecular markers from these loci can be used to distinguish F.sub.1 plants that are the result of a hybridization between the two parents (example: F.sub.1 #1) from plants that are the result of a self-pollination of Parent A (example: F.sub.1 #2) shown in Table 6.

(76) TABLE-US-00009 TABLE6 Self-pollinatingeventsinanF1generation. Line/ W T Td L2 R Plant locus locus locus locus locus Analysis Parent DD CC II AAGGAA TT A Parent II TT DD TTTTGG AA B F.sub.1#1 DI CT DI ATGTAG AT TrueF.sub.1 F.sub.1#2 DD CC II AAGGAA TT Self- polli- nation

Example 2. Early Selection of Seed Lot Purity Traits in a Soybean Breeding Program

(77) The seed lot purity traits of flower, pubescence, hilum, and pod wall color can be fixed as early as in the F2 generation in a breeding program. A breeder can use molecular marker assays to evaluate the progeny for the W1, T, Td, L2, and R loci. The breeder can maintain soybean plants that are homozygous at the W1, T, Td, L2, and R loci and discard soybean plants that are segregating for these seed lot purity traits. (Table 7).

(78) Two lines are used as parents (Parent A and Parent B) of an F.sub.2 population. A breeder can determine which plants are segregating for the traits associated with the W, T, Td, L2, and R loci to discard (e.g., F.sub.2 #1 and F.sub.2 #3 in Table 7) and which plants are homozygous for the traits to keep (e.g., F.sub.2 #2 and F.sub.2 #4 in Table 7).

(79) TABLE-US-00010 TABLE7 SelectionofSeedLotPurityTraitsofan F.sub.2population. Line/ W T Td L2 R Plant locus locus locus locus locus Analysis Parent DD CC II AAGGAA TT A Parent II TT DD TTTTGG AA B F.sub.2#1 DI TT DI ATGTAG TT Discard F.sub.2#2 II CC II AAGGAA AA Keep F.sub.2#3 DD CC DI TTTTGG AT Discard F.sub.2#4 DD TT II TTTTGG TT Keep

Example 3. Validation of Phenotype in the F6 Generation

(80) The environmental conditions, for example weather, can prevent an accurate determination of seed lot purity traits of F.sub.5:6 lines. Breeders can use molecular markers to validate their field observations or to confirm their breeder's notes as shown in Table 8.

(81) Markers for the traits associated with the W, T, Td, L2, and R loci can be used to validate visual observations of flower color, pubescence color, pod wall color, and hilum of F.sub.5:6 lines.

(82) TABLE-US-00011 TABLE 8 Validation of Phenotype in the F.sub.6 Generation Line Visual Observation Marker Data Result Action F.sub.5:6 #1 Pod wall is tan Pod wall is mixed Check the line in tan/brown the field again F.sub.5:6 #2 Flower color is Flower color is white Observation is white validated F.sub.5:6 #3 Pubescence is mixed Pubescence is gray Check the line in light tawny/gray the field again F.sub.5:6 #4 Hilum is brown Hilum is black Check the seed again

Example 4. Characterizing Flower Color Through the Use of Molecular Markers

(83) Soybean flower color is used as a classification characteristic to differentiate and describe soybean varieties. Flower colors are typically either purple or white, although there is some variation of color in wild perennial Glycine species and cultivars. Visual observation has been the gold standard used to determine flower color. Environmental factors such as temperature, moisture, and nutrient availability can contribute to phenotypic variation among soybean plant varieties; which can lead to errors in determining inherited traits and soybean plant variety purity. The gene flavonoid 35 hydroxylase controls flower pigmentation and is located within the W1 locus of linkage group F (chromosome 13). The two variant alleles of this gene were previously cloned and sequenced (Zabala & Vodkin, Crop Sci. 47(S2): S113-S124 (2007)). Alignment of the two genomic sequences suggested the mutation contains an insertion of 53 extra bases and a substitution of 10 nucleotides in the w1 allele relative to the W1 allele. Using this information, primers were designed to detect the variant alleles of flavonoid 35 hydroxylase among 16 soybean lines differing in flower color phenotype. Detection of a deletion (DD) (W1 allele-SEQ ID: 1) corresponds to purple flower color and the detection of an insertion (II) (w1 allele-SEQ ID: 2) corresponds to white flower color. The data is presented in Table 9 and shows an exact correlation between the genotype and flower color phenotype.

(84) TABLE-US-00012 TABLE 9 Soybean Lines Genotyped at the W1 Locus for Flower Color. Allelic Form Soybean Flower Associated with Line Phenotype Flower Color AG0801 Purple DD AG0808 White II DKB10-52 White II AG1102 Purple DD AG2605 White II AG2606 Purple DD AG2909 White II AG3505 Purple DD DKB35-52 White II AG4403 Purple DD DKB46-51 White II AG5301 White II AG5501 Purple DD AG6702 Purple DD AG7201 White II

Example 5. Characterizing Pubescence Color Through the Use of Molecular Markers

(85) Soybean pubescence color is controlled through the association of two loci, the T locus and the Td locus. The T locus is located on linkage group C2 (chromosome 6) and contains the flavonoid 3 hydroxylase gene. Within the gene is a molecular marker, M0243191 (SEQ ID: 8), which distinguishes tawny or light tawny pubescence color from a gray pubescence color. The identified polymorphic allele of marker M00243191 CC genotype can be associated with a tawny or light tawny pubescence color and a TT genotype can be associated with a gray pubescence color.

(86) The Td locus is located on linkage group is on linkage group N (chromosome 3). The detection of a 12 base pair deletion in the Td locus (see SEQ ID: 13 and SEQ ID: 14) can distinguish a light tawny pubescence color, (deletion genotype DD), from gray and tawny pubescence color (insertion genotype, II).

(87) In Table 10, 42 soybean lines were tested at the T and Td locus for pubescence color. In all tested soybean lines the T locus M0243191 marker, (SEQ ID: 8), distinguished tawny or light tawny pubescence color from a gray pubescence color. At the Td locus, the presence or absence of the 12 base pair deletion showed a near-perfect correlation between pubescence color genotypes and phenotypes.

(88) TABLE-US-00013 TABLE 10 Soybean Lines Genotyped at the T and Td loci for Pubescence Color. Allelic Form Allelic Form of Associated with Pubescence Marker Td Loci Color Associated with T (SEQ ID: 13 and Soybean Line Phenotype Loci (SEQ ID: 8) SEQ ID NO: 14) 98820-33 Light tawny CC DD A3525 Gray TT II AG0801 Tawny CC II AG0808 Tawny CC II AG1102 Tawny CC II AG1702 Light Tawny CC DD AG2106 Light tawny CC DD AG2107 Gray TT II AG2110 Gray TT DD AG2204 Light tawny CC DD AG2406 Tawny CC II AG2605 Light tawny CC DD AG2606 Light tawny CC DD AG2802 Gray TT II AG2909 Gray TT DD AG3101 Gray TT II AG3205 Gray TT II AG3402 Tawny CC II AG3505 Gray TT II AG3705 Tawny CC II AG4005 Tawny CC II AG4303 Light tawny CC DD AG4403 Light tawny CC DD AG4801 Tawny CC II AG4903 Light tawny CC DD AG4907 Light tawny CC DD AG5301 Gray TT II AG5501 Gray TT II AG5606 Tawny CC II AG5803 Gray TT II AG6702 Tawny CC II AG7201 Tawny CC II AG7501 Gray TT II AG7502 Tawny CC II CST353 Light tawny CC DD CSTX365N Light tawny CC DD Dennison Light tawny CC DD DKB10-52 Light tawny CC DD DKB24-52 Light tawny CC DD DKB35-52 Light tawny CC DD DKB38-52 Gray TT II DKB46-51 Tawny CC II

(89) In another study, 772 soybean line were tested at the T and Td locus for pubescence color. In all tested soybean lines in Table 18, the haplotype at the Td locus on linkage group is on linkage group N (chromosome 3) containing molecular markers M006200746 (SEQ ID NO: 53) and M006200926 (SEQ ID NO: 54), distinguished tawny or light tawny pubescence color from a gray pubescence color. At the Td locus, the presence or absence of the TT TT haplotype demonstrated a correlation between pubescence color genotypes and phenotypes for light tawny and tawny.

(90) TABLE-US-00014 TABLE 18 Soybean Lines Genotyped at the Td locus for Pubescence Color. Haplotype Pubescence M006200746 M006200926 Color (SEQ ID (SEQ ID Haplotype Phenotype NO: 53) NO: 54) # of Lines 1 Light TT TT 228 Tawny 2 Tawny GG TT 87 3 TT CC

Example 6. Characterizing Hilum Color Through the Use of Molecular Markers

(91) Soybean hilum color is a key classification characteristic used to describe soybean plant varieties. Hilum color can be used to identify a soybean plant variety and establish the purity of seed lots. Hilum color is classified as black, imperfect black, brown, reddish brown, gray, buff, or yellow and is determined by visual observations.

(92) Hilum is controlled by the interaction of five genes: pubescence color (T, t), flower color, (W1, w1) and genes controlling the distribution and color of pigmentation in the seed [(I, ii), (R, r), and (O, o)]. The molecular markers associated with pubescence color and flower color were described in earlier examples within this section.

(93) Marker-trait association studies were used to identify molecular markers that co-segregated with variation at the R locus. Genotypic variation among 177 lines segregating for hilum color was assessed in the region surrounding the R locus, which is on linkage group K (chromosome 9) of the public genomic map. The allelic variation at the marker SEQ ID NO: 19 co-segregated with differences in hilum color (Table 11).

(94) TABLE-US-00015 TABLE 11 Genotypic variation at SEQ ID NO: 19 co-segregates with variation in hilum color. Genotype at Genotype at SEQ ID NO: 19 = SEQ ID NO: 19 = Hilum Color AA TT Brown (BR) 1 10 Black (BL) 83 1 Imperfect black 60 0 (IB) Yellow (Y) 0 1 Buff (BF) 18 3

(95) The TT genotype is associated with brown, yellow, and buff hila colors. The TT genotype thus seems to be linked to the recessive r allele of the R locus, which has been demonstrated to influence these hila colors. The AA genotype is associated with black, imperfect black, and buff hila colors, and thus seems to be linked to the dominant R allele of the R locus. Buff hilum color can thus result from the presence of either the R or r allele, although more buff lines appear to possess the R allele. Based on flower color, pubescence color, and R locus, many of the classes of hilum color can be characterized through the use of molecular markers.

(96) In another marker-trait association study, genotypic variation among 211 soybean lines In another marker-trait association study, genotypic variation among 211 soybean lines segregating for hilum color identified molecular markers at the R locus (linkage group K-chromosome 9) (SEQ ID NO: 55-57) and the I locus (linkage group A2-chromosome 8) (SEQ ID NO: 58-62). The preferred haplotypes for hilum color identification (BL, IB, BF, BR) are shown in Table 19. The results also show the interaction of other genes controlling hilum color in soybean seed: pubescence color (T, t), flower color, (W1, w1) (Fehr, W. R., 1978. Breeding. In: A. G. Norman (Ed.), Soybean, Physiology, Agronomy and Utilization, pp. 119-155. Academic Press, New York.).

(97) TABLE-US-00016 TABLE 19 The relationship of haplotypes for the molecular markers SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62 to hilum color. Haplotype Number of Hilum SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID Pubescence Flower Soybean Haplotype Color NO: 55 NO: 56 NO: 57 NO: 58 NO: 59 NO: 60 NO: 61 NO: 62 color Color Lines 1 BL CC TT TT AA GG AA TT AA Tawny White 68 2 CC TT TT AA GG AA TT AA Light Tawny Purple 3 IB CC TT TT AA GG AA TT AA Gray Purple 110 4 BF CC TT TT AA GG AA TT AA Gray White 19 5 CC TT TT TT AA TT CC CC Gray Purple 6 BR CC TT TT TT AA TT CC CC Light Tawny Purple 14 7 CC TT TT TT AA TT CC CC Tawny White

Example 7. Characterizing Pod Wall Color Through the Use of Molecular Markers

(98) Pod wall color is a key classification characteristic used to describe soybean varieties. Pod wall color is generally classified as brown or tan. Visual observations are typically used to determine pod wall color. Approximately 5% of the selections advanced through to the first stage of elite yield testing are incorrectly identified as fixed for characteristic traits; many resources were invested in these soybean lines prior to realizing that the soybean lines were in fact segregating, often for pod wall color. Molecular marker associated with such purity marker traits will confirm or refute visual observations or discrepancies in the data.

(99) The L2 locus is associated with pod wall color. A marker-association study was conducted on the L2 locus for pod wall color on a set of 2371 soybean lines. An analysis of variance indicated that three molecular markers, SEQ ID NO: 26, SEQ ID NO: 33, and SEQ ID NO: 40 were highly significantly associated with pod wall color (P<0.0001). Pod color type was further rated for this study: Brown (BR)=1, Mixed (MX)=2, and Tan (TN)=3. When the three markers were combined into a haplotype, distinct haplotypes were significantly associated with Brown (BR) pod walls and other haplotypes were associated with Mixed (MX) and Tan (TN) pod walls (Table 12).

(100) TABLE-US-00017 TABLE 12 The relationship of haplotypes for the molecular markers SEQ ID NO: 26, SEQ ID NO: 33, and SEQ ID NO: 40 to pod wall color. Haplotype Number of Pod SEQ ID SEQ ID SEQ ID Soybean Haplotype Wall NO: 26 NO: 33 NO: 40 Lines Score 1 BR AA GG AA 1324 1.2 2 BR AA GG GG 87 1.1 3 MX AA GT AG 37 1.6 4 MX AA GT GG 21 1.3 5 MX AA TT AA 39 1.6 6 MX AA TT GG 573 2.2 7 MX TT GG AA 25 2.6 8 TN TT TT AA 171 2.9 9 TN TT TT GG 94 2.7

(101) A second marker-association study was conducted on the L2 locus for pod wall color on a set of 308 soybean lines. An analysis of variance indicated that six molecular markers, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, were highly significantly associated with pod wall color (P<0.001). When the three markers were combined into a haplotype, a distinct haplotype were associated with brown pod walls and another haplotype was associated with tan pod walls (Table 20).

(102) TABLE-US-00018 TABLE 20 The relationship of haplotypes for the molecular markers SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52 to pod wall color. Number of Pod Haplotype Soybean Wall SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID Lines Haplotype Color NO: 47 NO: 48 NO: 49 NO: 50 NO: 51 NO: 52 Evaluated 1 BR TT AA CC TT TT GG 190 2 TN AA TT AA GG CC AA 118 Haplotype 1 is with brown pod wall color. Haplotype 2 is associated with tan wall color.

Example 8. Uses for Molecular Markers Associated with Flower, Pubescence, Hilum, and Pod Wall Color

(103) The major morphological traits assess by seed certifying agencies are flower, pubescence, hilum, and pod wall color. As mentioned earlier, misclassification of these key seed lot purity traits in soybean can greatly delay the certification process and cost the seed producer financially. The invention is also useful in the process of soybean breeding.

(104) Having illustrated and described the principles of the present invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles.

APPENDIX TO THE SPECIFICATION

(105) TABLE-US-00019 TABLE 14 Linkage Group Locus/ (LG) Display (Chromosome) Start End Additional Locus Name (1) (2) Source (3) Base (4) Base (5) Information (6) AI973910 LG K (9) Glycine_max_release_2 42256806 42258847 COP8-like protein [Lilium longiflorum (Trumpet lily)] (SEQ ID: 20) BG045318 LG K (9) Glycine_soja_release_2 43505686 43506197 Hypothetical protein At2g44140 [Arabidopsis thaliana (Mouse- ear cress)] (SEQ ID: 21) AW459958 LG N (3) Glycine_max_release_2 460898 461355 Multi antimicrobial extrusion protein MatE [Medicago truncatula (Barrel medic)] (SEQ ID: 27) AW755424 LG N (3) Glycine_max_release_3 959341 961299 OSJNBa0065O17.11 protein [Oryza sativa (japonica cultivar-group)] (SEQ ID: 28) BF597543 LG N (3) Glycine_soja_release_2 557339 558689 Putative co- chaperone CGE1 isoform b [Oryza sativa (japonica cultivar-group)] (SEQ ID: 34) BU550813 LG N (3) Glycine_max_release_2 946496 947639 Calmodulin binding heat shock protein [Gossypium hirsutum (Upland cotton)] (SEQ ID: 35) BF597543 LG N (3) Glycine_soja_release_2 557339 558689 Putative co- chaperone CGE1 isoform b [Oryza sativa (japonica cultivar-group)] (SEQ ID: 41) TA53077_3847 LG N (3) Glycine_max_release_2 1713156 1716644 Splicing factor-like protein [Vitis riparia (Frost grape) (Vitis vulpina)] (SEQ ID: 42)