SNP (Single Nucleotide Polymorphism) marker related to soybean leucine content and application thereof

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker related to the content of soybean leucine and application of the SNP marker. The SNP marker is located at the S16-18737366 site of the No.16 chromosome of soybean, the nucleotide type is T or TC, and the TC type is significantly related to the content of homoleucine. The invention also provides a primer group for detecting the SNP site and a sequence of the primer group, a detection method for PCR amplification, sequencing or enzyme digestion analysis is established based on the primer group, and a matched kit is constructed. The SNP marker can be used for rapid detection of soybean leucine content and molecular marker-assisted breeding of homoleucine varieties, has the advantages of rapid detection, high accuracy, strong applicability and the like, and provides reliable technical support for improvement of soybean nutritional quality.

Description

Technical Field

[0001] This invention relates to the field of soybean molecular breeding technology, specifically to an SNP marker related to soybean leucine content and its application. Background Technology

[0002] Soybeans, as a globally important food and oilseed crop, are rich in high-quality protein and are one of the important sources of plant-based protein for humans. Leucine, as one of the essential amino acids for the human body, must be obtained from external sources and is an important indicator for measuring the nutritional value of soybean protein. In recent years, with the increasing attention to nutrition and health, increasing the leucine content in soybeans has become an important goal for improving soybean quality and promoting the breeding of functional varieties.

[0003] In conventional breeding processes, improving soybean leucine content typically relies on phenotypic determination and progeny selection. However, because leucine content is easily affected by various external conditions such as environmental factors and growth stage, traditional phenotypic identification suffers from significant problems such as long cycles, high testing costs, poor reproducibility, and low efficiency, making it difficult to meet the precision and high-throughput requirements of modern breeding. Therefore, there is an urgent need to develop molecular marker tools for early prediction and rapid identification of leucine content to improve the efficiency of superior germplasm screening and variety breeding.

[0004] In recent years, with the development of high-throughput genome sequencing technology, genome-wide association analysis (GWAS) based on SNPs (single nucleotide polymorphisms) has been widely used in target trait mapping studies of crops such as soybean. GWAS methods do not rely on candidate genes and can discover functional loci significantly associated with complex quantitative traits across the entire genome. They have high resolution, accurate mapping, and the results can be directly converted into molecular markers to serve molecular breeding practices.

[0005] Although previous studies have used GWAS analysis to identify some QTLs or SNPs related to soybean amino acid content, there is currently a lack of stable, functionally defined molecular markers for soybean leucine content that can be widely applied to different germplasm backgrounds. Especially in marker-assisted breeding of leucine content traits, the lack of easy-to-use and highly valuable functional SNP markers has become a technical bottleneck restricting the development of this field.

[0006] Therefore, it is urgent to combine soybean germplasm resources and leucine phenotypic data to develop functional SNP markers related to stable leucine content, and to establish corresponding rapid detection methods and application schemes. This will provide key technical support for molecular marker-assisted breeding of high-leucine soybean varieties and promote the refined and intelligent development of soybean nutritional quality breeding. Summary of the Invention

[0007] To overcome the problems of lack of molecular markers stably associated with soybean leucine content, low detection efficiency, and long breeding cycles in existing technologies, this invention provides an SNP marker related to soybean leucine content, along with its matching primer set, detection kit, and detection method. The following description, in conjunction with multiple embodiments and in accordance with the technical solutions defined in the claims, illustrates the structural composition, functional features, and optional implementation forms of this invention.

[0008] In one embodiment of the present invention, an SNP marker related to the leucine content of soybean is provided. The SNP marker is located at S16-18737366 on soybean chromosome 16, and the nucleotide at this site is T or TC.

[0009] Furthermore, when the nucleotide of the SNP marker site S16-18737366 is TC, the leucine content of soybeans is high; when the nucleotide of the SNP marker site is C, the leucine content of soybeans is low.

[0010] In one embodiment of the present invention, the SNP tag can be applied to any of the following purposes: (1) Molecular detection of leucine content in soybeans; (2) Molecular marker-assisted breeding of soybean varieties with high leucine content.

[0011] In one embodiment of the present invention, a primer set for detecting the leucine content of soybeans is provided, the primer set being used to amplify a DNA fragment containing the SNP marker site S16-18737366.

[0012] Preferably, the upstream primer sequence of the primer set is SEQ ID NO.1: 5'-TGGGCCATTGCGGAACTAAT-3', and the downstream primer sequence is SEQ ID NO.2: 5'-TAGGGTGCACTGAAAGAGGC-3'.

[0013] In one embodiment of the present invention, a kit for detecting the leucine content in soybeans is provided, the kit comprising the primer set.

[0014] Optionally, the kit may also include one or more of the following components required for the PCR reaction: DNA polymerase, dNTPs, and PCR buffer.

[0015] In one embodiment of the present invention, a method for detecting soybean leucine content based on the primer set is provided, comprising the following steps: (1) Extract genomic DNA from the soybean sample to be tested; (2) Using the DNA as a template, perform PCR amplification to obtain the amplification product; (3) Sequencing or enzyme digestion analysis of the amplified product to determine the nucleotide type of the SNP marker site S16-18737366; (4) Determine the leucine content of the soybean sample: if the nucleotide type is TC, the leucine content is high; if it is C, the leucine content is low.

[0016] Preferably, the determination of nucleotide type in step (3) is performed using Sanger sequencing.

[0017] Optionally, the determination of nucleotide type in step (3) can be performed using restriction endonuclease digestion analysis.

[0018] Based on the above technical solution, the present invention provides an SNP marker related to soybean leucine content and its detection application. By using the nucleotide variation of the SNP site S16-18737366 located on soybean chromosome 16, a stable association between the marker and soybean leucine content is established. This enables rapid and accurate detection of soybean leucine content, solving the problems of long phenotypic evaluation cycle and low efficiency in traditional breeding. It provides an efficient, low-cost, and non-destructive technical tool for molecular marker-assisted breeding of high-leucine soybean varieties.

[0019] The SNP markers provided by this invention have advantages such as precise localization, good genetic stability, and strong detection repeatability. Through GWAS analysis of soybean germplasm materials with different leucine content levels, functional loci significantly associated with the target trait were screened. Experiments confirmed that these loci can stably distinguish between high and low leucine content in different varieties, achieving precise molecular-level identification of soybean nutritional quality traits. This technology breaks through the limitations of traditional phenotypic selection methods that rely on environment and developmental stage, significantly shortening the breeding cycle of new soybean varieties and improving breeding efficiency.

[0020] Furthermore, this invention also provides a matching primer set and kit solution, which can be directly used for rapid amplification and detection of target SNP sites. It is applicable to various modes such as routine PCR detection, sequencing analysis, or enzyme digestion detection under laboratory conditions, and has good versatility and operability. The entire detection process is simple and fast, and can be completed in about 2 hours, significantly reducing the time and labor costs for breeding units and research institutions in the process of germplasm resource evaluation and material screening.

[0021] This invention is not only applicable to scientific research and breeding, but also has promising prospects for industrialization. In actual breeding processes, it can be applied to the early molecular screening stage of soybean varieties to help select materials with high leucine content potential as preferred parents for subsequent breeding and crossbreeding. At the same time, it is also applicable to field production scenarios such as variety authenticity identification and quality control, which is of great significance for promoting the development of the nutritional soybean industry.

[0022] In summary, the SNP markers, primer sets, kits, and detection methods of this invention provide a scientific and efficient technical means for molecular breeding of soybean nutritional quality traits, and have significant technological advancements and application value. Attached Figure Description

[0023] Figure 1 This is a Manhattan plot of genome-wide association analysis (GWAS) for soybean leucine content. Each point in the plot represents the association between a SNP locus and leucine content, with the ordinate being -LOG. 10 (P-value), the horizontal axis represents the physical location on the soybean genome chromosome. Among them, the SNP site S16-18737366 located on chromosome 16 reached the significance threshold and is a functional marker site significantly associated with leucine content. Detailed Implementation

[0024] To better understand the technical solution and beneficial effects of the present invention, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the embodiments described are for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention. Various modifications, equivalent substitutions, or improvements made by those skilled in the art based on the concept of the present invention without departing from its essential spirit should still be covered within the scope of protection of the present invention.

[0025] Example 1: Discovery and Validation of Leucine-Related SNP Sites in Soybeans This embodiment provides a genome-wide association analysis (GWAS)-based method for identifying functional SNP markers that are significantly associated with soybean leucine content and conducting experimental verification, thus providing a foundation for marker-assisted breeding.

[0026] 1. Material Preparation Fifty representative soybean germplasm materials were selected as the research subjects, including 25 materials with high leucine content and 25 materials with low leucine content. Each material was grown under the same environmental conditions, and its leucine content was measured after maturity to obtain accurate phenotypic data.

[0027] 2. SNP Data Acquisition and Preprocessing High-throughput resequencing technology was used to sequence the genomes of the aforementioned soybean materials, obtaining whole-genome SNP data. Using standard quality control procedures (such as filtering low-sequencing-quality sites and removing sites with high deletion rates), a high-quality SNP dataset suitable for GWAS analysis was obtained.

[0028] 3. GWAS Analysis Association analysis was performed between leucine content phenotypic data and SNP genotypic data. A Q+K mixed linear model was established using GCTA software to control for biases from population structure and kinship. A significance threshold of -LOG was set. 10 (P-value) ≥5, SNP sites that are significantly associated with leucine content are screened.

[0029] like Figure 1 As shown in the GWAS Manhattan plot, each point represents the degree of association between a SNP site and leucine content, with the vertical axis representing -LOG. 10 (P-value), the horizontal axis represents the physical location of the SNP on the soybean chromosome. The results showed that the SNP site S16-18737366 on soybean chromosome 16 reached the significance threshold, with a p-value of [value missing]. 10 The (P-value) is 6.731, indicating a functional site associated with stable leucine levels.

[0030] 4. Information on significant SNP sites The key functional SNP locus, S16-18737366, is located on soybean chromosome 16, with an allele expression of T / TC. Statistical analysis revealed a stable correlation between the genotype of this locus and leucine content: soybean materials with the TC genotype had significantly higher leucine content than those with the C genotype.

[0031] 5. SNP marker verification experiment Design a specific primer set based on the flanking sequence of the S16-18737366 site: Upstream primer (SEQ ID NO.1): 5'-TGGGCCATTGCGGAACTAAT-3'; Downstream primer (SEQ ID NO.2): 5'-TAGGGTGCACTGAAAGAGGC-3'.

[0032] Genomic DNA was extracted from the leaves of each soybean material using the CTAB method. The PCR amplification system consisted of 20 μL, including 2 μL of 10×PCR buffer, 1.6 μL of dNTPs (2.5 mmol / L), 0.8 μL of each primer, 0.2 μL of Taq enzyme, 2 μL of template DNA, and ddH2O to a final volume of 20 μL.

[0033] The PCR amplification procedure is as follows: Pre-denaturation at 94℃ for 5 min; Denaturation at 94℃ for 30 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 30 seconds, cycled 35 times; Final extension at 72℃ for 10 min, then store at 4℃.

[0034] The amplified products were subjected to Sanger sequencing to read the nucleotide types at the S16-18737366 locus. The results showed that the leucine content of samples with the TC genotype was generally in the range of 8.2-9.0, while the leucine content of samples with the C genotype was 7.4-7.8, verifying the effectiveness of this locus as a functional molecular marker for leucine content.

[0035] The amplified products were subjected to Sanger sequencing to read the nucleotide types at position S16-18737366. The obtained genotype information was statistically correlated with the leucine content of each material, and the results are shown in Table 1.

[0036] Table 1. Leucine content and locus information in soybean varieties Variety name Site type Leucine content Variety name Site type Leucine content PI 423954 T 7.4 PI 88502 TC 8.2 PI 438020 T 7.4 PI 153286 TC 8.2 PI 89055 T 7.4 PI 458018 TC 8.2 PI 437830 T 7.5 PI 561405 TC 8.2 PI 567686 T 7.5 PI 592951 TC 8.2 PI567522 B T 7.5 PI 547531 TC 8.21 PI 417298 T 7.5 PI 548417 TC 8.22 PI417324 A T 7.5 PI 59845 TC 8.3 PI 153293 T 7.5 PI 548430 TC 8.3 PI 548488 T 7.6 PI 547873 TC 8.3 PI 591435 T 7.6 PI 548593 TC 8.5 PI 438145 T 7.6 PI 547409 TC 8.5 PI 592975 T 7.6 PI 417269 TC 8.5 PI 553052 T 7.6 PI 547871 TC 8.5 PI 89773 T 7.6 PI 547522 TC 8.5 PI 437625 T 7.6 PI 547530 TC 8.5 PI 80837 T 7.7 PI 548180 TC 8.5 PI 467343 T 7.7 PI 88458 TC 8.6 PI 508266 T 7.7 PI 458060 B TC 8.6 PI407877 C T 7.7 PI 594022 TC 8.63 PI 548416 T 7.7 PI 548160 TC 8.7 PI 547777 T 7.7 PI 232991 TC 8.8 PI 86449 T 7.76 PI 548167 TC 8.8 PI 548604 T 7.79 PI 594448 B TC 8.82 PI 548318 T 7.8 PI 408196 B TC 9 The data results show that the leucine content of samples with genotype TC is generally higher than that of samples with genotype T, which verifies the reliability and stability of SNP site S16-18737366 as a functional molecular marker.

[0037] Example 2: A method for detecting soybean leucine content based on SNP markers and its application This embodiment provides a method for detecting soybean leucine content based on the functional SNP marker S16-18737366 described in Example 1, and illustrates its application process in the breeding of high-leucine soybean varieties.

[0038] 1. Construction of primer sets For the SNP marker site S16-18737366 verified in Example 1, a specific primer set was designed based on its upstream and downstream flanking sequences to amplify the target DNA fragment containing this SNP site. The primer set includes: Upstream primer: SEQ ID NO.1, sequence 5'-TGGGCCATTGCGGAACTAAT-3'; Downstream primer: SEQ ID NO.2, sequence 5'-TAGGGTGCACTGAAAGAGGC-3'.

[0039] The primer set is characterized by high amplification efficiency and high specificity, and is suitable for conventional PCR detection systems.

[0040] 2. Detection Method Based on the above primer set, a molecular detection method for soybean leucine content was established, including the following steps: (1) Genomic DNA extraction Young leaves or seed embryo tissues of soybean materials to be tested were selected, and genomic DNA was extracted using the CTAB method. The concentration and purity of the DNA were then detected and used as templates for PCR amplification.

[0041] (2) PCR amplification Using the extracted genomic DNA as a template, PCR amplification was performed using the primer set described above. The PCR reaction system and conditions were consistent with those in Example 1, yielding an amplification product containing SNP site S16-18737366.

[0042] (3) SNP site detection Nucleotide type analysis of PCR amplification products can be performed using one of the following methods, depending on the actual needs: The Sanger sequencing method was used to directly determine the sequence of the amplified fragment and read the nucleotide type of the SNP site; Alternatively, restriction endonuclease digestion analysis can be used to determine the genotype of the SNP site by the difference in the length of the digested fragments.

[0043] (4) Judgment of leucine content Based on the test results, the samples were judged as follows: when the nucleotide type of SNP site S16-18737366 was TC, the soybean material was judged to be a high-leucine potential material; when the nucleotide type was C, it was judged to be a low-leucine potential material.

[0044] The basis for this judgment is... Figure 1 The GWAS analysis results shown indicate that the S16-18737366 site is significantly correlated with soybean leucine content, and its -LOG 10 The p-value was 6.731, indicating strong significance and stable association.

[0045] 3. Application of Detection Methods Applying the above detection methods to soybean breeding practices, molecular testing is performed on a large number of breeding materials in the early generation stage of variety selection (such as F2 generation and later generations) to screen out superior single plants or lines carrying high leucine-related alleles, which can then be used as preferred materials for subsequent field identification and quality improvement.

[0046] Compared with traditional methods that rely on mature grains for amino acid determination, the detection method described in this embodiment does not require waiting for crop maturity, is not significantly affected by environmental factors, and can complete screening during the seedling stage, significantly shortening the breeding cycle, improving breeding efficiency, and reducing detection costs.

[0047] 4. Technical Effects The method described in Example 2 enables rapid, accurate, and stable detection of leucine content in soybeans, providing a reliable technical means for molecular marker-assisted breeding of high-leucine soybean varieties and possessing good application value.

[0048] Example 3: Soybean leucine detection kit based on SNP site S16-18737366 and its usage method This embodiment provides a molecular detection kit for detecting leucine content in soybeans. The kit is constructed based on the functional SNP site S16-18737366 verified in Examples 1 and 2, and is suitable for rapid detection needs in soybean variety breeding, germplasm evaluation and other scenarios.

[0049] 1. Kit Components The kit comprises the following components: (1) Specific primer set Upstream primer (SEQ ID NO.1): 5'-TGGGCCATTGCGGAACTAAT-3'; Downstream primer (SEQ ID NO.2): 5'-TAGGGTGCACTGAAAGAGGC-3'; The primer set was used to amplify a soybean DNA fragment containing the SNP site S16-18737366.

[0050] (2) PCR reaction system reagents dNTPs mixture (2.5 mmol / L); 10×PCR buffer (containing Mg²⁺) + ); Taq DNA polymerase (5 U / μL); Nuclease-free water (ddH2O) is used for fluid replacement.

[0051] (3) Positive control DNA template Provide one soybean DNA sample (genotype TC) known to have high leucine content for comparison and verification.

[0052] (4) Instruction manual and operation guide This includes operational procedures such as sample processing, reaction formulation, PCR program, product analysis methods, and result interpretation criteria.

[0053] Optionally, the kit may also include a restriction endonuclease for detection (used for enzyme digestion to determine the genotype of the site) and its buffer solution to accommodate different experimental conditions.

[0054] 2. How to use The recommended steps for using this kit are as follows: (1) Sample processing Genomic DNA was extracted from soybean materials (such as leaves or seed embryos) using the CTAB method and diluted to an appropriate concentration (50 ng / μL is recommended).

[0055] (2) PCR reaction setup Add the following components to a 20 μL PCR system: 10× buffer: 2 μL dNTP mixture: 1.6 μL Upstream primer: 0.8 μL Downstream primer: 0.8 μL Taq enzyme: 0.2 μL Template DNA: 2 μL ddH2O: Add to a final volume of 20 μL (3) PCR procedure Pre-denaturation at 94℃ for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, cycled 35 times; Final extension at 72℃ for 10 min, then store at 4℃.

[0056] (4) Analysis of amplification products Depending on the selected detection method, the following can be performed: Sanger sequencing: read the nucleotide type at site S16-18737366; or Enzyme digestion analysis: Fragment length was determined using specific restriction enzymes.

[0057] (5) Result interpretation If the nucleotide at this site is TC, it is determined that the leucine content is high; If the result is C, it indicates a low leucine content.

[0058] The interpretation is based on Example 1. Figure 1 The GWAS analysis results are shown.

[0059] 3. Technological Advantages The kit has the following advantages: The components are complete and highly standardized. The operation procedure is simple and suitable for routine conditions in molecular laboratories. The test is fast and accurate, and sample screening can be completed within 2 to 3 hours; Supports high-throughput detection, suitable for large-sample breeding processes in breeding units; It can be flexibly applied to different detection platforms, such as PCR amplification, enzyme digestion, electrophoresis, Sanger sequencing, etc.

[0060] In summary, this invention provides a SNP marker associated with soybean leucine content and its application. Combining GWAS association analysis and empirical verification methods, the functional SNP site S16-18737366 was screened, significantly associated with soybean leucine content. Based on this site, a matching primer set, detection kit, and molecular detection method were constructed. This technical solution has advantages such as rapid detection, high accuracy, and strong applicability, and is suitable for molecular marker-assisted breeding of soybean varieties with high leucine content, providing reliable technical support and practical tools for improving the nutritional quality of soybeans.

[0061] It should be understood that, for those skilled in the art, various modifications, substitutions or variations can be made to the specific embodiments of the present invention without departing from the principles and essence of the present invention. The above embodiments should not be construed as limiting the scope of protection of the present invention. All equivalent substitutions or variations made based on the technical solution concept of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A SNP marker related to soybean leucine content, characterized in that, The SNP marker is located at S16-18737366 on soybean chromosome 16, and the nucleotide at this site is either T or TC.

2. The SNP marker according to claim 1, characterized in that, When the nucleotide at the SNP marker site S16-18737366 is TC, the leucine content of soybeans is high; when the nucleotide at the SNP marker site S16-18737366 is C, the leucine content of soybeans is low.

3. The application of the SNP marker as described in claim 1 or 2 in any of the following uses: (1) Molecular detection of leucine content in soybeans; (2) Molecular marker-assisted breeding of soybean varieties with high leucine content.

4. A primer set for detecting the leucine content in soybeans, characterized in that, The primer set is used to amplify the DNA fragment containing the SNP marker site S16-18737366 as described in claim 1.

5. The primer set according to claim 4, characterized in that: The upstream primer sequence of the primer set is SEQ ID NO.1: 5'-TGGGCCATTGCGGAACTAAT-3'; The downstream primer sequence of the primer set is SEQ ID NO.2: 5'-TAGGGTGCACTGAAAGAGGC-3'.

6. A reagent kit for detecting the leucine content in soybeans, characterized in that, The kit contains the primer set as described in claim 4 or 5.

7. The reagent kit according to claim 6, characterized in that, The kit also includes one or more of the following components required for PCR reactions: DNA polymerase, dNTPs, and PCR buffer.

8. A method for detecting soybean leucine content based on the primer set described in claim 4 or 5, characterized in that, Includes the following steps: (1) Extract genomic DNA from the soybean sample to be tested; (2) Using the DNA as a template, perform PCR amplification to obtain the amplification product; (3) Sequencing or enzyme digestion analysis of the amplified product to determine the nucleotide type of the SNP marker site S16-18737366; (4) Determine the leucine content of the soybean sample: if the nucleotide type is TC, the leucine content is high; if it is C, the leucine content is low.

9. The method according to claim 8, characterized in that, The determination of the nucleotide type in step (3) was performed using Sanger sequencing.

10. The method according to claim 8, characterized in that, The determination of the nucleotide type in step (3) is performed using restriction endonuclease digestion analysis.

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