A SNP molecular marker related to soybean oil content and application thereof

Abstract

The present application relates to the technical field of molecular biology, and particularly relates to a SNP molecular marker related to soybean oil content and application thereof, a nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO:1, a nucleotide at the 359th position is a SNP site, and polymorphism is C / T. The present application carries out gene positioning and candidate gene analysis on the soybean oil content trait through whole genome association analysis, mines a related gene determining the soybean seed oil content trait and a SNP molecular marker associated with the soybean oil content trait, enriches a target resource library in the field of soybean high-oil breeding, and provides a new technical means for soybean molecular marker assisted breeding. Through detection of the SNP molecular marker, the soybean material can be identified for the oil content trait in the early breeding stage, and germplasm resources with high oil content are quickly screened out, so that the breeding cycle is effectively shortened, and the breeding process of new high-oil soybean varieties is accelerated.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a SNP molecular marker related to soybean oil content and its application. Background Technology

[0002] Soybeans are an important oilseed crop in my country. The oil content of soybean kernels is a core quality indicator that determines the processing value and industrial benefits of soybeans. The breeding of high-oil soybean varieties is directly related to the stability and security of domestic vegetable oil supply.

[0003] Genome-wide association analysis (GWAS) is a technique that uses genome-wide molecular markers to identify genetic loci associated with target traits. This method can efficiently locate key genes controlling complex quantitative traits, providing a foundation for marker-assisted breeding. Marker-assisted breeding utilizes molecular markers tightly linked to target genes to indirectly select for traits early in the breeding process, overcoming the limitations of traditional breeding methods such as long cycles, low efficiency, and susceptibility to environmental influences. Single nucleotide polymorphisms (SNPs), as third-generation molecular markers, are widely distributed and genetically stable in the genome, making them important tools for genetic diversity analysis, gene mapping, and molecular breeding research. Combining GWAS technology with SNP markers for the genetic analysis of high oil content in soybeans can accurately identify key loci controlling oil content, providing technical support for the breeding of new high-oil soybean varieties. Summary of the Invention

[0004] To address the lack of SNP markers in existing technologies for developing new high-oil soybean varieties, this invention provides an SNP molecular marker related to soybean oil content and its application.

[0005] The technical solution provided by this invention is: This invention utilizes resequencing technology to sequence mature soybean seed materials and perform GWAS analysis, obtaining a SNP locus significantly associated with soybean oil content. This SNP locus is located at chr17:5386219 in the Glycine max Wm82.a4.v1 genome. This SNP locus includes two genotypes, C and T. Statistical analysis revealed significant differences in the probability distribution of this SNP locus among soybean seeds with different oil contents. Specifically, C is the dominant allele in soybean seeds with high oil content, while T is the dominant allele in soybean seeds with low oil content. This SNP locus can be used as a molecular marker for the breeding of superior soybean seeds; and in soybean materials with low oil content, the oil content of seeds can be increased by selecting individuals with the C allele.

[0006] In a first aspect, the present invention provides a SNP molecular marker related to soybean oil content, wherein the nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO:1, the SNP site of the SNP molecular marker is located at position 359 of the nucleotide sequence shown in SEQ ID NO:1, and the polymorphism is C / T; the SNP site of the SNP molecular marker is located at position 5386219 of soybean chromosome 17, and the reference genome version number of soybean chromosome 17 is Glycine maxWm82.a4.v1.

[0007] According to the SNP molecular markers related to soybean oil content provided by the present invention, preferably, soybeans with polymorphism C at the SNP site have a higher oil content than soybeans with polymorphism T.

[0008] More preferably, soybean individuals with the SNP molecular marker genotype CC have a higher oil content than soybean individuals with the genotype TT.

[0009] Secondly, the present invention also provides a primer pair for detecting SNP molecular markers related to soybean oil content, comprising an upstream primer and a downstream primer, wherein the upstream primer comprises a nucleotide sequence as shown in SEQ ID NO:2; and the downstream primer comprises a nucleotide sequence as shown in SEQ ID NO:3.

[0010] According to the present invention, a primer pair for detecting SNP molecular markers related to soybean oil content is preferably provided, wherein the nucleotide sequence of the upstream primer has at least 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO:2.

[0011] More preferably, the nucleotide sequence of the upstream primer is shown in SEQ ID NO:2.

[0012] According to the present invention, a primer pair for detecting SNP molecular markers related to soybean oil content is preferably provided, wherein the nucleotide sequence of the downstream primer has at least 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO:3.

[0013] More preferably, the nucleotide sequence of the downstream primer is shown in SEQ ID NO:3.

[0014] Thirdly, the present invention provides a composition for detecting the SNP molecular marker, comprising the primer pair.

[0015] According to the present invention, a composition for detecting the SNP molecular marker is provided, preferably, the composition further comprising PCR reagents and / or sequencing reagents.

[0016] Fourthly, the present invention provides the application of the primer pair or the composition thereof in any of the following: (1) identifying the level of soybean oil content; (2) screening or identifying soybean varieties with high oil content; (3) soybean variety improvement; (4) soybean germplasm resource improvement; (5) molecular marker-assisted breeding of soybean.

[0017] According to the application of the primer pair or the composition provided by the present invention, preferably, the soybean sample with a polymorphism of C at the SNP site of the SNP molecular marker has a higher oil content than the soybean sample with a polymorphism of T.

[0018] More preferably, the soybeans tested with the SNP molecular marker having the genotype CC have a higher oil content than the soybeans tested with the genotype TT.

[0019] According to the application of the primer pair or the composition provided by the present invention, preferably, the oil content of the high oil content soybean variety is ≥20%.

[0020] In this invention, the oil content of soybeans, the oil content of soybean varieties, and the oil content of soybeans all refer to the percentage of crude fat in soybean seeds by their dry weight.

[0021] This invention also provides the application of a detection reagent for SNP molecular markers related to soybean oil content in the identification of soybean oil content. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO:1. The SNP site of the SNP molecular marker is located at position 359 of the nucleotide sequence shown in SEQ ID NO:1, and the polymorphism is C / T. The SNP site is located at position 5386219 of soybean chromosome 17, and the reference genome version number of soybean chromosome 17 is Glycine maxWm82.a4.v1.

[0022] According to the application of the detection reagent for SNP molecular markers related to soybean oil content provided by the present invention in identifying soybean oil content, preferably, soybean individuals with the SNP molecular marker genotype CC have a higher oil content than soybean individuals with the genotype TT.

[0023] The application of the detection reagent for SNP molecular markers related to soybean oil content provided by the present invention in identifying soybean oil content is preferably provided that the detection reagent includes the primer pair for detecting SNP molecular markers related to soybean oil content, or the composition for detecting SNP molecular markers related to soybean oil content.

[0024] Fifthly, the present invention provides a method for identifying the oil content of soybeans, comprising: detecting the polymorphism of the SNP site of the SNP molecular marker related to soybean oil content in the soybean to be tested, and determining the oil content of the soybean based on the detection results; the soybean to be tested with the polymorphism of SNP site C has a higher oil content than the soybean to be tested with the polymorphism of T.

[0025] According to the method for identifying the oil content of soybeans provided by the present invention, preferably, the soybeans tested with the SNP molecular marker genotype CC have a higher oil content than the soybeans tested with the genotype TT.

[0026] According to the present invention, a method for identifying the oil content of soybeans is provided. Preferably, the method for detecting the polymorphism of SNP sites of SNP molecular markers in the soybeans to be tested includes any one or more of PCR, liquid phase chip, microfluidics, mass spectrometry and sequencing.

[0027] More preferably, the method for detecting the polymorphism of SNP molecular markers in soybeans to be tested includes first performing PCR on the soybeans to be tested.

[0028] More preferably, the obtained PCR product is sequenced.

[0029] More preferably, PCR is performed using primer pairs for detecting the SNP molecular marker.

[0030] More preferably, PCR is performed using the genomic DNA of the soybean to be tested as a template.

[0031] More preferably, the PCR reaction system includes: the upstream primer at a working concentration of 0.08 μM to 0.12 μM, and the downstream primer at a working concentration of 0.08 μM to 0.12 μM.

[0032] More preferably, the PCR reaction system includes the upstream primer at a working concentration of 0.08 μM, 0.09 μM, 0.1 μM, 0.11 μM or 0.12 μM.

[0033] More preferably, the PCR reaction system includes the downstream primers at working concentrations of 0.08 μM, 0.09 μM, 0.1 μM, 0.11 μM, or 0.12 μM.

[0034] More preferably, the PCR reaction system includes: the upstream primer at a working concentration of 0.1 μM and the downstream primer at a working concentration of 0.1 μM.

[0035] More preferably, the PCR reaction program includes: pre-denaturation at 94℃~96℃ for 2 min~4 min; amplification reaction for 28 to 35 cycles, each cycle including denaturation at 94℃~96℃ for 14 s~16 s, annealing at 54℃~56℃ for 14 s~16 s and extension at 71℃~73℃ for 38 s~42 s; and final extension at 71℃~73℃ for 4 min~6 min.

[0036] More preferably, the PCR reaction program includes: pre-denaturation at 95°C for 3 min; 30 cycles of amplification reaction, each cycle including denaturation at 95°C for 15 s, annealing at 55°C for 15 s and extension at 72°C for 40 s; and final extension at 72°C for 5 min.

[0037] Sixthly, the present invention provides a method for breeding soybeans with high oil content, comprising: during soybean breeding, selecting soybeans with the genotype CC that possess the SNP molecular marker related to soybean oil content in the offspring. Soybean individuals with the genotype CC have a higher oil content than soybean individuals with the genotype TT.

[0038] The present invention has the following beneficial effects: This invention utilizes genome-wide association analysis (GWIA) to locate and analyze candidate genes for soybean oil content, identifying the relevant gene GmbHLH1, which determines soybean seed oil content, and the SNP molecular markers associated with this trait within the gene. This invention provides new germplasm resources for breeding high-oil-content soybean varieties, enriches the target resource library in the field of high-oil-content soybean breeding, and offers a new technical means for marker-assisted breeding of soybeans. By detecting these SNP molecular markers, the oil content trait of soybean materials can be identified in the early stages of breeding, rapidly screening high-oil-content germplasm resources, thereby effectively shortening the breeding cycle and accelerating the breeding process of new high-oil-content soybean varieties. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0040] Figure 1 This is the Manhattan plot of genome-wide association analysis provided in Embodiment 1 of the present invention.

[0041] Figure 2This is a box plot of oil content of soybean materials with different SNP locus genotypes provided in Example 3 of the present invention; CC represents soybean material with SNP locus genotype CC; TT represents soybean material with SNP locus genotype TT. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0043] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0044] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0045] Example 1: Genome-wide association analysis of oil content in soybean seed materials This invention uses 521 natural soybean populations as research objects, mainly including varieties such as Dongsheng 112, Dongsheng 137, and Dongsheng 124. These materials were collected from Gongzhuling, Changchun City and preserved at the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences. The public can obtain these biological materials from the applicant. The whole genome resequencing has been completed, and the data is highly representative.

[0046] Genome-wide association analysis was performed using the Glycine max Wm82.a4.v1 genome as a reference genome, such as... Figure 1 As shown, a total of 21,752,494 SNPs were obtained after analysis, covering all 20 chromosomes. Genome-wide association analysis was performed on the soybean oil content phenotype using rMVP software and the GLM model. SNP-associated signal sites were then screened based on P=0.05 / n (where n is the number of effective SNPs in the population), identifying candidate genes and their SNP sites significantly associated with soybean oil content.

[0047] The GmbHLH1 gene was identified as a candidate gene associated with oil content, and its partial sequence is shown below: (SEQ ID NO:1).

[0048] In-depth analysis of the GmbHLH1 gene revealed a key SNP site highly correlated with oil content, located at position 359 of the nucleotide sequence shown in SEQ ID NO:1, with a polymorphism of C or T. This SNP site is located at position 5386219 on chromosome 17 and has been named chr17:5386219.

[0049] Example 2: Method for Identifying Soybean SNP Sites 1. PCR detection Based on the sequence of the molecular marker (SEQ ID NO:1) to which the SNP site (chr17:5386219) belongs, primer pairs for amplifying the SNP site were designed, and the specific sequences are as follows: Upstream primer: 5'-ACAGTACTGCAATGACACGGA-3' (SEQ ID NO:2); Downstream primer: 5'-GGGAACGGATCCATCCATTAAA-3' (SEQ ID NO:3).

[0050] Using the genomic DNA of the soybean sample to be tested as a template, PCR amplification was performed using the primer pairs described above.

[0051] The PCR amplification reaction system (in 20 μL) includes the following components: 2 μL of genomic DNA at a concentration of 70 ng / μL, 1 μL of upstream primer at a concentration of 2 μM (SEQ ID NO:2), 1 μL of downstream primer at a concentration of 2 μM (SEQ ID NO:3), 10 μL of 2×Rapid Taq Master Mix, and 6 μL of ddH2O.

[0052] The PCR reaction program was as follows: pre-denaturation at 95℃ for 3 min; 30 cycles of amplification reaction, each cycle including denaturation at 95℃ for 15 s, annealing at 55℃ for 15 s and extension at 72℃ for 40 s; final extension at 72℃ for 5 min; and maintenance at 4℃ until removal.

[0053] 2. First-generation sequencing The PCR amplification products were subjected to first-generation sequencing. The sequencing results were compared with the sequence of the molecular marker (SEQ ID NO:1) to analyze the genotype of the SNP site (chr17:5386219) in the soybean material to be tested.

[0054] Example 3: Population Validation of the Association Between Soybean SNP Sites and Soybean Oil Content 1. Soybean Material Information The study analyzed 497 natural soybean populations (not overlapping with the soybean materials in Example 1), including the main varieties Dongsheng 112, Dongsheng 137, and Dongsheng 124. These materials were collected from Gongzhuling, Changchun City, and are preserved at the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences. The public can obtain these biological materials from the applicant.

[0055] 2. Identification of soybean SNP sites Genomic DNA was extracted from the soybean material and used as a template. Following the method in Example 2, under the condition that each soybean sample was homozygous, the genotype of the SNP site (chr17:5386219) in the soybean material was counted.

[0056] 3. Determination of soybean oil content The oil content (OC) of all soybean materials was determined by Fourier transform near-infrared spectroscopy, which is the percentage of crude fat in the dry weight of the soybean material (%).

[0057] 4. Correlation analysis between soybean SNP sites and oil content The T-test was used to statistically analyze the differences in oil content among different SNP loci genotypes.

[0058] like Figure 2 As shown, 457 soybean accessions had the genotype CC at their SNP loci, with a mean OC value of 20%, meeting the high-oil soybean standard (≥20%) specified in the national standard GB 1352-2009. The remaining 40 soybean accessions had the genotype TT at their SNP loci, with a mean OC value of 17.5%. T-test analysis showed that the average oil content of soybean accessions with the CC genotype at their SNP loci was significantly higher than that of soybean accessions with the TT genotype.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of primer pairs for detecting SNP molecular markers associated with soybean oil content, or compositions comprising said primer pairs, in any of the following: (1) To determine the level of soybean oil content; (2) Screening or identifying soybean varieties with high oil content; (3) Improvement of soybean varieties related to oil content traits; (4) Improvement of soybean germplasm resources related to oil content traits; The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO:

1. The SNP site of the SNP molecular marker is located at position 359 of the nucleotide sequence shown in SEQ ID NO:1, and the polymorphism is C / T. The SNP site is located at position 5386219 of soybean chromosome 17, and the reference genome version number of soybean chromosome 17 is Glycine maxWm82.a4.v1. Soybean individuals with the SNP molecular marker genotype CC have a higher oil content than soybean individuals with the genotype TT. The primer pair includes an upstream primer and a downstream primer, wherein the upstream primer includes the nucleotide sequence shown in SEQ ID NO:2; and the downstream primer includes the nucleotide sequence shown in SEQ ID NO:

3. The soybean varieties mentioned are Dongsheng 112, Dongsheng 137, or Dongsheng 124.

2. The application according to claim 1, characterized in that, The composition also includes PCR reagents and / or sequencing reagents.

3. A method for determining the oil content of soybeans, characterized in that, include: The polymorphism of the SNP site of the SNP molecular marker described in claim 1 in the soybean to be tested is detected, and the oil content of the soybean is determined according to the detection results; the soybean to be tested with the genotype CC of the SNP molecular marker has a higher oil content than the soybean to be tested with the genotype TT. The soybean varieties mentioned are Dongsheng 112, Dongsheng 137, or Dongsheng 124.

4. The identification method according to claim 3, characterized in that, The methods for detecting the polymorphism of SNP sites of SNP molecular markers in the soybean to be tested include any one or more of PCR, microfluidics, mass spectrometry, and sequencing.

5. The identification method according to claim 4, characterized in that, PCR is performed using primer pairs, the primer pairs comprising an upstream primer and a downstream primer, the upstream primer comprising the nucleotide sequence shown in SEQ ID NO:2; and the downstream primer comprising the nucleotide sequence shown in SEQ ID NO:

3.

6. A method for breeding soybeans with high oil content, characterized in that, include: During soybean breeding, the offspring are selected from soybeans with the genotype CC that possesses the SNP molecular marker described in claim 1; The soybean varieties mentioned are Dongsheng 112, Dongsheng 137, or Dongsheng 124.

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