Methods and compositions for selecting soybean plants with favorable combinations of stem top and mature alleles

Abstract

The present disclosure relates to compositions and methods for identifying, selecting and / or producing soybean plants with limited stem top and late maturing. The disclosure also relates to compositions and methods for identifying and deselecting soybean plants having infinite stem tops and / or prematurity.

Description

Technical Field

[0001] The present disclosure relates to compositions and methods for identifying, selecting and / or producing soybean plants having a desired combination of alleles for late maturity and determinate stem apex. Sequence Listing

[0002] A sequence listing in XML format, named 82466-US-L-ORG-NAT-1.xml, generated on June 30, 2022, and approximately 64 kb in size, filed pursuant to 37 C.FR §1.831-1.835, is filed with this document. This sequence listing is hereby incorporated by reference into this specification for its disclosure. Background Art

[0003] Soybean (soybean / Glycine max L. Merr) is a major cash crop and investment commodity in North America and other regions. Soybean oil is one of the most widely used edible oils, and soybeans are used for both animal feed and human food production worldwide. Soybean cultivars can be classified based on a variety of phenotypic traits. One such trait is the stem top, which affects the growth habit of the plant stem. Another such trait is soybean maturity, which affects the number of days to flowering.

[0004] Most soybean cultivars can be divided into two major categories of stem apices: determinate and indeterminate. In indeterminate cultivars, the apical meristem at the tips of the stems and branches maintains vegetative activity until the demand for photosynthates from the developing seeds causes cessation of vegetative dry matter production. In contrast, for determinate cultivars, the apical meristem ceases vegetative activity at or shortly after photoperiod-induced floral induction, and the meristem then becomes a reproductive inflorescence. The stem apices are known to have an effect on plant height, node production, water use efficiency, and soybean yield. One of the genes that plays a role in the stem apices is the Dt1 gene on chromosome 19 of the soybean genome. Classical genetic analysis of soybean stem apices habit revealed a recessive allele at the determinate stem (Dt1) locus. Using pea orthologs to extract coding sequences, expression data, and gene silencing, Dt1 has been identified as an ortholog of Arabidopsis TERMINAL FLOWER1 (GmTFL1b) (Liu et al., Plant Physiology, 153:198-210 (2010)). Indefinite cultivars are more widely adapted to adverse environmental conditions and are more stable. Molecular markers associated with the indefinite allele at the Dt1 locus have been disclosed in U.S. Pat. No. 10,517,242, the contents of which are incorporated herein by reference in their entirety.

[0005] Soybean maturity refers to a growth trait involved in controlling flowering time (that is, photoperiod-induced floral induction) and plant maturation, with early maturity associated with shorter flowering days and late maturity associated with more flowering days. It has been reported that at least eleven major E genes are involved in controlling flowering time and maturity of soybean, of which E1-E4 are major genes whose dominant alleles can delay maturity (Tsubokura et al., Annuals of Botany, 113(3):429-441(2014)). The E1 locus is confined to a 17.4 kb region containing an intronless gene and identified by positional cloning (Xia et al., PNAS, 109:E2155-E2164, (2012)). This gene (E1 gene) encodes a protein containing a putative two-component nuclear localization signal and a region distant from the B3 domain, indicating that the protein is a novel transcription factor. The E1 gene is believed to be a repressor of the expression of GmFT2a and GmFT5a (both orthologs of Arabidopsis FLOWERING LOCUS T (FT)) under the regulation of the E3 and E4 loci (Xia et al., PNAS, 109: E2155-E2164, (2012)) (Kong et al., Plant Physiology, 154: 1220-1231 (2010)). Xia et al. also disclosed allelic variation of the E1 gene. The E1 maturation gene regulates the number of days required for photoperiod-induced floral induction.

[0006] Typically, stem apex and soybean maturity are evaluated by phenotypic analysis, which can be time consuming, subjective, and can only be performed late in a breeding program. When evaluating segregating populations developed from breeding crosses involving parental lines with different allele combinations for stem apex and maturity genes, errors in the selection of the desired phenotype (due to variation in growth habit and flowering traits) can lead to breeding selection errors. Summary of the invention

[0007] This paper discloses a combination of molecular markers associated with the E1 mature locus and the Dt1 stem top locus to provide a more consistent and reliable method for selecting plants with favorable or desired phenotypes. In some instances, the method disclosed herein can be used to identify and / or select soybean plants with a favorable allele combination of dominant alleles (E1 / E1) at the E1 locus and recessive alleles (dt1 / dt1) at the Dt1 locus, thereby obtaining plants with a favorable phenotypic combination of limited stem growth and delayed maturity (or more flowering days). Plants identified and / or selected by this method can be advanced in the early stages of a breeding process. In other examples, the methods disclosed herein can be used to identify and / or select soybean plants having unfavorable allele combinations such as recessive alleles (e1 / e1) at the E1 locus and recessive alleles (dt1 / dt1) at the Dt1 locus, thereby obtaining plants having unfavorable phenotypic combinations such as a combination of limited stem growth and early maturity (or fewer days to flowering). Other unfavorable phenotypic combinations can also be identified and selected. Plants identified and / or selected by this method can be removed at an early stage of the breeding process.

[0008] This paper also discloses a composition comprising a nucleic acid-based molecular marker associated with a favorable allele combination, which can also be used to select a plant with a favorable phenotype produced by the favorable allele combination. In some instances, the composition comprises a marker associated with a favorable allele combination of a dominant allele (E1 / E1) at the E1 locus and a recessive allele (dt1 / dt1) at the Dt1 locus, thereby allowing the selection of a soybean plant with a favorable phenotypic combination of limited stem growth and delayed maturity (or more days to bloom). The composition can be used to select a plant with a favorable allele combination and / or to select a plant with an unfavorable allele combination.

[0009] The use of molecular markers associated with soybean stem apex and maturity will support soybean breeding programs in early breeding stages for selection, and will ensure that only plants with the desired alleles are advanced to later testing. Moreover, by eliminating the need for expensive low-throughput phenotyping in the early breeding stages, it is expected to improve cost efficiency, thereby increasing the accuracy of selection and maximizing the investment value of field trials. From the perspective of trait introgression, molecular marker combinations can be deployed for rapid introgression of both GM and natural traits; and the expansion of allele frequencies of natural traits in a broad germplasm base.

[0010] Embodiments of the method include identifying and / or selecting soybean plants having a genotype associated with a late-determinate growth phenotype, the method comprising obtaining a nucleic acid (e.g., DNA or RNA) sample from a soybean plant or a portion thereof; detecting a molecular marker combination in the obtained nucleic acid, the molecular marker combination comprising: (1) a G at position 335 of SEQ ID NO:1; and (2) a T at position 260 of SEQ ID NO:2; and selecting at least one soybean plant based on the presence of the molecular marker combination, wherein the molecular marker combination is associated with a desired late-determinate growth phenotype.

[0011] The present disclosure also encompasses a method of introgressing a favorable allele combination of a dominant allele at the E1 locus and a recessive allele at the Dt1 locus into the germplasm of a soybean plant that does not contain the favorable allele combination by crossing a donor parent soybean plant containing the favorable allele combination with a recurrent parent soybean plant that does not contain the favorable allele combination to produce progeny plants; selecting progeny plants for backcrossing with the recurrent parent soybean plant based on the presence of a first marker associated with the dominant allele at the E1 locus and a second marker associated with the recessive allele at the Dt1 locus in the progeny plant genome, wherein the first marker comprises a G at position 335 of SEQ ID NO:1 and the second marker comprises a T at position 260 of SEQ ID NO:2; and backcrossing the selected progeny plants with the recurrent parent soybean plant to generate further progeny plants having the desired allele combination.

[0012] Also disclosed are compositions and / or kits comprising at least two amplification primer pairs capable of initiating DNA polymerization on a soybean nucleic acid template by a DNA polymerase and amplifying nucleic acid from the template to generate soybean marker amplicons, wherein the soybean amplicons can be used to identify the presence of a combination of molecular markers associated with a dominant allele at the E1 locus and a recessive allele at the Dt1 locus. Such compositions comprise (i) a first primer pair comprising a nucleotide sequence as shown in SEQ ID NOs: 1 and 2; and (ii) a second primer pair comprising a nucleotide sequence as shown in SEQ ID NOs: 3 and 4. In additional embodiments, the composition further comprises a label probe pair for identifying the label amplicon, wherein the label probe comprises SEQ ID NO: 7 and SEQ ID NO: 9 or SEQ ID NO: 8 and SEQ ID NO: 10.

[0013] These and other aspects of the invention are set forth in more detail in the description of the invention below. Sequence Description

[0014] SEQ ID NO: 1 is the DNA sequence of molecular marker SY3970 associated with the stem apex allele at the Dtl1 locus of the soybean genome.

[0015] SEQ ID NO: 2 is the DNA sequence of the molecular marker SY0768BQ associated with the early maturity allele at the E1 locus of the soybean genome.

[0016] SEQ ID NO: 3 is the forward primer of SY3970 labeling.

[0017] SEQ ID NO: 4 is the reverse primer of SY3970 labeling.

[0018] SEQ ID NO: 5 is the SY0768BQ labeled forward primer.

[0019] SEQ ID NO: 6 is the SY0768BQ labeled reverse primer.

[0020] SEQ ID NO: 7 is the first labeled probe labeled with SY03970.

[0021] SEQ ID NO: 8 is the second labeled probe labeled with SY03970.

[0022] SEQ ID NO: 9 is the SY0768BQ labeled first label probe.

[0023] SEQ ID NO: 10 is a SY0768BQ labeled second label probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Different developmental stages of a soybean plant are shown. definition

[0025] While it is believed that the following terms are well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate understanding of the subject matter of the present disclosure.

[0026] Compositions and methods are provided for identifying, selecting, and producing soybean plants having favorable allele combinations for determinate stem apex and late maturity.

[0027] As used herein, the term "limited type" refers to a favorable phenotype, in which the soybean apical meristem stops vegetative activity during or shortly after the flowering induction induced by the photoperiod, and then the meristem becomes a reproductive inflorescence. Therefore, "limited growth habit" or "limited stem top phenotype" refers to the cessation of vegetative growth after the main stem terminates in a cluster of flowers. In contrast, the term "unlimited type" refers to an unfavorable phenotype, in which the soybean apical meristem at the stem and branch top maintains vegetative activity until the demand for photosynthetic products of the developing seeds causes the nutrient dry matter production to stop. Therefore, "unlimited growth habit" or "unlimited stem top phenotype" refers to the simultaneous development of leaves and flowers in a part of its entire reproductive period, with one to three pods at the terminal apex. In an embodiment of the present invention, the marker disclosed herein can be used to select, identify or produce a soybean plant with a limited type phenotype, or a plant breeder can use a marker as disclosed herein to eliminate a plant line with an unlimited type phenotype.

[0028] As used herein, "Dt1 gene", "Dt1 locus", or its alleles are referenced to GmDt1 or Glyma.19G194300 (www.soybase.org), which is associated with the stem apex and is located on chromosome 19. Dt1 affects the stem apex, which in turn has a significant effect on plant height, flowering time, node production, maturity time, water use efficiency, and soybean yield. In general, indeterminate cultivars are more adaptable and more stable to adverse environmental conditions.

[0029] As used herein, "E1 gene", "E1 locus" or its alleles are referenced to GmE1 or Glyma.06G207800 (www.soybase.org), which is associated with flowering time and maturity and is located in the pericentromeric region of chromosome 6. The E1 gene is intronless and encodes an E1 protein that contains a putative two-component nuclear localization signal (NLS) and a domain (B3-like domain) that is distantly related to the plant-specific B3 domain (Xia, ZJ et al. Proc Natl Acad Sci USA. [Proceedings of the National Academy of Sciences of the United States] 109, E2155-E2164 (2012)). E1 is a transcription factor. E1 is considered to be a contributor to flowering time variation in soybean cultivars. In addition to its effects on post-flowering responses, E1 also has an effect on pre-flowering development. E1 is expressed in a dual mode, with expression under long day (LD) conditions being higher than expression under short day (SD) conditions. E1 is a putative transcription factor (TF) that negatively controls GmFT2a and GmFT5a to delay flowering in a background with functional PHYA genes (E3, E4) and LD conditions.

[0030] In an embodiment of the present invention, a marker associated with the Dt1 locus of the soybean genome, particularly a marker associated with a recessive allele at the Dt1 locus, can be used in combination with a marker associated with the E1 locus, particularly a marker associated with a dominant allele at the E1 locus, to identify or select plants having a favorable late-maturing determinate phenotype. In a specific embodiment, the marker associated with the recessive allele at the Dt1 locus and responsible for the favorable determinate phenotype is a marker of SEQ ID NO: 1 with a G at position 335 (also referred to herein as molecular marker SY3970), and the marker associated with the dominant allele at the E1 locus and responsible for the favorable late-maturing phenotype is a marker of SEQ ID NO: 2 with a T at position 260 (also referred to herein as molecular marker SY0768BQ). The use of a molecule associated with the Dt1 locus and a molecule associated with the E1 mature locus in combination enables one to select, identify or produce a soybean plant having a favorable late-maturing determinate phenotype. Additionally or alternatively, the use of molecules associated with the Dt1 locus in combination with molecular markers associated with the El maturity locus enables one to select, identify and eliminate soybean plants having an unfavorable early determinate phenotype, an unfavorable early indeterminate phenotype, or an unfavorable late indeterminate phenotype.

[0031] As used herein, the general phrase "late maturity" refers to a phenotype in which a soybean plant has an increased "flowering time" or number of days to flowering compared to a soybean plant that does not have the phenotype, while "early maturity" refers to a phenotype in which a soybean plant has a decreased flowering time or number of days to flowering compared to a soybean plant that does not have the phenotype. The exact number of days increased or decreased relative to a control plant comprising a wild-type allele at the relevant locus may vary.

[0032] For example, the markers disclosed herein can be used to select, identify or generate soybean plants with a late maturing phenotype, or plant breeders can use markers as disclosed herein to eliminate plant lines that have an early maturing phenotype compared to wild-type plants. In addition to flowering time, other aspects of the maturity phenotype can be described using time after flowering, relative maturity stage, maturity time, maturity stage group, and the number of days from flowering to the onset of maturity in soybean plants. Examples of different growth stages are provided in Figure 1 middle.

[0033] As used herein, "flowering time" or "days to flowering" is an estimate of the duration (e.g., in hours, days, weeks, etc.) that elapses between the onset of first flowering and seed emergence. In specific embodiments, flowering time is defined as the number of days that elapse for a soybean plant to transition from the VE stage (e.g., seed emergence, where cotyledons have pushed through the soil surface for at least 50% of the seeds) to the R1 stage (e.g., the onset of flowering, where at least 50% of the plants have at least one flower at any node). In specific embodiments, "maturity time" or post-flowering time is defined as the number of days that elapse for a soybean plant to transition from the R1 stage (e.g., the onset of flowering, where there is one open flower at any node on the main stem) to the R7 stage (where any pod has reached its mature pod color) or from the R1 stage to the R8 stage (where 95% of the pods have reached their mature pod color). Descriptions of the various developmental stages of soybean plants and mature pod colors are provided in Figure 1 Provided as a reference.

[0034] In an embodiment, a plant having an allele associated with a late maturing phenotype has a flowering time that is between 1 and 10 days longer or later than a control plant (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days longer than a control plant). In an embodiment, a plant having an allele associated with an early maturing phenotype has a flowering time that is between 1 and 10 days shorter or earlier than a control plant (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days shorter than a control plant).

[0035] As used herein, the term "a / an" or "the" can refer to one or more than one. For example, "a" marker (e.g., SNP, QTL, haplotype) can mean one marker or multiple markers (e.g., 2, 3, 4, 5, 6, etc.).

[0036] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0037] As used herein, the term "about" when used with respect to a measurable value (such as an amount of mass, dose, time, temperature, etc.) is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0038] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim will be interpreted as covering the specified materials or steps mentioned in the claim and those materials or steps that do not materially affect one or more basic and novel features of the claimed invention. Therefore, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising".

[0039] As used herein, the term "allele" refers to a variant or alternative nucleotide sequence at a gene or a specific genetic locus. Such an allele can be considered to be (i) wild type or (ii) mutant if there are one or more mutations or edits in the nucleotide sequence of the mutant allele relative to the wild-type allele. In diploids, a single allele at each locus is inherited by the offspring individual from each parent, respectively. Although one of ordinary skill in the art understands that the allele in any particular individual does not necessarily represent all alleles present in the species, the two alleles present in a given locus in a diploid organism occupy a corresponding position on a pair of homologous chromosomes.

[0040] As used herein, the terms "desired allele," "favorable allele," "target allele," and / or "purpose allele" are used interchangeably to refer to an allele associated with a desired trait. In some embodiments, the desired allele may be associated with an increase or decrease (relative to a control) in a given trait, depending on the nature of the desired phenotype. In some embodiments of the invention, the phrases "desired allele," "target allele," or "purpose allele" refer to one or more alleles in a soybean plant that are associated with a late maturing and / or determinate stem trait (relative to a control soybean plant that does not have the one or more target alleles).

[0041] With respect to wild-type allele, the mutant allele of gene can have gene activity or expression level that reduces or eliminates.For diploid organisms (such as corn and soybean), the first allele can appear on a chromosome, and the second allele can appear at the same locus on the second homologous chromosome.If an allele at the locus on a chromosome of plant is a mutant allele, and another corresponding allele on the homologous chromosome of plant is wild-type, then plant is described as heterozygous to mutant allele.But if two alleles at the locus are mutant alleles, then plant is described as homozygous to mutant allele.To the mutant allele at the locus, be homozygous plant can comprise identical mutant allele or different mutant allele (if heteroallele or double allele).

[0042] "Allelic variation" refers to the variation phenomenon of allele sequence form on a given genetic locus. Allelic variation results in the production of two or more allele variants. Variant can be naturally occurring and reflects the genetic differences between individuals of the same species. Such natural variation may occur due to natural breeding patterns. Alternatively, variant can be non-naturally occurring and is artificially (e.g., created by breeders or scientists), such as using mutagenesis and / or gene editing techniques. "Dominant allele" is an allele that affects the corresponding trait when present in a single copy (heterozygous) or double copy (homozygous). Therefore, "dominant allele at E1 locus" is an E1 allele that causes a late-maturing phenotype when present in a single copy or double copy. "Recessive mature allele at E1 locus" is an allele that affects the maturation of plants only when present in double copies (homozygous), thereby causing an early-maturing phenotype, and does not cause an early-maturing phenotype when present in a single copy (heterozygous). A "dominant allele at the Dt1 locus" is a Dt1 allele that results in an indeterminate stem top phenotype when present in either single or double copies. A "recessive maturity allele at the Dt1 locus" is an allele that affects plant maturity only when present in double copies (homozygous), resulting in a determinate stem top phenotype.

[0043] As used herein, "allele combination" refers to a specific combination of alleles present at more than one specific position or locus. Embodiments of the present invention include methods for identifying the presence of favorable or desired allele combinations at the E1 and Dt1 loci in the soybean plant genome using molecular markers. Such desired allele combinations include recessive alleles at the Dt1 locus that result in a limited stem top phenotype, and dominant alleles at the E1 locus that result in a late maturing phenotype. Embodiments of the present invention also include methods for identifying the absence of favorable allele combinations and / or the presence of unfavorable or undesirable allele combinations at the E1 and Dt1 loci in the soybean plant genome using molecular markers. Non-limiting examples of undesirable allele combinations at the E1 and Dt1 loci include: (i) a recessive allele at the Dt1 locus that causes a determinate stem top phenotype, and a recessive allele at the E1 locus that causes an early maturing phenotype; (ii) a dominant allele at the Dt1 locus that causes an indeterminate stem top phenotype, and a dominant allele at the E1 locus that causes a late maturing phenotype; and (iii) a dominant allele at the Dt1 locus that causes an indeterminate stem top phenotype, and a recessive allele at the E1 locus that causes an early maturing phenotype.

[0044] In an embodiment of the present invention, the allele combination of a plant at the E1 and Dt1 loci can be determined via a molecular marker-based assay, such as a first assay of plant DNA indicating the presence of a recessive allele at the Dt1 locus and a second assay of DNA indicating the presence of a dominant allele at the E1 locus. The first assay can be performed using a primer pair for a molecular marker associated with a recessive allele at the Dt1 locus, such as a primer pair of SEQ ID NOs: 3-4 for detecting molecular marker SY3970 (SEQ ID NO: 1 with a G at position 335). Alternatively, the first assay can be performed using a probe for a molecular marker associated with a recessive allele at the Dt1 locus, such as a probe of SEQ ID NOs: 7 or 8 for detecting molecular marker SY3970 (SEQ ID NO: 1 with a G at position 335). The second assay can be performed using a primer pair for a molecular marker associated with a dominant allele at the E1 locus, such as a primer pair of SEQ ID NOs: 5-6 for detecting the molecular marker SY0768BQ (SEQ ID NO: 2 with a T at position 260). Alternatively, the second assay can be performed using a probe for a molecular marker associated with a recessive allele at the E1 locus, such as a probe of SEQ ID NOs: 9 or 10 for detecting the molecular marker SY0768BQ (SEQ ID NO: 2 with a T at position 260).

[0045] A "locus" is a position on a chromosome where a gene or marker or allele is located. In some embodiments, a locus can encompass one or more nucleotides. In a specific embodiment of the invention, a locus is defined with reference to the 8X public construct of the Williams 82 soybean genome on the SoyBase internet resource (soybase.org / SequenceIntro.php) or on the USDA (bfgl.anri.barc.usda.gov / cgi-bin / soybean / Linkage.pl).

[0046] When a trait is linked to a marker and when the presence of the marker indicates whether and / or to what extent the desired trait or trait form will occur in a plant / germplasm containing the marker, the marker is "associated" with the trait. Similarly, when a marker is linked to an allele or a chromosome interval and when the presence of the marker indicates whether the allele or chromosome interval is present in a plant / germplasm containing the marker, the marker is "associated" with the allele or chromosome interval. For example, a "marker associated with late maturity" refers to a marker whose presence or absence can be used to predict whether a soybean plant has a late maturing phenotype with an increased number of days before flowering. Similarly, a "marker associated with a finite trait" refers to a marker whose presence or absence can be used to predict whether a soybean plant has a finite stem phenotype (in which the apical meristem stops vegetative activity at or shortly after photoperiod-induced floral induction and then becomes a reproductive inflorescence).

[0047] As used herein, the terms "backcross" and "backcrossing" refer to a process whereby a progeny plant is backcrossed to one of its parents one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8 times, etc.). In a backcrossing protocol, the "donor" parent refers to the parent plant that has the desired gene or locus to be introgressed. The "recipient" parent (used one or more times) or the "recurrent" parent (used two or more times) refers to the parent plant into which the gene or locus is introgressed. See, for example, Ragot, M. et al., Marker-assisted Backcrossing: A Practical Example, Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted Selection in Backcross Breeding, Proceedings of the Symposium "Analysis of Molecular Marker Data", pp. 41-43 (1994). The initial cross produces the F1 generation. The term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on.

[0048] As used herein, the term "cross" or "crossed" refers to the fusion of gametes through pollination to produce progeny (e.g., cells, seeds, or plants). The term encompasses both sexual crossing (one plant being pollinated by another) and selfing (self-pollination, e.g., when the pollen and ovules are from the same plant). The term "crossing" refers to the act of fusing gametes through pollination to produce progeny.

[0049] As used herein, the terms "cultivar" and "variety" refer to a group of similar plants that can be distinguished from other varieties within the same species by structural or genetic characteristics and / or performance.

[0050] As used herein, the term "elite" and / or "elite line" refers to any line that is essentially homozygous and is the result of breeding and selection for desired agronomic performance.

[0051] As used herein, the terms "exotic," "exotic strains," and "exotic germplasm" refer to any plant, strain, or germplasm that is not superior. Typically, an exotic plant / germplasm is not derived from any known superior plant or germplasm, but is selected to introduce one or more desired genetic elements into a breeding program (e.g., to introduce novel alleles into a breeding program).

[0052] As used herein, the term "dominant" refers to a variant or allele of a gene that masks or overshadows the phenotypic effect of a different variant (allele) or gene on another copy of the chromosome. As generally understood in the art, this definition may also specifically encompass incomplete dominance or co-dominance.

[0053] A "genetic map" is a description of the genetic linkage relationships between loci on one or more chromosomes within a given species, usually depicted in a graphical or tabular form. For each genetic map, the distances between loci are measured by the frequencies of recombination between them. Recombination between loci can be detected using a variety of markers. A genetic map is the product of the mapping population, the types of markers used, and the polymorphic potential of each marker between different populations. The order and genetic distances between loci can differ from one genetic map to another.

[0054] As used herein, the term " genotype " refers to the genetic composition of the individual (or individual population) at one or more genetic loci in contrast to observable and / or detectable and / or demonstrated proterties (phenotype). Genotype is defined by the combination of one or more alleles of one or more known loci of its parent by individual inheritance. The term genotype can be used to refer to the genetic composition of the individual at a single locus, a plurality of loci, or more generally, the term genotype can be used to refer to the individual inheritance of all genes in its genome. Genotype can be, for example, indirectly characterized using a mark and / or directly characterized by nucleic acid sequencing.

[0055] As used herein, the term "germplasm" refers to genetic material belonging to or coming from an individual (e.g., a plant), a population of individuals (e.g., a plant strain, variety, or family), or a clone derived from a strain, variety, species, or culture. Germplasm can be a part of an organism or a cell, or can be separated from the organism or cell. In general, germplasm provides genetic material with a specific genetic makeup that provides a basis for some or all of the genetic qualities of an organism or cell culture. As used herein, germplasm includes cells, seeds, or tissues from which new plants can be grown, as well as plant parts (e.g., leaves, stems, buds, roots, pollen, cells, etc.) that can be cultured into whole plants.

[0056] "Haplotype" is the genotype of an individual at a plurality of genetic loci, i.e., a combination of alleles. In certain embodiments, the genetic loci defining a haplotype are physically and genetically linked, i.e., on the same chromosome segment, but this is not necessarily the case. If the various combinations of alleles are not physically and genetically linked, the haplotype combination is usually determined by the ability of the haplotype to provide a desired phenotype, as currently described for the late-maturing limited type phenotype. The term "haplotype" may refer to a polymorphism at a specific locus (e.g., a single marker locus), or a polymorphism at a plurality of loci along one or more chromosome segments.

[0057] As used herein, the term "heterozygous" refers to a genetic state in which different alleles are located at corresponding loci on homologous chromosomes.

[0058] As used herein, the term "homozygous" refers to a genetic state in which identical alleles are located at corresponding loci on homologous chromosomes.

[0059] As used herein, the term "hybrid" in the context of plant breeding refers to a plant that is the offspring of genetically distinct parents produced by crossing plants of different lines or varieties or species, including but not limited to a cross between two inbred lines.

[0060] As used herein, the term "inbred" refers to a substantially homozygous plant or variety. The term can refer to a plant or plant variety that is substantially homozygous throughout the genome, or a plant or plant variety that is substantially homozygous for a particular portion of the genome of interest.

[0061] As used herein, the term "indel" refers to an insertion or deletion in a pair of nucleotide sequences, wherein a first sequence may be referred to as having an insertion relative to a second sequence, or the second sequence may be referred to as having a deletion relative to the first sequence.

[0062] As used herein, the terms "introgression," "introgressing," and "introgressed" refer to the natural and artificial transfer of a desired allele or combination of desired alleles of one or more genetic loci from one genetic background to another. For example, a desired allele at a given locus can be transferred to at least one progeny by sexual hybridization between two parents of the same species, wherein at least one of the parents has the desired allele within its genome. Alternatively, for example, the transfer of the allele can occur by recombination between two donor genomes, such as in fused protoplasts, wherein at least one donor protoplast has the desired allele in its genome. The desired allele can be a selected allele of a marker, a QTL, a transgenic, etc. The allelic offspring that comprises expectation can be backcrossed once or many (for example 1,2,3,4 or more times) with the strain with the genetic background of expectation, selects for the allelic of expectation, and as a result, the allelic of this expectation is fixed in the genetic background of expectation.For example, the mark that is associated with late maturity or the mark that is associated with limited type can be infiltrated from donor gene to show the recurrent parent of precocious, average maturity or unlimited type stem trait.Then the offspring that obtains can be backcrossed once or many and selected, until filial generation has one or more genetic markers that are associated with late maturity and / or unlimited type in this recurrent parent background.

[0063] As used herein, the term "linkage" refers to the degree of association of a marker locus with another marker locus or some other marker loci. The linkage relationship between a genetic marker and a phenotype can be given as a "probability" or "adjusted probability". Linkage can be expressed as a desired limit or range. For example, in some embodiments, when a marker is separated by less than about 50, 40, 30, 25, 20 or 15 map units (or cM), any marker is linked to any other marker (genetically and physically).

[0064] The centimorgan ("cM") or genetic map unit (mu) is a measure of recombination frequency and is defined as the distance between genes for which one out of 100 meiotic products is recombinant. One cM equals a 1% chance that a marker at one genetic locus will segregate with a marker at a second locus due to hybridization in a single generation. Thus, a recombination frequency (RF) of 1% is equivalent to 1 m.u.

[0065] As used herein, phrase " linkage group " refers to all genes or hereditary traits that are located on the same chromosome. In linkage group, those loci that are close enough can show linkage in genetic hybridization. Because the probability of exchange increases with the physical distance between the loci on the chromosome, the loci that are located far away from each other in linkage group may not show any detectable linkage in direct gene testing. The term " linkage group " is mainly used to refer to following genetic loci, and it shows linkage behavior in the genetic system that has not yet carried out chromosome localization. Therefore, the term " linkage group " is synonymous with the physical entity of chromosome, but those of ordinary skill in the art will understand that linkage group can also be defined as the region corresponding to a given chromosome (that is, less than whole).

[0066] As used herein, the term "linkage disequilibrium" refers to the non-random separation of genetic loci or proteus (or both). In either case, linkage disequilibrium means that the related loci are physically close enough along a section of chromosome so that they are separated together with a frequency higher than random (i.e. non-random) (in the case of co-segregated proteus, the loci controlling these proteus are close enough to each other). Markers showing linkage disequilibrium are considered to be linked. The linked loci are co-segregated more than 50% of the time (e.g., from about 51% to about 100% of the time). In other words, two co-segregated markers have a recombination frequency less than 50% (and, by definition, separated less than 50cM on the same chromosome). As used herein, linkage can be present between two markers, or alternatively, between marker and phenotype. Marker loci can be "associated" (linkage) with proteus (e.g., late maturing and / or limited type). For example, the linkage degree of genetic markers and phenotypic proteus is measured as the statistical probability that, for example, the marker is co-segregated with the phenotype.

[0067] Linkage disequilibrium is most commonly measured using the measure r 2 Evaluation, the measure r 2 The calculation was performed using the formula described in Hill and Robertson, Theor. Appl. Genet. 38:226 (1968). 2 =1, there is complete linkage disequilibrium between the two marker loci, meaning that the markers have not yet separated by recombination and have the same allele frequency. 2Values ​​greater than 1 / 3 indicate sufficiently strong linkage disequilibrium to be useful for mapping. Ardlie et al., Nature Reviews Genetics 3:299 (2002). Thus, when r between paired marker loci is 2 When the value is greater than or equal to about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0, the alleles are in linkage disequilibrium.

[0068] As used herein, the term "linkage equilibrium" describes a situation in which two markers segregate independently, ie, randomly distribute among progeny. Markers that exhibit linkage equilibrium are considered unlinked (regardless of whether they are located on the same chromosome or not).

[0069] As used herein, the terms "marker" and "genetic marker" are used interchangeably to refer to nucleotides and / or nucleotide sequences that have been associated with a phenotype and / or a trait. Markers can be, but are not limited to, alleles, genes, haplotypes, chromosomal intervals, restriction fragment length polymorphisms (RFLPs), simple sequence repeats (SSRs), randomly amplified polymorphic DNA (RAPDs), cleaved amplified polymorphic sequences (CAPS) (Rafalski and Tingey, Trends in Genetics 9:275 (1993)), amplified fragment length polymorphisms (AFLPs) (Vos et al., Nucleic Acids Res. [Nucleic Acids Research] 23:4407 (1995)), single nucleotide polymorphism (SNP) (Brookes, Gene [Gene] 234:177 (1993)), sequence-characterized amplified region (SCAR) (Paran and Michelmore, Theor. Appl. Genet. [Theoretical and Applied Genetics] 85:985 (1993)), sequence-tagged site (STS) (Onozaki et al., Euphytica [Netherlands Plant Breeding Journal] 138:255 (2004)), single-strand conformation polymorphism (SSCP) (Orita et al., Proc. Nat. USA [Proceedings of the National Academy of Sciences of the United States of America] 86:2766 (1989)), intersimple sequence repeats (ISSR) (Blair et al., Theor. Appl. Genet. [Theoretical and applied genetics] 98:780 (1999)), retrotransposon inter-amplified polymorphisms (IRAP), retrotransposon microsatellite amplified polymorphisms (REMAP) (Kalendar et al., Theor. Appl. Genet. [Theoretical and applied genetics] 98:704 (1999)), isozyme markers, RNA cleavage products (such as Lynx tags), or any combination of markers described herein. The marker can be present in a genomic nucleic acid or an expressed nucleic acid (e.g., EST). A large number of soybean genetic markers are known in the art and are published or can be obtained from various sources such as the SoyBase Internet resource (www.soybase.org). In some embodiments, the genetic markers of the invention are SNP alleles, SNP alleles located in a chromosomal interval, and / or haplotypes (combinations of SNP alleles) associated with a late-maturing determinate phenotype.

[0070] Can detect the mark corresponding to the genetic polymorphism between colony members by the method generally recognized in the art.These methods include but are not limited to, nucleic acid sequencing, hybridization method, amplification method (for example sequence-specific amplification method based on PCR), restriction fragment length polymorphism (RFLP) detection, isozyme marker detection, polynucleotide polymorphism detection carried out by allele-specific hybridization (ASH), amplification variable sequence detection of plant genome, self-sustaining sequence replication detection, simple sequence repeat (SSR) detection, random amplified polymorphic DNA (RAPD) detection, single nucleotide polymorphism (SNP) detection and / or amplified fragment length polymorphism (AFLP) detection.Therefore, in some embodiments of the present invention, these well-known methods can be used to detect SNP alleles as defined herein (see for example Table 1-3).

[0071] Thus, in some embodiments of the invention, markers are detected by amplifying Glycine max nucleic acid using, for example, polymerase chain reaction (PCR) with two oligonucleotide primers.

[0072] A "marker allele," also described as an "allele of a marker locus," may refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus that are polymorphic for the marker locus in a population.

[0073] "Marker assisted selection" (MAS) is a method of selecting phenotypes based on marker genotypes. Marker assisted selection involves the use of marker genotypes to identify plants for inclusion in and / or removal from a breeding program or planting.

[0074] As used herein, the term "marker locus (marker locus and marker loci)" refers to one or more specific chromosomal locations in an organism's genome in which one or more specific markers can be found. The marker locus can be used to track the presence of a second linked locus (e.g., a linked locus that encodes or contributes to the expression of a phenotypic trait). For example, the marker locus can be used to monitor the separation of alleles at a locus (e.g., a QTL or a single gene) that are genetically or physically linked to the marker locus.

[0075] As used herein, the terms "label probe" and "probe" refer to nucleotide sequences or nucleic acid molecules (e.g., nucleic acid probes complementary to all or part of the marker or marker locus) that can be used to detect the presence of one or more specific alleles in the marker locus. Label probes comprising about 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more continuous nucleotides can be used for nucleic acid hybridization. Alternatively, in some aspects, label probes refer to probes of any type that can distinguish (i.e., genotyping) specific alleles present at the marker locus. Typically, the probe is a single-stranded oligonucleotide sequence that will form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative. Non-limiting examples of the probes disclosed herein include those listed in Table 4.

[0076] As used herein, when identifying linked loci, the term "molecular marker" can be used to refer to genetic markers as defined above, or its coded product (e.g., protein) as a reference point. Molecular markers can be derived from genomic nucleotide sequences or expressed nucleotide sequences (e.g., RNA, cDNA, etc. from splicing). The term also refers to a nucleotide sequence complementary to a marker sequence or to a nucleotide sequence located on its flank, such as a nucleotide sequence used as a probe and / or primer capable of amplifying a marker sequence. For example, according to the Watson-Crick base pairing principle, when a nucleotide sequence specifically hybridizes in a solution, these nucleotide sequences are "complementary". When located on an indel region, some of the markers described herein are also referred to as hybridization markers. This is because, by definition, the insertion region is a polymorphism about a plant that does not have the insertion. Therefore, the marker only needs to indicate whether the indel region exists. Any suitable marker detection technology (e.g., SNP technology) can be used to identify this hybridization marker.

[0077] As used herein, the term "primer" refers to an oligonucleotide that can anneal to a nucleic acid target and serve as a starting point for DNA synthesis when placed under conditions that induce synthesis of primer extension products (e.g., in the presence of nucleotides and reagents for polymerization (e.g., DNA polymerase) and at a suitable temperature and pH). In order to obtain maximum efficiency in extension and / or amplification, in some embodiments, primers (in some embodiments, extension primers, and in some embodiments, amplification primers) are single-stranded. In some embodiments, primers are oligodeoxyribonucleotides. Primers are typically long enough to initiate the synthesis of extension and / or amplification products in the presence of reagents for polymerization. The minimum length of a primer can depend on many factors, including but not limited to the temperature and composition (A / T vs. G / C content) of the primer. In the case of amplification primers, these amplification primers are typically provided as a pair of bidirectional primers consisting of a forward and a reverse primer, or as a pair of forward primers commonly used in the field of DNA amplification (e.g., in PCR amplification). Thus, it should be understood that the term "primer" as used herein may refer to more than one primer, particularly where there is some ambiguity in the information about one or more terminal sequences of a target region to be amplified. Therefore, a "primer" may include a set of primer oligonucleotides containing sequences representing possible variations in the sequence, or may include nucleotides that allow typical base pairing. Non-limiting examples of primers for amplifying marker loci and generating amplicons in this disclosure include those listed in Table 3.

[0078] Primer can be prepared by any suitable method.The method for preparing the oligonucleotide of specific sequence is known in the art, and comprises clone and restriction and direct chemical synthesis of for example suitable sequence.Chemical synthesis method can comprise for example phosphodiester or triester method, diethylphosphoramidate method and solid support method disclosed in U.S. Patent number 4,458,066.If necessary, can come labelled primer by mixing detectable part, for example spectral part, fluorescent part, photochemical part, biochemical part, immunochemical part or chemical part.

[0079] The PCR method is well described in the manual and is known to the technician. After PCR amplification, the target polynucleotide can be detected by hybridizing with a probe polynucleotide, which forms a stable hybrid with the target sequence under strict to moderately strict hybridization and washing conditions. If the probe is expected to be substantially completely complementary to the target sequence (that is, about 99% or more), stringent conditions can be used. If it is expected that there are some mismatches, for example, if it is expected that variant varieties will cause incomplete complementarity of the probe, the stringency of hybridization can be reduced. In certain embodiments, conditions are selected to exclude non-specific / accidental binding. Conditions affecting hybridization and conditions selected for non-specific binding are known in the art and are described in, for example, Sambrook and Russell (2001). MolecularCloning:ALaboratory Manual, ThirdEdition , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, United States of America [Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, United States of America]. Generally, lower salt concentrations and higher temperatures for hybridization and / or washing increase the stringency of hybridization conditions.

[0080] Different nucleotide sequences or polypeptide sequences with homology are referred to herein as "homologs". The term homolog includes homologous sequences from the same species and other species as well as orthologous sequences from the same species and other species. "Homology" refers to the level of similarity between two or more nucleotide sequences and / or amino acid sequences in terms of percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids, amino acids, and / or proteins.

[0081] As used herein, the phrase "nucleotide sequence homology" refers to the presence of homology between two polynucleotides. When comparison is performed to obtain maximum correspondence, if the sequence of the nucleotides in the two sequences is identical, the polynucleotide has a "homologous" sequence. The "percentage of sequence homology" of a polynucleotide, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology, can be determined by comparing the sequences of two best comparisons on a comparison window (e.g., about 20-200 continuous nucleotides), wherein for the best comparison of the two sequences, compared to the reference sequence, the part of the polynucleotide sequence in the comparison window can include additions or deletions (i.e., spaces). The best comparison of the sequence for comparison can be performed by computerized implementations of known algorithms or by visual inspection. Readily available algorithms for sequence comparison and multiple sequence alignment are the Basic Local Alignment Search Tool (BLAST; Altschul et al. (1990) J Mol Biol 215:403-10; Altschul et al. (1997) Nucleic Acids Res 25:3389-3402) and ClustalX (Chenna et al. (2003) Nucleic Acids Res 31:3497-3500) programs, both of which are available on the Internet. Other suitable programs include, but are not limited to, GAP, BestFit, PlotSimilarity, and FASTA, which are part of the Accelrys GCG software package, available from Accelrys Software, Inc., San Diego, California, USA.

[0082] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide sequences or polypeptide sequences are invariant over the entire comparison window of components (eg, nucleotides or amino acids). "Identity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton, N.J. Stockton Press, New York (1991).

[0083] As used herein, the term "substantially identical" or "corresponding" refers to two nucleotide sequences having at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity. In some embodiments, the two nucleotide sequences can have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0084] The "identity score" of the aligned segments of the test and reference sequences is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment (i.e., the complete reference sequence or a smaller defined portion of the reference sequence). The percentage of sequence identity is expressed as the identity score multiplied by 100. As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (there are less than 20% of the appropriate nucleotide insertions, deletions, or gaps within the comparison window) . In some embodiments, "percent identity" can refer to the percentage of identical amino acids in an amino acid sequence.

[0085] Optimal alignment of sequences for comparison windows is well known to those skilled in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and optionally by computerized implementations of these algorithms, such as Wisconsin The comparison of one or more polynucleotide sequences can be with respect to the full-length polynucleotide sequence or its part, or with respect to a longer polynucleotide sequence. For purposes of the present invention, "percent identity" can also be determined using 2.0 versions of BLASTX for the nucleotide sequence translated and 2.0 versions of BLASTN for the polynucleotide sequence.

[0086] You can use the Sequence Analysis Software Package TMThe percent sequence identity is determined by the "Best Fit" or "Gap" programs of Genetics Computer Group (Version 10; Genetics Computer Group, Inc., Madison, Wisconsin). "Gap" uses the algorithm of Needleman and Wunsch (Needleman and Wunsch, J Mol. Biol. [Journal of Molecular Biology] 48:443-453, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. "BestFit" performs the best alignment of the best similarity segments between two sequences and inserts gaps using the local homology algorithm of Smith and Waterman to maximize the number of matches (Smith and Waterman, Adv. Appl. Math. [Progress in Applied Mathematics], 2:482-489, 1981; Smith et al., Nucleic Acids Res. [Nucleic Acids Research] 11:2205-2220, 1983).

[0087] Useful methods for determining sequence identity are also disclosed in: Guide to Huge Computers [Supercomputer Guide] (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo et al. (Applied Math 48:1073 (1988)). More specifically, preferred computer programs for determining sequence identity include, but are not limited to: the Basic Local Alignment Search Tool (BLAST) program publicly available from the National Center Biotechnology Information (NCBI), which is located at the National Institutes of Health (Bethesda, Maryland, 20894) National Medical Library; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; (Altschul et al., J. Mol. Biol. [Journal of Molecular Biology] 215:403-410 (1990)); version 2.0 or higher of the BLAST program allows gaps (deletions and insertions) to be introduced into the alignment; for peptide sequences, BLASTX can be used to determine sequence identity; and for polynucleotide sequences, BLASTN can be used to determine sequence identity.

[0088] As used herein, the terms "phenotype", "phenotypic trait" or "trait" refer to one or more traits of an organism. A phenotype is observable by the naked eye or by any other means of evaluation known in the art (e.g., microscopy, biochemical analysis, or electromechanical assay). In some cases, a phenotype is directly controlled by a single gene or genetic locus, i.e., a "monogenic trait". In other cases, a phenotype is the result of multiple genes.

[0089] As used herein, the term "polymorphism" refers to the variation in the nucleotide sequence at the locus, wherein the variation is too common, and not just due to spontaneous mutation. Polymorphism must have a frequency of at least about 1% in a colony. Polymorphism can be a single nucleotide polymorphism (SNP) or an insertion / deletion polymorphism (also referred to as "indel" herein). In addition, the variation can be in a transcriptional profile or a methylation pattern. One or more polymorphic sites of a nucleotide sequence can be determined by carrying out a nucleotide sequence comparison at one or more loci in two or more germplasm entries.

[0090] As used herein, the term "plant" may refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term "plant" may refer to a whole plant, a plant component or plant organ (i.e., leaves, stems, roots, etc.), a plant tissue, a seed, and / or a plant cell. A plant cell is a plant cell obtained from a plant, or a plant cell derived from a cell taken from a plant by culturing.

[0091] As used herein, the term "soybean" refers to plants of the genus Glycine max, including but not limited to soybean, and any part thereof.

[0092] As used herein, the term "plant part" includes, but is not limited to, embryos, pollen, seeds, leaves, flowers (including but not limited to anthers, ovules, etc.), fruits, stems or branches, roots, root tips, cells (including intact cells in plants and / or plant parts), protoplasts, plant cell tissue cultures, plant callus, plant masses, etc. Thus, plant parts include soybean tissue cultures that can be regenerated into soybean plants. Additionally, as used herein, "plant cell" refers to the structural and physiological unit of a plant, including a cell wall and may also refer to a protoplast. The plant cell of the present invention may be in the form of an isolated single cell, or may be a cultured cell, or may be a part of a higher organized unit (e.g., such as a plant tissue or a plant organ).

[0093] As used herein, the term "population" refers to a genetically heterogeneous collection of plants sharing a common genetic derivation.

[0094] As used herein, the term "progeny", "progeny plant" and / or "offspring" refers to a plant produced by one or more parental plants nutrition or sexual reproduction. Progeny plant can be obtained by cloning or selfing of a single parental plant or by the hybridization of two parental plants, and includes selfing bodies and F1 or F2 or even further generations. F1 is the first generation offspring produced from a parent (at least one of the parents is used as a donor of a trait for the first time), and the offspring of the second generation (F2) or subsequent generations (F3, F4, etc.) is a sample produced from the selfing or hybridization of F1, F2, etc. Therefore F1 can (and in some embodiments) be a hybrid produced by hybridization between two true breeding parents (the phrase "true breeding" refers to an individual that is homozygous for one or more traits), and F2 can (and in some embodiments) be an offspring produced by self-pollination of F1 hybrids.

[0095] As used herein, the term "recessive" refers to a variant or allele of a gene that is masked or overshadowed by the phenotypic effects of a different variant (allele) or gene (which is usually described as "dominant") on another copy of the chromosome. As is generally understood in the art, this definition may also encompass incomplete recessiveness.

[0096] As used herein, the term "reference sequence" refers to a determined nucleotide sequence used as a basis for nucleotide sequence comparison. For example, a reference sequence of a marker can be obtained by genotyping multiple strains at one or more target loci, aligning these nucleotide sequences in a sequence alignment program, and then obtaining a consensus sequence for the alignment. Therefore, the reference sequence identifies the polymorphism in the allele at the locus. The reference sequence may not be a copy of the actual nucleic acid sequence from any particular organism; however, it is useful for designing primers and probes for the actual polymorphism in the actual nucleic acid sequence. DETAILED DESCRIPTION

[0097] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art. References to the technology employed herein are intended to reference technology commonly understood in the art, including variations of those technologies that are clear to those skilled in the art or replacement of equivalent technologies.

[0098] All patents, patent publications, non-patent publications, and sequences cited herein are incorporated by reference in their entirety.

[0099] Disclosed herein are compositions for combinations of genetic markers (e.g., combinations of SNPs) for identifying soybean plants having a desired or favorable allele combination at the E1 and Dt1 loci; and methods for identifying soybean plants having a desired or favorable allele combination at the E1 and Dt1 loci using a combination of genetic markers (e.g., a combination of SNPs), the favorable allele combination being associated with a late-maturing determinate phenotype in soybean plants. A combination of alleles of these two genes is disclosed in a specific haplotype that provides a desired late-maturing determinate growth phenotype. By using molecular markers to identify plants having a favorable allele combination, plants that are expected to have a late-maturing determinate phenotype can be identified and promoted in the early stages of the breeding process. Also disclosed are methods for identifying plants having an undesirable or unfavorable allele combination at the E1 and Dt1 loci using a combination of genetic markers (e.g., a combination of SNPs), the unfavorable allele combination being associated with an early-maturing and / or determinate phenotype in soybean plants. By using molecular markers to identify plants with unfavorable allele combinations, plants expected to have an early and / or indeterminate phenotype can be identified and removed in the early stages of the breeding process. This method supports soybean germplasm evaluation, correct progeny selection during population development, and assists in soybean product development. By selecting the correct plant type required for late-maturing determinate soybeans based on the genetic information provided by the markers disclosed in the present invention, the cost efficiency of developing late-maturing determinate soybean products is improved because the correct plant type is identified earlier in the development process. Overall, the time and labor involved in phenotyping each plant in a segregating population is significantly reduced. Markers associated with late maturity and determinate stem growth

[0100] The genetic locus relevant to a particular phenotype (such as late maturing and / or limited stem growth) can be located in the genome of an organism. By identifying a marker or a marker cluster that is co-separated with the combination of a target proterties or a target proterties, the breeder can rapidly select a desired phenotype or a phenotypic combination by selecting one or more suitable markers (being called the method for marker-assisted selection or MAS). The breeder can also use such a marker to simulate the design genotype on a computer and implement full genome selection.

[0101] Molecular markers are used to visualize the differences of nucleic acid sequences. This visualization can be attributed to DNA-DNA hybridization techniques after digestion with restriction endonucleases (e.g., RFLP) and / or to techniques using polymerase chain reaction (e.g., SNP, STS, SSR / microsatellite, AFLP, etc.). In certain embodiments, based on the hybridization of these parental genotypes, all differences between the two parental genotypes are separated in the mapping population. The separation of different markers can be compared and the recombination frequency can be calculated. The method for locating markers in plants is disclosed in, for example, Glick and Thompson (1993) Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Florida, USA; Zietkiewicz et al., (1994) Genomics [genomics] 20: 176-183. Methods for identifying markers in soybean are disclosed, inter alia, in Yuan et al., Plant Genetics, Genomics, and Biotechnology, 2(1): 90-94 (2014).

[0102] The recombination frequency of the genetic markers on different chromosomes and / or in different linkage groups is generally 50%.Being positioned between the genetic markers on the same chromosome or in the same linkage group, the recombination frequency depends on the physical distance between the mark on the chromosome usually.Low recombination frequency typically corresponds to the low genetic distance between the mark on the chromosome.The comparison of all recombination frequencies between one group of genetic markers draws the most logical order of genetic markers on the chromosome or in the linkage group.This most logical order can be described with linkage map.One group of adjacent or continuous markers on the linkage map that is associated with late maturing and / or infinite type phenotype can provide the position of the loci associated with those phenotypes.

[0103] The present invention provides combinations of SNP markers that can be used in various aspects of the disclosed subject matter as set forth herein. The SNP markers provided herein can be used to detect the presence of a combination of alleles in a soybean plant or germplasm that results in a desired late-maturing determinate phenotype, and thus can be used in methods involving marker-assisted breeding and selecting soybean plants / soybean plants having a combination of alleles that results in a desired late-maturing determinate phenotype.

[0104] In particular, methods are disclosed that rely on a combination of markers associated with two independent genes, one marker (e.g., a SNP marker) associated with the stem apex Dt1 gene on Gm 19 (LG L), and another marker (e.g., another SNP marker) associated with the E1 maturity gene on Gm 6 (LG C2). The marker combination is used to select for the correct plant type consisting of a determinate growth habit (dt1 / dt1) and a correct E1 genotype (E1 / E1), while also deselecting for incorrect plant types (dt1 / dt1, e1 / e1) that result in a deleterious early determinate growth habit (consisting of reduced plant height, early maturity, and lower yield). The association of the markers used herein has been determined by genome-wide association mapping studies, such as the association of marker SY3970 with the Dt1 stem apex locus and the association of marker SY0768BQ with the E1 maturity locus. Thus, in a particular embodiment, a combination of the E1 maturity locus marker SY0768BQ and the stem apex marker SY3970 is used to identify the correct plants in a segregating population developed from a breeding cross of control parental lines involving the stem apex and E1 maturity genes (Dt1 / Dt1 e1 / e1×dt1 / dt1 E1E1), thereby allowing the breeding process to rapidly identify and select progeny based on the need to fix either allele (Dt1 / e1 or dt1 / E1).

[0105] In an embodiment, the present invention provides a composition comprising a combination of the following items: (1) at least one marker associated with the stem apex allele at the Dt1 locus of the soybean genome and associated with a determinate stem growth habit; and (2) at least one marker associated with the E1 locus of the soybean genome and associated with late maturity. In an embodiment, the detection of a combination of markers and / or other linked markers can be used in a method for identifying, selecting and / or producing plants with a combination of determinate stem apex alleles and late maturity alleles. The detection of a combination of these markers and / or other linked markers can also be used to identify and select plants with determinate stem apex alleles and / or early maturity alleles, thereby enabling early elimination of such plants from a breeding program.

[0106] In some embodiments, the method for detecting the presence of a combination of SNPs in a soybean plant or germplasm may include providing two, or at least two, oligonucleotides or polynucleotides capable of hybridizing with the nucleotide sequences of the two SNPs disclosed herein under stringent hybridization conditions; contacting the oligonucleotides or polynucleotides with a genomic nucleic acid (or a fragment thereof, including but not limited to a restriction fragment thereof) of a soybean plant or germplasm; and determining the presence of each SNP by specific hybridization of the oligonucleotides or polynucleotides with a soybean genomic nucleic acid (or a fragment thereof) at the corresponding locus. Here, the two SNPs include a first SNP associated with a late maturing allele and a second SNP associated with a limited stem top allele.

[0107] Table 1 provides information about the combination of the first molecular marker associated with the late-maturing phenotype and the second molecular marker associated with the limited type stem top phenotype. For every kind of marker, Table 1 includes the favorable allele in the favorable allele combination associated with the desired late-maturing unlimited type phenotype, and the nucleotide position of the favorable allele in the isolated SNP sequence. The position of the marker locus in the 8X public construct (Williams82_V3 reference genome) of the Williams82 soybean genome on the SoyBase internet resource (www.soybase.org / SequenceIntro.php) or the USDA (bfgl.anri.barc.usda.gov / cgi-bin / soybean / Linkage.pl) can be described in this article to mark of the present invention. Table 1: Description of molecular markers associated with desirable shoot apex and maturity phenotypes

[0108] In a specific embodiment, the marker associated with late maturity comprises SY0768BQ (SEQ ID NO: 2), and the marker associated with limited stem apex comprises SY3970 (SEQ ID NO: 1). In another embodiment, the marker associated with late maturity may include any marker linked to marker SY0768BQ (SEQ ID NO: 2). Similarly, the marker associated with limited stem apex may include any marker linked to marker SY3970 (SEQ ID NO: 1). For example, linked markers can be determined by using the available resources on the SoyBase Internet resource (soybase (.) org). Therefore, in an embodiment, the compositions and methods for detecting the presence of favorable allele combinations in soybean plants or germplasm may include detecting

[0109] In the case of soybean maturity genes (such as E1), the phenotypic traits include one or more or a combination of flowering time, time after flowering, relative maturity stage, maturity time, maturity stage group, and the number of days from flowering to the onset of maturity in the soybean plant. In a specific embodiment, the phenotypic trait measured is flowering time, and includes the difference between the VE and R1 stages of the modified soybean plant relative to the control plant (for different stages, see Figure 1 In another embodiment, the phenotypic trait measured is time to maturity and includes the time between the R1 and R7 stages or the R1 and R8 stages of the modified soybean plant relative to the control plant (see Figure 1 ) is a measure of the time that passes between two loci. Relative to a control plant containing wild-type alleles at two loci, the number of days can vary based on the specific allele combination of the plant. For more descriptions of the different growth stages of soybean plants, see Endres, G., An overview of soybean plant growth stages [Summary of soybean plant growth stages], Soybean Growth and Management Quick Guide [Soybean Growth and Management Quick Guide], publication A1174, North Dakota State University [North Dakota State University], revised November 2021.

[0110] As used herein, a modified plant having a later flowering time and / or maturity time compared to a control plant has a flowering time and / or maturity time that is between 1 day and 10 days later than a control plant (e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days later than a control plant). In embodiments, if the flowering time of the modified plant is 1-2 days, 1-3 days, 1-4 days, 1-5 days, 1-6 days, 1-7 days, 1-8 days, 1-9 days or 1-10 days later than a control plant, the flowering time of the modified plant is later than that of the control plant.

[0111] In embodiments, the progeny plant has a later flowering time and / or maturity time relative to the control plant, and wherein the control plant comprises a wild-type allele at the E1 locus. In some instances, the change in the specified flowering time and / or maturity time comprises specifying a shortened number of days for the flowering time and / or maturity time of the progeny plant relative to the control plant. In particular embodiments, the change in the specified flowering time comprises an increase in the number of days between the VE stage and the R1 stage of the progeny plant relative to the control plant, and / or wherein the change in the specified maturity time comprises an increase in the number of days between the R1 stage and the R7 (or R8) stage of the progeny plant relative to the control plant.

[0112] A "dominant determinate allele" is an allele that, when present in a single copy (heterozygous) or in two copies (homozygous), affects the determinate phenotype, that is, whether the plant ceases vegetative activity at or shortly after photoperiod-induced floral induction, and whether the meristem becomes a reproductive inflorescence. A "recessive determinate allele" is an allele that affects the determinate phenotype of a plant only when present in two copies (homozygous), but does not affect the maturity of the plant when present in a single copy (heterozygous).

[0113] The present disclosure provides specific recessive determinate alleles that, when present in a homozygous state, provide a desired stem-top growth habit, thereby exhibiting a determinate phenotype, that is, vegetative activity ceases at or shortly after photoperiod-induced floral induction, and the meristem then becomes a reproductive inflorescence. In other words, once reproductive growth (flowering) begins, determinate plants essentially cease vegetative growth, while indeterminate plants continue to grow after flowering begins. For more information on the determinate phenotype, see Pyle, A Comparison of Determinate and Indeterminate Soybean Lines for Double Cropping in Virginia, Dissertation, Virginia Polytechnic Institute and State University, 1982.

[0114] Therefore, the present disclosure theme also relates to the method for identifying, selecting and / or producing the soybean plant with one or more late maturing finite allelotypes, the method is included in the existence of one or more genetic markers associated with one or more late maturing finite allelotypes as described herein and / or associated with the late maturing finite phenotype in the donor soybean plant, and the nucleotide sequence comprising at least one genetic marker thus detected is transferred to the recipient soybean plant from the donor soybean plant.It should be noted that the recipient soybean plant can be late maturing or precocious, or shows a finite or indefinite type stem phenotype.Typically, the recipient soybean plant carries at least one or more undesirable proterties of precocious or indefinite type stem phenotype.In other embodiments, the recipient soybean plant can have these two undesirable phenotypes.The transfer of nucleotide sequence can be performed by any method as described herein.

[0115] Thus, methods for identifying, selecting and / or producing soybean plants or germplasm comprising a late maturing determinate phenotype may include detecting the presence of a genetic marker associated with a late maturing and / or determinate phenotype. The SNP marker may be detected in any sample obtained from a soybean plant or germplasm, including but not limited to a whole plant or germplasm, a portion of the plant or germplasm (e.g., a cell, leaf, seed, etc. from the plant or germplasm), or a nucleotide sequence from the plant or germplasm.

[0116] As discussed herein, in some embodiments of the invention, amplification products generated by amplifying Glycine max nucleic acid with two oligonucleotide primers can be used to identify markers. In some embodiments, amplification is performed by PCR, and these primers are PCR primers designed to hybridize to opposite strands of Glycine max genomic DNA (e.g., chromosome 19) so as to amplify Glycine max genomic DNA sequences that are present between the sequences to which the PCR primers hybridize. Methods for amplifying nucleic acids are well known in the art.

[0117] Therefore, in some embodiments of the present invention, a method for identifying and / or selecting soybean plants or germplasm having a combination of late maturity and determinate phenotypes is provided, the method comprising: detecting the presence of one or more genetic markers associated with each of the late maturity and determinate stem apex alleles in the soybean plant or germplasm, wherein the markers are detected using probes in an amplification product of a nucleic acid sample isolated from the soybean plant or germplasm, the amplification product being produced using an amplification primer pair, wherein the amplification primers and probes are as described in the provided sequence listing. Marker-Assisted Selection

[0118] The subject matter disclosed herein also relates to a method for producing a pathogen-resistant soybean plant, the method comprising detecting the presence of a genetic marker associated with the late indefinite type in a donor soybean plant according to the method described herein, and transferring a nucleic acid sequence comprising at least one genetic marker thus detected from the donor plant to a recipient soybean plant. The transfer of the nucleic acid sequence can be performed by any method known in the art.

[0119] Thus, the present invention encompasses methods of plant breeding and methods of selecting / identifying plants, particularly soybean plants, particularly cultivated soybean plants as breeding plants for use in breeding programs, or cultivated soybean plants having desirable genotypic properties or potential phenotypic properties, particularly associated with producing valuable soybeans (also referred to herein as commercially valuable plants). Cultivated plants are defined herein as intentionally selected plants, or plants derived from plants intentionally selected in agricultural or horticultural practice for having desirable genotypic or potential phenotypic properties, such as plants obtained by inbreeding.

[0120] The present disclosure subject matter therefore also provides a method for selecting a soybean plant having a late maturing and determinate phenotype, the method comprising detecting the presence of one or more late maturing and / or determinate alleles as defined herein in the plant. In an exemplary embodiment of the method for selecting such a plant of the present disclosure, the method comprises providing a genomic DNA sample from a soybean plant; and (b) detecting at least one genetic marker associated with a late maturing and / or determinate phenotype in the genomic DNA sample. In some embodiments, the detection comprises detecting one or more SNPs associated with a late maturing or determinate phenotype.

[0121] Providing genomic DNA samples from soybean plants can be performed by standard DNA isolation methods well known in the art.

[0122] In certain embodiments, the detection of genetic markers (e.g., SNP, combination of SNP) can include using one or more groups of primer pairs (SNP assay), which can be used to produce one or more amplified products that can be used to detect genetic markers (SNP). In certain embodiments, such a group of primers can include the nucleotide sequence as shown in the sequence table.

[0123] In certain embodiments, the detection of genetic markers can include the use of a nucleic acid probe having a nucleotide base sequence that is substantially complementary to the nucleic acid sequence that defines the genetic marker, and the nucleic acid probe hybridizes with the nucleic acid sequence that defines the genetic marker under stringent conditions. Suitable nucleic acid probes can, for example, be single strands corresponding to the amplified product of the marker. In certain embodiments, the detection of genetic markers is designed to determine whether a specific allele of a SNP in a specific plant exists.

[0124] Therefore the present disclosure theme also relates to the method for producing soybean plant, the method comprises the existence of the genetic marker associated with late maturing and / or determinate stem proterties in the donor soybean plant according to the present disclosure theme detection as described herein, and the part of the nucleotide sequence of the nucleotide sequence that comprises at least a genetic marker thus detected or the nucleotide sequence that grants late maturing and / or determinate proterties is transferred to the recipient soybean plant from the donor plant.In a specific embodiment, the nucleotide sequence of recipient soybean plant maturation and / or determinate stem proterties is changed by the described transfer.The transfer of nucleotide sequence can be performed by any method as described herein.

[0125] Exemplary embodiments of such methods include transferring nucleic acid sequences from late maturing limited type donor soybean plants to recipient soybean plants by hybridizing plants through gene introgression. This transfer can be accomplished using traditional breeding techniques. In certain embodiments, late maturing and / or limited type locus genes are introgressed into commercial soybean varieties using marker assisted selection (MAS) or marker assisted breeding (MAB). MAS and MAB involve the use of one or more molecular markers that are identified as having significant associations with desired proterties, and are used to identify and select those offspring plants containing one or more genes encoding desired proterties. As disclosed herein, such identification and selection are based on the selection of SNP alleles of the present invention or markers associated therewith. MAB can also be used to develop near isogenic lines (NILs) comprising one or more target pathogen resistance alleles, thereby allowing the effects of such one or more alleles to be studied in more detail. MAB is also an effective method for developing backcross inbred lines (BIL) populations. In certain embodiments, the soybean plants developed according to these embodiments can obtain most of their proterties from recipient plants, and obtain late maturing and / or limited type proterties from donor plants. MAB / MAS technology improves the efficiency of backcrossing and introgression genes using marker-assisted selection (MAS) or marker-assisted breeding (MAB).

[0126] Therefore, traditional breeding techniques can be used for infiltration of nucleic acid sequence genes associated with late maturing and / or limited type traits into recipient soybean plants.In some embodiments of the present invention, recipient soybean does not have late maturing or limited type traits, and these traits are given by transferring the described nucleic acid sequence associated with late maturing limited type.Therefore, for example, recurrent selection and backcrossing, selfing and / or double haploid technology or any other technology for preparing parental lines can be used to develop inbred late maturing limited type soybean plant lines.In the method for recurrent selection and backcrossing, by making the recurrent parent hybridize with the first donor plant (that is, non-recurrent parent) that is different from the recurrent parent, the late maturing limited type genes can be infiltrated into the target recipient plant (recurrent parent).However, in some embodiments, the recurrent parent is a plant with commercially desired characteristics (such as but not limited to (extra) disease and / or insect resistance, valuable nutritional characteristics, valuable abiotic stress tolerance (including but not limited to drought tolerance, salt tolerance) etc.). In some embodiments, the non-recurrent parent exhibits a late indeterminate phenotype and comprises a nucleic acid sequence associated with a late indeterminate phenotype.The non-recurrent parent can be any plant variety or inbred line that is fertile when crossed with the recurrent parent.

[0127] In certain embodiments, the progeny produced by the hybridization between the recurrent parent and the non-recurrent parent are backcrossed with the recurrent parent. Then, the resulting plant population is screened for the desired characteristics, and the screening can be performed in a variety of different ways. For example, a phenotypic pathology screening or a quantitative bioassay as known in the art can be used to screen the population. Alternatively, as an alternative to using a bioassay, one or more of the molecular markers, hybridization probes, or polynucleotides described above can be used to perform MAB to identify those progeny comprising nucleic acid sequences encoding, for example, late maturing and / or limited type combination phenotypes (e.g., SNPs and SNP combinations described herein). Moreover, MAB can be used to confirm the results obtained from the quantitative bioassay. In certain embodiments, the markers defined herein are suitable for selecting appropriate offspring plants by genotypic screening.

[0128] After screening, F1 hybrid plants that exhibit a pathogen resistance phenotype or (in some embodiments) a genotype and thus contain the necessary nucleic acid sequences associated with the late maturing indefinite type are then selected and backcrossed to the recurrent parent so that the soybean plants become increasingly inbred. The process of selection and backcrossing can be repeated for many generations (e.g., one, two, three, four, five, six, seven, eight, or more generations).

[0129] The availability of an integrated linkage map of the soybean genome containing an enhanced density of public soybean markers facilitates soybean genetic mapping and MAS. See, for example, soybeanbreederstoolbox.org, which is available at the SoyBase internet resource (www.soybase.org).

[0130] Of the available genetic marker types, SNPs are some of the most abundant and have the potential to provide the highest genetic map resolution (Bhattramakki et al., Plant Molec. Biol. 48:539 (2002)). SNPs can be assayed in a so-called "ultra-high throughput" manner because they do not require large amounts of nucleic acid and automation of the assay is straightforward. SNPs also have the benefit of being a relatively low-cost system. These three factors together make the use of SNPs in MAS highly attractive. Several methods can be used for SNP genotyping, including but not limited to: hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, microsequencing, and coded spheres. Such methods have been reviewed in various publications: Gut, Hum. Mutat. 17:475 (2001); Shi, Clin. Chem. 47:164 (2001); Kwok, Pharmacogenomics 1:95 (2000); Bhattramakki and Rafalski, Discovery and application of single nucleotide polymorphism markers in plants, PLANT GENOTYPING: THE DNA FINGERPRINTING OF PLANTS, CABI Press, Wallingford (2001). A wide range of commercially available technologies utilize these and other methods to interrogate SNPs, including Masscode TM (Qiagen, Germantown, Maryland), (Hologic, Madison, Wisconsin), (Applied Biosystems, Foster City, CA), (Applied Biosystems, Foster City, CA) and Beadarrays TM (Illumina, San Diego, CA). Soybean plants, parts thereof, and germplasm having a late maturing determinate haplotype

[0131] This disclosure provides soybean plants and germplasm with late-maturing limited type haplotype.As discussed above, the method of this disclosure can be used for identification, generation and / or selection of soybean plants or germplasm with late-maturing and / or limited type alleles.In addition to the above method, soybean plants or germplasm with late-maturing and / or limited type alleles can also be produced by including but not limited to the following any method, thus late-maturing and / or limited type alleles are introduced in soybean plants or germplasm by such method: conversion (including but not limited to bacterial-mediated nucleic acid delivery (for example, via Agrobacterium)), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, electroporation, supersound process, infiltration, PEG-mediated nucleic acid uptake, and any other electricity, chemistry, physics (mechanics) and / or biological mechanism (or any combination thereof) causing nucleic acid to be introduced in the plant cell, protoplast transformation or fusion, double haploid technology, embryo rescue or by any other nucleic acid transfer system.

[0132] In the context of plant cells, plants and / or plant parts, "introduction" refers to contacting a nucleic acid molecule with the plant, plant part and / or plant cell in such a way that the nucleic acid molecule is able to enter the interior of the plant cell and / or the cell of the plant and / or plant part. When more than one nucleic acid molecule is introduced, these nucleic acid molecules can be assembled into a part of a single polynucleotide or nucleic acid construct, or assembled into separate polynucleotides or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Therefore, these polynucleotides can be introduced into plant cells in a single transformation event, in a separate transformation event, or, for example, as part of a breeding program. Therefore, the term "transformation" as used herein refers to the introduction of heterologous nucleic acids into cells.

[0133] Thus, soybean plants or parts thereof having haplotypes providing late maturing and / or determinate traits obtainable by the methods of the presently disclosed subject matter (i.e., soybean plants or parts thereof) are aspects of the presently disclosed subject matter. In some embodiments, soybean plants of the present invention have more than one haplotype providing late maturing determinate traits as described herein.

[0134] The soybean plant or part thereof of the present disclosure having a haplotype providing late maturity and / or determinate type can be homozygous for various allele combinations. In some embodiments of the present invention, the plant can be heterozygous.

[0135] The soybean plant or germplasm can be the progeny of a cross between one soybean variety and a second soybean variety comprising a haplotype that provides for late maturing and / or determinate traits.

[0136] The soybean plant or germplasm can be the progeny of an introgression, where the recurrent parent is a soybean variety and the donor comprises a haplotype that provides for late maturing and / or determinate traits.

[0137] The soybean plant or germplasm can be the progeny of a cross between a first soybean variety (e.g., a tester line), and the progeny of a cross between a second soybean variety (e.g., a recurrent parent) and a soybean variety of a haplotype that provides late maturing and / or determinate traits (e.g., a donor).

[0138] The soybean plant or germplasm can be the progeny of a cross between a first soybean variety and an introgressed progeny, wherein the recurrent parent is a second soybean variety and the donor comprises a haplotype that provides for late maturing and / or determinate traits.

[0139] Another aspect of the disclosed subject matter relates to a method of producing seeds that can be grown into soybean plants having a haplotype that provides late maturing and / or determinate traits. In some embodiments, the method comprises providing a soybean plant of the present specification, crossing the plant with another soybean plant, and collecting seeds produced by the cross that, when planted, produce plants having late maturing and / or determinate traits.

[0140] Thus, the present invention provides improved soybean plants, seeds, and / or tissue cultures produced by the methods described herein.In further embodiments, the present invention provides introgressed soybean plants and / or germplasm produced by the methods described herein. Composition for analyzing soybean genome

[0141] In some embodiments, the subject matter of the present disclosure provides methods for analyzing the genome of soybean plants / germplasm to identify those that include a desired marker and a combination of markers associated with a haplotype associated with late maturity and / or determinate type. In some embodiments, the analysis method comprises amplifying a subsequence of the soybean plant / germplasm genome and determining the nucleotides present in one, some, or all positions of the amplified subsequence.

[0142] Therefore, in some embodiments, the present invention provides a composition comprising one or more amplification primer pairs, which can initiate DNA polymerization on a soybean nucleic acid template by a DNA polymerase to generate a soybean marker amplicon. In some embodiments, the soybean amplicon can be used to identify a soybean marker comprising a nucleotide sequence of any one of SEQ ID NOs: 1-2. In view of the disclosure that SEQ ID NOs: 1-2 can be combined to provide a haplotype that leads to a desired late-maturing determinate type, a person of ordinary skill in the art will know the various techniques that can be used to analyze the sequence of the corresponding soybean nucleic acid. The representative sequences involved are provided in Table 2 below. Table 2: Sequence

[0143] A representative amplification primer pair may comprise the nucleotide sequence of a forward primer and a corresponding reverse primer as shown in the sequence listing provided in Table 3. A representative probe may comprise the nucleotide sequence as shown in the sequence listing provided in Table 4. Table 3: Example amplification primers Table 4: Example labeled probes Non-limiting Examples

[0144] The following provides non-limiting example embodiments of the present invention. Embodiment 1. A method for identifying and / or selecting soybean plants containing an allele combination associated with a late-maturing, determinate stem growth phenotype, the method comprising: a. isolating nucleic acid from a soybean plant part; b) detecting a first molecular marker associated with late maturity and a second molecular marker associated with determinate stem growth in the nucleic acid of a), wherein the first molecular marker associated with determinate stem growth contains a G at position 335 of SEQ ID NO:1, and wherein the second molecular marker associated with late maturity contains a T at position 260 of SEQ ID NO:2; and c) based on the presence of the first molecular marker and the second molecular marker of b), selecting or identifying soybean plants containing the allele combination associated with the late-maturing, determinate stem growth phenotype.

[0145] Embodiment 2. The method of embodiment 1, wherein the detecting comprises amplifying each of the first label and the second label, or a portion thereof, and detecting the resulting amplified label amplicons.

[0146] Embodiment 3. A method as described in Embodiment 2, wherein the amplification comprises: a) mixing an amplification primer pair with a nucleic acid isolated from a first soybean plant or its germplasm, wherein the primer pair is complementary or partially complementary to at least a portion of the first molecular marker associated with determinate stem growth and is capable of initiating DNA polymerization by a DNA polymerase using the soybean nucleic acid as a template; b) mixing another amplification primer or amplification primer pair with the nucleic acid isolated from the first soybean plant or its germplasm, wherein the primer pair is complementary or partially complementary to at least a portion of the second molecular marker associated with late maturity and is capable of initiating DNA polymerization by a DNA polymerase using the soybean nucleic acid as a template, and c) extending each primer pair in a DNA polymerization reaction comprising a DNA polymerase and a template nucleic acid to generate a corresponding amplicon.

[0147] Embodiment 4. A method as described in Embodiment 3, wherein the primer pair that is complementary or partially complementary to at least a portion of the first molecular marker associated with limited stem growth comprises SEQ ID NOs: 3 and 4, and wherein the primer pair that is complementary or partially complementary to at least a portion of the second molecular marker associated with limited stem growth comprises SEQ ID NOs: 5 and 6.

[0148] Embodiment 5. The method of embodiment 2, wherein the amplification comprises employing polymerase chain reaction (PCR) or ligase chain reaction (LCR), using nucleic acid isolated from soybean plants or their germplasm as a template in the PCR or LCR.

[0149] Embodiment 6. A method as described in Embodiment 2, wherein the detection of the amplified marker amplicon obtained includes detection using a marker probe, wherein the marker amplicon generated by amplifying the first marker associated with limited stem growth comprises SEQ ID NO: 7 or 9, and wherein the marker amplicon generated by amplifying the second marker associated with late maturity comprises SEQ ID NO: 8 or 10.

[0150] Example 7. A method of producing a soybean plant having a favorable combination of alleles associated with a late maturing determinate stem growth phenotype, the method comprising: a. isolating nucleic acid from a soybean plant part; b. detecting a first molecular marker associated with late maturity and a second molecular marker associated with determinate stem growth in the nucleic acid of a), wherein the first molecular marker associated with determinate stem growth comprises a G at position 335 of SEQ ID NO:1, and wherein the second molecular marker associated with late maturity comprises a T at position 260 of SEQ ID NO:2; c. selecting a first soybean plant comprising the favorable allele combination based on the presence of the first molecular marker and the second molecular marker of b); d. hybridizing the first soybean plant of c) with a second soybean plant that does not comprise the molecular marker of b); and e. producing progeny plants from the hybridization of d), thereby producing a soybean plant having the favorable allele combination associated with the late-maturing determinate stem growth phenotype.

[0151] Embodiment 8. The method of Embodiment 7, wherein the progeny plant comprises each of the molecular markers of b).

[0152] Embodiment 9. The method of Embodiment 7, wherein the first soybean plant or the second soybean plant is an elite soybean plant.

[0153] Embodiment 10. The method of embodiment 7, wherein the progeny plants are backcrossed for one or more generations.

[0154] Embodiment 11. A method as described in Embodiment 7, wherein the molecular marker is a single nucleotide polymorphism (SNP), a quantitative trait locus (QTL), an amplified fragment length polymorphism (AFLP), a randomly amplified polymorphic DNA (RAPD), a restriction fragment length polymorphism (RFLP) or a microsatellite.

[0155] Embodiment 12. The method of embodiment 7, wherein the detecting comprises amplifying each of the first label and the second label, or a portion thereof, and detecting the resulting amplified label amplicons.

[0156] Embodiment 13. A method as described in Embodiment 12, wherein the amplification includes: a) mixing an amplification primer pair with a nucleic acid isolated from a first soybean plant or its germplasm, wherein the primer pair is complementary or partially complementary to at least a portion of the first molecular marker associated with determinate stem growth and is capable of initiating DNA polymerization by a DNA polymerase using the soybean nucleic acid as a template; b) mixing another amplification primer or amplification primer pair with the nucleic acid isolated from the first soybean plant or its germplasm, wherein the primer pair is complementary or partially complementary to at least a portion of the second molecular marker associated with late maturity and is capable of initiating DNA polymerization by a DNA polymerase using the soybean nucleic acid as a template, and c) extending each primer pair in a DNA polymerization reaction comprising a DNA polymerase and a template nucleic acid to generate a corresponding amplicon.

[0157] Embodiment 14. A method as described in Embodiment 13, wherein the primer pair that is complementary or partially complementary to at least a portion of the first molecular marker associated with limited stem growth comprises SEQ ID NOs: 3 and 4, and wherein the primer pair that is complementary or partially complementary to at least a portion of the second molecular marker associated with limited stem growth comprises SEQ ID NOs: 5 and 6.

[0158] Embodiment 15. The method of embodiment 12, wherein the amplifying comprises employing polymerase chain reaction (PCR) or ligase chain reaction (LCR), using nucleic acid isolated from a soybean plant or its germplasm as a template in the PCR or LCR.

[0159] Embodiment 16. A method as described in Embodiment 12, wherein the detection of the amplified marker amplicon obtained includes detection using a marker probe, wherein the marker amplicon generated by amplifying the first marker associated with limited stem growth comprises SEQ ID NO:7 or 9, and wherein the marker amplicon generated by amplifying the second marker associated with late maturity comprises SEQ ID NO:8 or 10.

[0160] Embodiment 17. A composition comprising at least two amplification primer pairs, wherein the at least two amplification primer pairs are capable of initiating DNA polymerization on a soybean nucleic acid template by a DNA polymerase to generate at least two soybean marker amplicons to detect the presence of an allele combination associated with a late maturing determinate stem growth phenotype, wherein the at least two soybean marker amplicons comprise SEQ ID NO: 1 and SEQ ID NO: 2.

[0161] Embodiment 18. The composition of embodiment 17, wherein the at least two amplification primer pairs comprise (i) a primer pair of SEQ ID NO:3 and SEQ ID NO:4, and (ii) a primer pair of SEQ ID NO:5 and SEQ ID NO:6.

[0162] Embodiment 19. The composition of embodiment 17, further comprising at least two labeled probes for identifying the at least two amplicons, wherein the at least two labeled probes are: (i) SEQ ID NOs: 7 and 9; or (ii) SEQ ID NOs: 8 and 10.

[0163] Embodiment 20. The composition of embodiment 19, wherein the amplification primer pair comprises SEQ ID NOs: 3 and 4 and the label probe comprises SEQ ID NOs: 7 and 9, or the amplification primer pair comprises SEQ ID NOs: 5 and 6 and the label probe comprises SEQ ID NOs: 8 and 10. Examples

[0164] Average plant height (in centimeters) was used as an indicator to determine the effects of different allele combinations of stem top (SY3970) and E1 maturity gene (SY0768BQ) on soybean plants. Data from 62 individuals of 5 separate populations are recorded in Table 5 below. The results indicate that TTGG is the desired haplotype for the correct plant type of the late maturing determinate phenotype, where AAGG is highly undesirable. AACC is the correct haplotype for the mid-maturing indeterminate type, while TTCC is the correct haplotype for the late maturing indeterminate type. Table 5

Claims

1. A method of identifying and / or selecting a soybean plant comprising a combination of alleles associated with a late maturing determinate stem growth phenotype, the method comprising: a) isolating nucleic acids from soybean plant parts; b) detecting in the nucleic acid of a) a first molecular marker associated with late maturity and a second molecular marker associated with determinate stem growth, wherein the first molecular marker associated with determinate stem growth comprises a G at position 335 of SEQ ID NO: 1, and wherein the second molecular marker associated with late maturity comprises a T at position 260 of SEQ ID NO: 2; and c) selecting or identifying soybean plants comprising said combination of alleles associated with a late maturing determinate stem growth phenotype based on the presence of said first molecular marker and said second molecular marker of b).

2. The method of claim 1, wherein the detecting comprises amplifying each of the first marker and the second marker, or a portion thereof, and detecting the resulting amplified marker amplicons.

3. The method of claim 2, wherein the amplifying comprises: i) mixing an amplification primer pair with nucleic acid isolated from a first soybean plant or germplasm thereof, wherein the primer pair is complementary or partially complementary to at least a portion of the first molecular marker associated with determinate stem growth and is capable of initiating DNA polymerization by a DNA polymerase using the soybean nucleic acid as a template; ii) mixing another amplification primer or amplification primer pair with the nucleic acid isolated from the first soybean plant or its germplasm, wherein the primer pair is complementary or partially complementary to at least a portion of the second molecular marker associated with late maturity and is capable of initiating DNA polymerization by a DNA polymerase using the soybean nucleic acid as a template, and iii) extending each primer pair in a DNA polymerization reaction comprising a DNA polymerase and a template nucleic acid to generate a corresponding amplicon.

4. A method as claimed in claim 3, wherein the primer pair that is complementary or partially complementary to at least a portion of the first molecular marker associated with limited stem growth comprises SEQ ID NOs: 3 and 4, and wherein the primer pair that is complementary or partially complementary to at least a portion of the second molecular marker associated with late maturity comprises SEQ ID NOs: 5 and 6.

5. The method of claim 2, wherein the amplifying comprises employing polymerase chain reaction (PCR) or ligase chain reaction (LCR), using nucleic acid isolated from a soybean plant or its germplasm as a template in the PCR or LCR.

6. A method as claimed in claim 2, wherein detecting the obtained amplified labeled amplicon comprises detecting using a labeled probe, wherein the labeled amplicon generated by amplifying the first marker associated with limited stem growth comprises SEQ ID NO:7 or 9, and wherein the labeled amplicon generated by amplifying the second marker associated with late maturity comprises SEQ ID NO:8 or 10.

7. A method of producing a soybean plant having a favorable combination of alleles associated with a late maturing determinate stem growth phenotype, the method comprising: a) isolating nucleic acids from soybean plant parts; b) detecting in the nucleic acid of a) a first molecular marker associated with determinate stem growth and a second molecular marker associated with late maturity, wherein the first molecular marker associated with determinate stem growth comprises a G at position 335 of SEQ ID NO: 1, and wherein the second molecular marker associated with late maturity comprises a T at position 260 of SEQ ID NO: 2; c) selecting a first soybean plant comprising the favorable allele combination based on the presence of the first molecular marker and the second molecular marker of b); d) crossing said first soybean plant of c) with a second soybean plant that does not contain said molecular marker of b); and e) producing progeny plants from said cross of d), thereby producing soybean plants having said favorable combination of alleles associated with a late maturing determinate stem growth phenotype.

8. The method of claim 7, wherein the progeny plant comprises each of the first molecular marker and the second molecular marker of b).

9. The method of claim 7, wherein the first soybean plant or the second soybean plant is an elite soybean plant.

10. The method of claim 7, wherein the progeny plants are backcrossed for one or more generations.

11. The method of claim 7, wherein the molecular marker is a single nucleotide polymorphism (SNP), a quantitative trait locus (QTL), an amplified fragment length polymorphism (AFLP), a randomly amplified polymorphic DNA (RAPD), a restriction fragment length polymorphism (RFLP) or a microsatellite.

12. The method of claim 7, wherein the detecting comprises amplifying each of the first marker and the second marker, or a portion thereof, and detecting the resulting amplified marker amplicons.

13. The method of claim 12, wherein the amplifying comprises: i) mixing an amplification primer pair with nucleic acid isolated from said first soybean plant or germplasm thereof, wherein said primer pair is complementary or partially complementary to at least a portion of said first molecular marker associated with determinate stem growth and is capable of initiating DNA polymerization by a DNA polymerase using said soybean nucleic acid as a template; ii) mixing another amplification primer or amplification primer pair with the nucleic acid isolated from the first soybean plant or its germplasm, wherein the another primer pair is complementary or partially complementary to at least a portion of the second molecular marker associated with late maturity and is capable of initiating DNA polymerization by a DNA polymerase using the soybean nucleic acid as a template; and iii) extending each primer pair in a DNA polymerization reaction comprising a DNA polymerase and a template nucleic acid to generate a corresponding amplicon.

14. A method as claimed in claim 13, wherein the primer pair that is complementary or partially complementary to at least a portion of the first molecular marker associated with determinate stem growth comprises SEQ ID NOs: 3 and 4, and wherein the other primer pair that is complementary or partially complementary to at least a portion of the second molecular marker associated with determinate stem growth comprises SEQ ID NOs: 5 and 6.

15. The method of claim 12, wherein the amplifying comprises employing polymerase chain reaction (PCR) or ligase chain reaction (LCR), using nucleic acid isolated from a soybean plant or germplasm thereof as a template in the PCR or LCR.

16. A method as claimed in claim 12, wherein the detection of the amplified labeled amplicon comprises detection using a labeled probe, wherein the labeled amplicon generated by amplifying the first marker associated with limited stem growth comprises SEQ ID NO:7 or 9, and wherein the labeled amplicon generated by amplifying the second marker associated with late maturity comprises SEQ ID NO:8 or 10.

17. A composition comprising at least two amplification primer pairs, wherein the at least two amplification primer pairs are capable of initiating DNA polymerization on a soybean nucleic acid template by a DNA polymerase to generate at least two soybean marker amplicons to detect the presence of an allele combination associated with a late maturing determinate stem growth phenotype, wherein the at least two soybean marker amplicons comprise SEQ ID NO: 1 and SEQ ID NO:

2.

18. The composition of claim 17, wherein the at least two amplification primer pairs comprise (i) a primer pair of SEQ ID NO:3 and SEQ ID NO:4, and (ii) a primer pair of SEQ ID NO:5 and SEQ ID NO:

6.

19. The composition of claim 17 or 18, further comprising at least two labeled probes for identifying the at least two amplicons, wherein the at least two labeled probes are: (i) SEQ ID NOs: 7 and 9; or (ii) SEQ ID NOs: 8 and 10.

20. The composition of claim 19, wherein the amplification primer pair comprises SEQ ID NOs: 3 and 4 and the label probe comprises SEQ ID NOs: 7 and 9, and / or wherein the amplification primer pair comprises SEQ ID NOs: 5 and 6 and the label probe comprises SEQ ID NOs: 8 and 10.

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