SNP molecular markers related to soybean seed oil content, protein content and hundred seed weight and application

Abstract

The application provides a SNP molecular marker related to soybean seed oil content, protein content and hundred-seed weight and application. The SNP molecular marker contains a nucleotide sequence with C / T polymorphism at the 201th position of the sequence shown in SEQ ID NO:1. The molecular marker of the application has very important guiding significance in soybean molecular identification and assisted breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a SNP molecular marker and its application related to soybean seed oil content, protein content, and 100-seed weight. Background Technology

[0002] Soybean, as an economic crop, is one of the world's most important sources of oil and protein. Currently, the genetic mechanisms underlying soybean seed oil and protein content and 100-seed weight remain unclear. Genetic analysis of soybean seed oil and protein content, as well as 100-seed weight, holds promise for providing a theoretical basis for breeding high-yielding and high-quality soybeans. Developing varieties with high oil and protein content and high 100-seed weight using traditional breeding methods is not only time-consuming but also difficult to succeed, failing to meet current high-yield breeding needs. There is an urgent need for a more accurate and efficient method to identify superior soybean varieties in terms of seed oil and protein content and 100-seed weight. With the development and use of linkage or associated molecular markers for important yield traits (QTLs), marker-assisted selection has become an important tool in rapid breeding (Cregan et al., 1999). The greatest advantage of marker-assisted selection is that it can identify plants with the target phenotype by confirming the presence of the target genotype without requiring an assessment of phenotypic characteristics. Summary of the Invention

[0003] The purpose of this invention is to provide an SNP molecular marker and its application related to soybean seed oil content, protein content, and 100-seed weight.

[0004] To achieve the objectives of this invention, in a first aspect, this invention provides an SNP molecular marker related to soybean seed oil content, protein content, and 100-seed weight, wherein the SNP molecular marker contains a nucleotide sequence with a polymorphism of C / T at position 201 of the sequence shown in SEQ ID NO:1.

[0005] The genotype of the site with the aforementioned polymorphism is CC, corresponding to a phenotype of high oil content, high 100-grain weight, and low protein content; the genotype of the site with the aforementioned polymorphism is TT, corresponding to a phenotype of low oil content, low 100-grain weight, and high protein content.

[0006] Secondly, the present invention provides KASP primers for amplifying the SNP molecular marker, including forward primer 1, forward primer 2 and universal reverse primer, the sequences of which are shown in SEQ ID NO:2-4 respectively.

[0007] Thirdly, the present invention provides detection reagents or kits containing the primers.

[0008] Fourthly, the present invention provides the application of the SNP molecular marker, the primer, the detection reagent, or the kit in identifying soybean seed oil and protein content, as well as the 100-seed weight phenotype.

[0009] Fifthly, the present invention provides the application of the SNP molecular marker, the primer, the detection reagent, or the kit in the identification of soybean germplasm resources.

[0010] Sixthly, the present invention provides the application of the SNP molecular marker, the primer, the detection reagent, or the kit in soybean directional breeding.

[0011] In a seventh aspect, the present invention provides the application of the SNP molecular marker, the primer, the detection reagent, or the kit in the cultivation of high-quality, large-grained soybean varieties.

[0012] Eighthly, the present invention provides the application of the SNP molecular marker, the primer, the detection reagent, or the kit in marker-assisted breeding of soybean.

[0013] Ninthly, the present invention provides the SNP molecular marker, the primer, the detection reagent, or the kit in soybeans. SOC5 Gene( SOC5 The application of gene-encoded proteins (NCBI ID: XP_006579876.1) in typing.

[0014] The SNP marker of this invention is located at SOC5 1595bp downstream of the gene start codon.

[0015] In a tenth aspect, the present invention provides a method for identifying the oil and protein content of soybean seeds, as well as the 100-seed weight phenotype, comprising the following steps:

[0016] 1) Detect the genotype of the polymorphic sites of the SNP molecular marker in the soybean to be tested;

[0017] 2) Determine the seed oil and protein content and 100-seed weight of the soybean to be tested based on the genotype obtained in step 1).

[0018] The specific determination method for step 2) is as follows: if the genotype is CC, then the soybean to be tested is a candidate seed with high oil content, high 100-seed weight, and low protein content; if the genotype is TT, then the soybean to be tested is a candidate seed with high protein content, low oil content, and low 100-seed weight.

[0019] Further, step 1) includes:

[0020] a) Extract soybean genomic DNA for testing;

[0021] b) Using DNA as a template, PCR amplification was performed using the primers shown in SEQ ID NO:2-4, and the amplification products were sequenced to identify the variant type of the target site.

[0022] The amplification system used was as follows: 20 μL of PCR reaction system, including 30-60 ng of genomic DNA, 2 μL of 10×PCR reaction buffer, 1.5 μL of 2 mmol / L dNTPs, 2 μL each of 2 mmol / L upstream and downstream primers, and 1 U Taq DNA polymerase.

[0023] The amplification program used was as follows: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 s, annealing at the optimized annealing temperature of 55 °C for 30 s, extension at 72 °C for 50 s, for 36 cycles; and finally extension at 72 °C for 10 min, followed by storage at 4 °C.

[0024] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0025] (I) This invention utilizes soybeans SOC5 Genetic variation analysis revealed that the SNP site Chr05:15709660 was significantly associated with soybean seed oil and protein content, as well as 100-seed weight. This SNP site has significant guiding value for soybean molecular identification and assisted breeding.

[0026] (ii) The method for detecting soybean seed oil and protein content and 100-seed weight provided by the present invention is simple, fast and accurate. Attached Figure Description

[0027] Figure 1 In a preferred embodiment of the present invention, the PCR amplification product contains SOC5 The variant type of the SNP site Chr05:15709660 was identified by sequencing. (A) William82, a high-oil, large-grain, low-protein variety; (B) Xiaoheidou, a high-protein, small-grain, low-oil variety. Detailed Implementation

[0028] This invention provides SNP molecular markers and applications associated with soybean seed oil and protein content, as well as 100-seed weight, enabling rapid detection of soybean seed oil and protein content, as well as 100-seed weight, providing strong technical support for soybean molecular identification and assisted breeding.

[0029] The present invention adopts the following technical solution:

[0030] The present invention provides an SNP molecular marker associated with soybean seed oil content, protein content and 100-seed weight, wherein the SNP molecular marker contains a nucleotide sequence (n is c or t) with a polymorphism of C or T at position 201 of the sequence shown in SEQ ID NO:1.

[0031] According to the present invention, a SNP molecular marker associated with soybean seed oil content, protein content, and 100-seed weight is provided. The genotype of the site with the polymorphism is CC, which corresponds to a phenotype with high seed oil content and 100-seed weight, and low protein content; the genotype of the site with the polymorphism is TT, which corresponds to a phenotype with high seed protein content, and low oil content and 100-seed weight.

[0032] The present invention also provides a candidate primer for amplifying the above-mentioned SNP molecular marker.

[0033] Preferably, the candidate primers used to amplify the above-mentioned SNP molecular markers are KASP primer combinations.

[0034] More preferably, the KASP primer combination for amplifying the above-mentioned SNP molecular markers includes the following primers: allele forward primer 1 as shown in SEQ ID NO:2: 5′-aagcaagaagcagaacaatttC-3′; allele forward primer 2 as shown in SEQ ID NO:3: 5′-aagcaagaagcagaacaatttT-3′; and universal reverse primer as shown in SEQ ID NO:4: 5′-gcacaatgaaattcttgtgca-3′.

[0035] Preferably, allele forward primer 1, as shown in SEQ ID NO:2, and allele forward primer 2, as shown in SEQ ID NO:3, contain different fluorescent groups.

[0036] More preferably, allele forward primer 1, as shown in SEQ ID NO:2, contains the fluorescent group FAM, and allele forward primer 2, as shown in SEQ ID NO:3, contains the fluorescent group HEX.

[0037] The present invention provides a kit comprising the primers described above.

[0038] This invention provides the application of the above-mentioned SNP molecular markers, primers, and kits in identifying soybean seed oil and protein content, as well as 100-seed weight phenotype.

[0039] This invention provides the application of the above-mentioned SNP molecular markers, primers, and kits in the identification, improvement, or marker-assisted breeding of soybean germplasm resources. For example, by crossing and backcrossing soybean materials carrying superior alleles with other materials, soybean materials carrying superior alleles can be created.

[0040] This invention provides the application of the above-mentioned SNP molecular markers, primers, and kits in screening high-quality large-grained soybean varieties.

[0041] According to the application described, the soybean can be any soybean variety. The molecular markers, primers, and kits described in this invention are universal and can be applied to any soybean variety grown in any environment.

[0042] This invention provides a method for identifying the oil and protein content of soybean seeds, as well as the weight of 100 seeds, the method comprising the following steps:

[0043] 1) Detect the genotypes of the polymorphic sites of the above-mentioned SNP molecular markers in soybean samples;

[0044] 2) Determine the seed oil content, protein content, and 100-seed weight of the sample based on the genotype obtained in step 1).

[0045] The specific determination method is as follows: if the obtained genotype is CC, then the soybean to be tested is a candidate seed with high oil content and high 100-seed weight; if the obtained genotype is TT, then the soybean to be tested is a candidate seed with high protein content.

[0046] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0047] Example 1: Identification of SNP sites

[0048] This invention, through whole-genome sequencing and association analysis, identified a single SNP locus that is significantly associated with soybean seed oil content, protein content, and 100-seed weight. This SNP locus is a genomic version. Glycine max The allele at the Chr05:15709660 locus of Wm82.a2.v1 (Soybean, http: / / www.phytozome.net) is C or T, with two main genotypes: CC and TT (Table 1).

[0049] Table 1. Marker site information

[0050]

[0051] Example 2: Application of SNP sites in identifying or assisting in the identification of soybean seed oil and protein content and 100-seed weight

[0052] 1. The Chr05:15709660 locus genotype was significantly associated with soybean seed oil and protein content as well as 100-seed weight.

[0053] (1) Identify the seed oil and protein content and 100-seed weight of soybean varieties.

[0054] To verify the reliability of SNP loci, seed oil and protein content, as well as 100-seed weight, were investigated in 279 soybean varieties in Shijiazhuang, Hebei Province. Row length was 2 meters (two rows), plant spacing was 0.1 meters, and row spacing was 0.5 meters. Local standard management practices were followed to ensure normal growth and maturity of the experimental materials. Seeds harvested from the field were analyzed for seed oil and protein content, as well as 100-seed weight.

[0055] The genotypes of the 279 soybean samples were detected using the genotype identification method provided in Example 1. The results are shown in Tables 2 to 8.

[0056] Table 2. Genotypes of 279 soybean Chr05:15709660 loci, seed oil and protein content, and 100-seed weight.

[0057]

[0058] Table 3. Genotypes of 279 soybean Chr05:15709660 locus, seed oil and protein content, and 100-seed weight.

[0059]

[0060] Table 4. Genotypes of 279 soybean Chr05:15709660 locus, seed oil and protein content, and 100-seed weight.

[0061]

[0062] Table 5. Genotypes of 279 soybean Chr05:15709660 locus, seed oil and protein content, and 100-seed weight.

[0063]

[0064] Table 6. Genotypes of 279 soybean Chr05:15709660 locus, seed oil and protein content, and 100-seed weight.

[0065]

[0066] Table 7. Genotypes of 279 soybean Chr05:15709660 locus, seed oil and protein content, and 100-seed weight.

[0067]

[0068] Table 8. Genotypes of 279 soybean Chr05:15709660 locus, seed oil and protein content, and 100-seed weight.

[0069]

[0070] Note: The soybean varieties mentioned in Tables 2 to 8 can be found in the following references: Wang Guoxun. Catalogue of Chinese Soybean Variety Resources. Beijing: China Agriculture Press, 1982; Chang Ruzhen and Sun Jianying. Catalogue of Chinese Soybean Variety Resources: Continued Volume 1. Beijing: Agriculture Press, 1991; Chang Ruzhen, Sun Jianying, Qiu Lijuan, and Chen Yiwu. Catalogue of Chinese Soybean Variety Resources: Continued Volume 2. Beijing: China Agriculture Press, 1996.

[0071] The genotypes of the Chr05:15709660 locus in 279 materials were detected using whole-genome resequencing, including 262 CC genotypes and 17 TT genotypes. In the Shijiazhuang environment, the average seed oil content of materials carrying the CC genotype was (19.19 ± 1.38)%, the protein content was (42.23 ± 1.10)%, and the 100-seed weight was (14.72 ± 3.02 g); the average seed oil content of materials carrying the TT genotype was (15.67 ± 0.59)%, the protein content was (43.78 ± 0.60)%, and the 100-seed weight was (9.72 ± 0.80) g. The Wilcoxon Rank Sum Test showed that the oil content (P < 0.001) and 100-seed weight (P < 0.001) of the CC genotype materials were significantly higher than those of the TT genotype materials. The protein content of the CC genotype materials was significantly lower than that of the TT genotype materials (P < 0.001).

[0072] The above experimental results show that it is feasible and accurate to use the SNP sites provided by this invention to detect the oil and protein content and 100-seed weight of soybean seeds.

[0073] The specific method for detecting soybean seed oil and protein content and 100-seed weight of soybeans using the SNP sites provided by the present invention is as follows: The whole genome resequencing method provided in Example 1 is used to identify the genomic DNA of the soybeans to be tested and obtain the sample genotype: if the obtained genotype is CC, then the soybeans to be tested are candidate materials with high oil content and high 100-seed weight; if the obtained genotype is TT, then the soybeans to be tested are candidate materials with high protein content.

[0074] For example, using the primers shown in SEQ ID NO:2-4, PCR amplification was performed on the high-oil, low-protein, large-grain variety Williams82 and the high-protein, low-oil, small-grain variety Xiaoheidou. After sequencing the amplification products, Williams82 carried the CC genotype, and Xiaoheidou carried the TT genotype. Figure 1 ).

[0075] The genotype at the Chr05:15709660 locus in soybean can serve as a new genetic resource for marker-assisted selection in high-yield and high-quality soybean breeding, and has very important guiding significance.

[0076] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of SNP molecular markers related to soybean seed oil content, protein content and 100-seed weight, or KASP primers for amplifying said SNP molecular markers, or detection reagents or kits containing said primers in identifying soybean seed oil and protein content, as well as 100-seed weight phenotype; The SNP molecular marker contains a nucleotide sequence with a polymorphism of C / T at position 201 of the sequence shown in SEQ ID NO:1; the genotype of the site with the polymorphism is CC, corresponding to a phenotype of high oil content, high 100-grain weight, and low protein content; the genotype of the site with the polymorphism is TT, corresponding to a phenotype of low oil content, low 100-grain weight, and high protein content.

2. Application of SNP molecular markers related to soybean seed oil content, protein content, and 100-seed weight, or KASP primers for amplifying the SNP molecular markers, or detection reagents or kits containing the primers, in the identification of soybean germplasm resources related to soybean seed oil and protein content, as well as 100-seed weight phenotype; The SNP molecular marker contains a nucleotide sequence with a polymorphism of C / T at position 201 of the sequence shown in SEQ ID NO:1; the genotype of the site with the polymorphism is CC, corresponding to a phenotype of high oil content, high 100-grain weight, and low protein content; the genotype of the site with the polymorphism is TT, corresponding to a phenotype of low oil content, low 100-grain weight, and high protein content.

3. Application of SNP molecular markers related to soybean seed oil content, protein content, and 100-seed weight, or KASP primers for amplifying the SNP molecular markers, or detection reagents or kits containing the primers, in targeted breeding of soybean seed oil and protein content, as well as 100-seed weight phenotype; The SNP molecular marker contains a nucleotide sequence with a polymorphism of C / T at position 201 of the sequence shown in SEQ ID NO:1; the genotype of the site with the polymorphism is CC, corresponding to a phenotype of high oil content, high 100-grain weight, and low protein content; the genotype of the site with the polymorphism is TT, corresponding to a phenotype of low oil content, low 100-grain weight, and high protein content.

4. Application of SNP molecular markers related to soybean seed oil content, protein content, and 100-seed weight, or KASP primers for amplifying the SNP molecular markers, or detection reagents or kits containing the primers in the cultivation of high-quality large-seed soybean varieties with phenotypes related to soybean seed oil and protein content and 100-seed weight; The SNP molecular marker contains a nucleotide sequence with a polymorphism of C / T at position 201 of the sequence shown in SEQ ID NO:1; the genotype of the site with the polymorphism is CC, corresponding to a phenotype of high oil content, high 100-grain weight, and low protein content; the genotype of the site with the polymorphism is TT, corresponding to a phenotype of low oil content, low 100-grain weight, and high protein content.

5. Application of SNP molecular markers related to soybean seed oil content, protein content, and 100-seed weight, or KASP primers for amplifying said SNP molecular markers, or detection reagents or kits containing said primers in soybean molecular marker-assisted breeding related to soybean seed oil and protein content, as well as 100-seed weight phenotype; The SNP molecular marker contains a nucleotide sequence with a polymorphism of C / T at position 201 of the sequence shown in SEQ ID NO:1; the genotype of the site with the polymorphism is CC, corresponding to a phenotype of high oil content, high 100-grain weight, and low protein content; the genotype of the site with the polymorphism is TT, corresponding to a phenotype of low oil content, low 100-grain weight, and high protein content.

6. The application according to any one of claims 1-5, characterized in that, The KASP primers include forward primer 1, forward primer 2, and a universal reverse primer, the sequences of which are shown in SEQ ID NO:2-4, respectively.

7. A method of identifying soybean seed oil and protein content, and hundred kernel weight phenotype, characterized by, Includes the following steps: 1) Detect the genotype of polymorphic sites of SNP molecular markers related to soybean seed oil content, protein content and 100-seed weight in soybeans to be tested; the SNP molecular markers contain a nucleotide sequence with a polymorphism of C / T at position 201 as shown in SEQ ID NO:1; 2) Determine the seed oil and protein content and 100-seed weight of the soybeans to be tested based on the genotype obtained in step 1); The specific determination method is as follows: if the genotype is CC, then the soybean to be tested is a candidate seed with high oil content, high 100-seed weight, and low protein content; if the genotype is TT, then the soybean to be tested is a candidate seed with high protein content, low oil content, and low 100-seed weight.

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