Single nucleotide mutation site SNP and KASP markers significantly associated with soybean hundred-grain weight and application thereof

Abstract

The invention discloses a single nucleotide mutation site SNP (Single Nucleotide Polymorphism) and KASP (Kinase Associated Specific Protein) marker obviously associated with soybean hundred-grain weight and application thereof. The SNP molecular marker is located at the position of 40068386bp of the 15th chromosome of the soybean, the basic group is C or T, the SNP molecular marker is remarkably related to the soybean hundred-grain weight phenotype, and the hundred-grain weight of a soybean variety with the locus genotype of TT is remarkably lower than that of a soybean variety with the genotype of CC; three KASP primers which are respectively SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 are developed according to the SNP locus, PCR amplification and genetic typing are performed on to-be-detected soybeans by utilizing the primers, and if a detection result shows that the basic group type at the marker position is T, the hundred-grain weight of the soybean variety is small; if the detection result is C, the hundred-grain weight is judged to be large. The SNP molecular marker disclosed by the invention can be used as an auxiliary selection marker for the hundred-grain weight character in the soybean breeding process, so that the selection accuracy is improved, and the breeding process related to the hundred-grain weight character of the soybean is accelerated.

Description

Technical Field

[0001] The present invention belongs to the field of molecular genetic breeding, and provides a single nucleotide mutation site SNP, a KASP marker significantly associated with the 100-seed weight of soybean and their applications, which can be used for early molecular assisted selection of the 100-seed weight trait of soybean to improve the breeding efficiency. Background Art

[0002] Soybean, as a major source of high-quality protein and edible oil, is crucial for global food security. At the same time, it is also an important industrial raw material. Although China is the origin and major consumer market of soybean, the yield per unit area is lower than the world advanced level. Increasing soybean yield can not only enhance self-sufficiency and reduce import dependence, but also contribute to ensuring national food security and promoting sustainable agricultural development.

[0003] Traditional soybean breeding usually selects individual plants based on the 100-seed weight of the offspring plants. This method is not only time-consuming and labor-intensive, but also easily affected by external conditions, thus reducing the accuracy of selection. To improve the selection efficiency of 100-seed weight, constructing specific molecular markers based on the base differences in target genes for assisted selection is considered the most effective strategy. Such molecular markers have multiple advantages in the genetic improvement of crops, such as early screening, independence from environmental factors, accuracy, rapidity and high efficiency, and have developed into an important technical means. In particular, Kompetitive Allele-Specific PCR (KASP), which is a new single nucleotide polymorphism (SNP) genotyping method based on allele-specific amplification technology and highly sensitive fluorescence detection. The core of this technology is to design two forward primers and a common reverse primer for a specific allele SNP position. Each forward primer contains a specific sequence that can be linked to a specific fluorescent label. Through the combined action of these forward primers with specific fluorescent labels and the common reverse primer, PCR amplification of the sample DNA is carried out, and the allelic variation can be presented by different fluorescent signals.

[0004] The 100-seed weight of soybeans is a complex trait, regulated by multiple quantitative trait loci (QTLs), and involves the combined effects of various factors such as the number of seeds per plant, 100-seed weight, the number of pods per plant, and the number of nodes. This trait has relatively high genetic stability but is also significantly affected by the external environment. In recent years, studying the associations between quantitative traits such as the number of seeds per plant, 100-seed weight, the number of pods per plant, and the number of nodes and yield has become a hot topic in the domestic and international scientific research fields. Research shows that the yield of soybeans is closely related to the number of pods per plant, the number of seeds per pod, and the seed weight per plant. Generally, higher yields can be achieved when the yield per plant is high, the number of seeds per plant is large, the number of pods per plant is large, and the 100-seed weight is moderate. These QTLs are mainly identified through linkage analysis or genome-wide association (GWAS) methods. As an efficient gene mapping technology, GWAS can quickly and accurately identify SNP loci significantly associated with the 100-seed weight of soybeans. Therefore, by using SNPs significantly associated with the 100-seed weight of soybeans to develop closely related KASP markers for early selection (i.e., at the low-generation stage) in the breeding process, it can effectively reduce the burden of breeding work, accelerate the breeding process, and bring significant economic benefits. Mining SNPs significantly associated with the 100-seed weight of soybeans and developing corresponding KASP molecular markers for early molecular selection in assisted breeding are of extremely important significance for improving breeding efficiency. Summary of the Invention

[0005] The object of the present invention is to identify single nucleotide mutation sites (SNPs) significantly associated with the 100-seed weight of soybeans, and develop KASP molecular markers and their primer pairs based on the SNP locus information, so as to provide a molecular-assisted selection technical means for early identification and screening of this trait.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] As a first aspect, a SNP molecular marker related to the 100-seed weight trait of soybeans is provided. The SNP S15_40068386 significantly associated with the 100-seed weight of soybeans is located at the 40068386 bp position on chromosome 15 of the soybean genome (version number: Glycine max Wm82.a2.v1), with a base substitution from C to T. The CC genotype corresponds to a soybean variety with a large 100-seed weight, and the TT genotype corresponds to a soybean variety with a small 100-seed weight.

[0008] As a second aspect, the application of the above-mentioned SNP molecular marker related to the 100-seed weight trait of soybeans in identifying the 100-seed weight trait of soybeans is provided.

[0009] The application specifically is: detecting the base type of the SNP molecular marker related to the 100-seed weight trait of the soybean sample to be tested. If the detected result shows that the base type here is T, it is determined that the 100-seed weight of this soybean variety is small; if the detected result is C, it is determined that the 100-seed weight of this soybean variety is large.

[0010] As a third aspect, there is provided an application of the SNP molecular marker related to the above-mentioned 100-seed weight trait of soybean in genetic breeding.

[0011] As a fourth aspect, there is provided a KASP specific primer for the SNP molecular marker related to the 100-seed weight trait of soybean, which consists of three primers, including two specific primers designed for the base differences at the target site, namely upstream primer F1 (SEQ ID NO.1) and upstream primer F2 (SEQ ID NO.2), and a common downstream primer R (SEQ ID NO.3). The 3'-ends of these two specific primers correspond to the variant bases of the alleles, and the 5'-ends are linked with the specific FAM and HEX fluorescent tag sequences required for the KASP reaction provided by Chengdu Hanchen Opto-Wing Biotechnology Co., Ltd.

[0012] The sequence of the KASP marker upstream primer F1 is

[0013] 5’-GAAGGTGACCAAGTTCATGCTAAAATGCAAGCAGAACCAAACCAC-3’(SEQ ID NO.1);

[0014] The sequence of the KASP marker upstream primer F2 is

[0015] 5’-GAAGGTCGGAGTCAACGGATTAAAATGCAAGCAGAACCAAACCAT-3’(SEQ ID NO.2);

[0016] The sequence of the KASP marker downstream primer R is

[0017] 5’-AACAGTGACTCAAACCAAACCTTG-3’(SEQ ID NO.3).

[0018] In the process of synthesizing the above KASP molecular marker primers, carboxyfluorescein FAM was added to the 5'-end of the forward primer F1 as a fluorescent signal marker (the first 21 positions of the primer sequence); while hexachlorofluorescein phosphoramidite HEX was added to the 5'-end of the forward primer F2 as a fluorescent signal marker (also shown as the first 21 positions of the primer sequence).

[0019] As a fifth aspect, there is provided the use of the above KASP specific primers in identifying the 100-seed weight trait of soybeans. For the SNP locus significantly associated with the 100-seed weight of soybeans, the developed KASP molecular marker is used for the method of identification or assisted screening. Specifically, it is to detect the deoxyribonucleotide genotype at the position of 40068386 bp on chromosome 15 of soybeans and determine whether it is TT or CC. Among them, the TT genotype corresponds to a smaller 100-seed weight, while the CC genotype corresponds to a larger 100-seed weight. This molecular marker-assisted technology contributes to the genetic improvement of the 100-seed weight trait of soybeans.

[0020] In the above method, the KASP primer set consists of upstream primer F1, upstream primer F2, and downstream primer R. A 384-well microplate PCR reaction system is prepared using a Matrix Arrayer 3250 reaction plate preparation instrument, and the PCR amplification process is performed using a MatrixCycler 2010 high-throughput water bath thermal cycler. After the amplification is completed, a Matrix Scanner2100 high-speed fluorescence scanner is used for fluorescence signal scanning, and then the genotype analysis is completed with the accompanying Matrix Master software. If the initial genotyping result is not satisfactory, an additional amplification step will be added, and the genotyping effect will be checked every 5 cycles until the complete genotyping standard is reached.

[0021] The specific operation steps are as follows:

[0022] (1) Extraction of genomic DNA from soybean plants;

[0023] (2) Using the above PCR specific amplification primers to perform PCR amplification on the genomic DNA of biological samples to obtain amplified product fragments, and performing KASP genotyping detection on the PCR amplified product fragments. If the detection result shows that the base type here is T, it is determined that the 100-seed weight of this soybean variety is smaller; if the detection result is C, it is determined that the 100-seed weight of this soybean variety is larger:

[0024] Add the above molecular marker primers to the same PCR reaction system, and set up 3 blank controls with ultrapure water replacing the sample template DNA. Subsequently, the DNA of soybean germplasm resources is amplified on a Gene Matrix high-throughput genotyping system;

[0025] 2 μl reaction system: Soybean sample DNA template, 5 ng / μl, 1 μl; 2x Master Mix for ASPCR V1 1 μl; KASP Assay Mix, F1:F2:R = 1:1:3, 0.02 μl. The reaction conditions include pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 sec, annealing at 61 - 55°C for 40 sec, with a decrease of 0.6°C for each cycle, for 10 cycles; denaturation at 95°C for 20 sec, annealing at 55°C for 40 sec, for 30 cycles.

[0026] After the reaction, fluorescence scanning is performed on a Matrix Scanner 2100 high-speed fluorescence scanner, and genotyping analysis is carried out using the supporting Matrix Master software. This molecular marker primer can clearly distinguish two genotypes: the red dots near the X-axis represent individuals carrying the C allele variation, with the genotype CC; while the blue dots near the Y-axis indicate individuals carrying the T allele variation, with the genotype TT.

[0027] (3) Select the desired soybean single plants or lines with 100-seed weight according to the genotype in different segregation generations.

[0028] The beneficial effects of the present invention are as follows:

[0029] (1) The SNP significantly associated with the 100-seed weight of soybeans in the present invention, S15_40068386, is derived from the whole-genome resequencing information of 270 cultivated soybean varieties and related GWAS loci. The SNP locus S15_40068386 significantly associated with the 100-seed weight of soybeans can be detected, which is located at the position of 40068386 bp on chromosome 15 of the soybean genome (version number Glycine max Wm82.a2.v1). Select CC or TT genotype soybean low-generation breeding materials according to the need, providing technical support for molecular marker-assisted breeding of the 100-seed weight trait of soybeans.

[0030] (2) The present invention identified an SNP locus located on chromosome 15 of soybeans that affects the 100-seed weight of soybeans, and developed a KASP molecular marker based on this. This marker can directly and specifically identify and detect the C or T base at the SNP locus. This KASP molecular marker has significant application potential and can be used in the early selection and molecular-assisted breeding of the 100-seed weight trait of soybeans.

[0031] (3) Using KASP molecular marker primers, 185 soybean samples were amplified and genotyped on the Gene Matrix high-throughput genotyping system. The results showed that the molecular marker primers could clearly distinguish two genotypes: the red dots near the X-axis represented individuals carrying the C allele variation, with a genotype of CC, a total of 157, and an average 100-seed weight of 20.05 grams; the blue dots near the Y-axis represented individuals carrying the T allele variation, with a genotype of TT, a total of 26, and an average 100-seed weight of 18.56 grams. The dots near the origin of the XY-axis represented the blank control; the samples marked with red crosses were those that failed to be successfully genotyped, a total of 2. Statistical analysis showed that there were significant differences in the 100-seed weight of the two genotypes of soybean samples, which proved the practical application value of the marker described in the present invention. Description of the Drawings

[0032] Figure 1 It is the Manhattan and QQ-plot of the association analysis results between the genotypes of 207 SNP loci and the 100-seed weight phenotype of soybeans; among them, (a) is the Manhattan plot of the association analysis results, and (b) is the QQ-plot of the association analysis results;

[0033] Figure 2 It is the genotyping result map of different soybean varieties by KASP markers; among them, (a) is the genotyping result map of soybean materials SPBX001 - SPBX093 (excluding SPBX052), and (b) is the genotyping result map of soybean materials SPBX097 - SPBX189; the black squares near the origin represent the blank control without template DNA; the blue dots near the Y-axis and the red dots near the X-axis represent soybean varieties carrying the T allele variation site and soybean varieties carrying the C allele variation site respectively, and the red crosses represent the materials that failed to be successfully genotyped. Detailed Embodiments

[0034] Here, the exemplary embodiments will be described in detail, and the examples are shown in the drawings. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application. On the contrary, they are only examples of methods consistent with some aspects of the present application as detailed in the appended claims.

[0035] The terms used in the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0036] The singular forms "a", "the", and "said" used in the present application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term "and / or" used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

[0037] It should be understood that although the terms first, second, third, etc. may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from each other. For example, without departing from the scope of this application, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "when" or "while" or "in response to a determination".

[0038] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. Unless otherwise specified, the methods used are all conventional methods.

[0039] Example 1: Obtaining nucleotide mutation sites (SNPs) significantly associated with 100-seed weight of soybean

[0040] In the present invention, the SNP significantly associated with 100-seed weight of soybean, S15_40068386, is derived from the whole-genome resequencing information of 270 cultivated soybean varieties and related GWAS loci. The method for obtaining it includes the following steps:

[0041] (1) Sampling and obtaining of samples: 270 soybean core germplasm resources were sown in the experimental fields of the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences in 2018, and conventional field management was carried out. At the same time, the 100-seed weight data of these germplasm resources were recorded. Determination of 100-seed weight: Randomly take 100 well-developed seeds, accurately weigh them to 0.01 g, and convert them to the weight at a moisture content of 13%.

[0042] (2) SNP detection: Young leaves were collected during the V4 growth stage of soybean, and high-quality soybean genomic DNA was extracted using the CTAB method for whole-genome resequencing. The sequencing generated a total of 8100 Gb of high-quality clean data, with an average of 30 Gb per sample and a sequencing depth of approximately 30 times. The sequencing data were aligned to the soybean reference genome (version Glycine max Wm82.a2.v1) using the BWA software, and duplicate reads were removed using the PICARD software. Then, high-quality SNP data were obtained using the GATK software. Finally, the ANNORVAR software was used to perform functional annotation on the SNP detection results.

[0043] (3) Genome-wide association analysis: Genome-wide association analysis was performed on the obtained SNP marker loci and the measured 100-seed weight phenotypic information. The analysis software was TASSEL, and the mixed linear model was used for analysis.

[0044] (4)Obtaining of 20K Loci: To obtain 20K marker loci, among 270 cultivated soybean samples, the genotype missing rate was required to be lower than 20%, the proportion of heterozygous genotypes not exceeding 30%, the maximum allele frequency lower than 95%, and the minimum allele frequency higher than 5%. GWAS marker loci were preferentially selected, followed by marker loci in the gene coding region, and one marker was selected every 25 kb; if there were no such two types of markers in a continuous 75 kb region, markers in the non-coding region were selected. Finally, 20,648 SNP marker loci were screened out, among which 17,588 markers were located on functional genes, covering 31% of soybean coding genes. The SNP molecular markers were evenly distributed, with an average interval of 46 kb.

[0045] (5)Obtaining of 207 SNP Loci Evenly Distributed on Soybean Chromosomes: 61 SNP loci were selected from published literatures or patents, and 146 SNP loci were selected from the 20K loci. A total of 207 SNP loci could be evenly distributed on soybean chromosomes, and the physical position distance between loci was about 5 Mbp.

[0046] (6)Gene-Phenotype Association Analysis: The genotypes of 207 SNP loci were associated with the 100-seed weight phenotype. The analysis software was TASSEL, and the mixed linear model was used for analysis. The SNP locus S15_40068386 significantly associated with soybean 100-seed weight could be detected, which was located at the position of 40068386 bp on chromosome 15 of the soybean genome (version number Glycine max Wm82.a2.v1).

[0047] Example 2: Development of KASP Marker Specific Primers

[0048] Using the Primer-BLAST function of NCBI (https: / / www.ncbi.nlm.nih.gov / ), three primers were designed based on the nucleotide sequences before and after the S15_40068386 locus, the upstream primer F1 (SEQ ID NO.1), the upstream primer F2 (SEQ ID NO.2), and the downstream primer R (SEQ ID NO.3). Among them, F1 and F2 respectively contained FAM and HEX fluorescence linker sequences (shown in the first 21 positions of the primer sequences), and the sequences were as follows:

[0049] The sequence of KASP marker upstream primer F1 is

[0050] 5’-GAAGGTGACCAAGTTCATGCTAAAATGCAAGCAGAACCAAACCAC-3’(SEQ ID NO.1);

[0051] The sequence of KASP marker upstream primer F2 is

[0052] 5’-GAAGGTCGGAGTCAACGGATTAAAATGCAAGCAGAACCAAACCAT-3’(SEQ ID NO.2);

[0053] The downstream primer R of the KASP marker: 5’-AACAGTGACTCAAACCAAACCTTG-3’(SEQ ID NO.3).

[0054] Example 3: Genotyping of SNP Loci in 185 Different Varieties of Soybeans and Its Application

[0055] Genomic DNA was extracted from 185 different varieties of soybean samples respectively. Using the genomic DNA as a template, the DNA of soybean samples was amplified on the Gene Matrix high-throughput genotyping system with the specific primers of KASP markers. The amplification system was a 2 μl reaction system: 1 μl of soybean sample DNA template at 5 ng / μl; 1 μl of 2x Master Mix for ASPCR V1; 0.02 μl of KASP Assay Mix with F1:F2:R = 1:1:3. The reaction conditions included pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 20 sec, annealing at 61 - 55 °C for 40 sec, with a decrease of 0.6 °C for each cycle, for 10 cycles; denaturation at 95 °C for 20 sec, annealing at 55 °C for 40 sec, for 30 cycles.

[0056] After the reaction, fluorescence scanning was performed on the Matrix Scanner 2100 high-speed fluorescence scanner, and genotyping analysis was carried out through the supporting Matrix Master software. The results are as Figure 2 . Using the KASP molecular marker primers, 185 soybean samples were amplified and genotyped on the GeneMatrix high-throughput genotyping system. The results showed that this molecular marker primer could clearly distinguish two genotypes: the red dots near the X-axis represented individuals carrying the C allele variation with the genotype CC, a total of 157, and the average 100-seed weight was 20.05 grams; the blue dots close to the Y-axis represented individuals carrying the T allele variation with the genotype TT, a total of 26, and the average 100-seed weight was 18.56 grams (see Tables 1 and 2). The points close to the origin of the XY-axis represented the blank control (see Figure 2 ); the samples marked with red crosses were those that failed to be genotyped successfully, a total of 2. Statistical analysis showed that there were significant differences in the 100-seed weight of the two genotypes of soybean samples (see Table 2), which proved the practical application value of the markers described in the present invention.

[0057] Table 1. 100-seed weight of 185 different soybean varieties

[0058]

[0059]

[0060]

[0061] Table 2. Comparison of mean values among groups of 185 soybean varieties

[0062]

[0063] The embodiments of the present disclosure have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to technologies in the market, or to enable other ordinary skill in the art to understand the disclosed embodiments.

[0064] The above are only optional embodiments of the present disclosure and are not used to limit the present disclosure. For those skilled in the art, the present disclosure may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included within the protection scope of the present disclosure.

Claims

1. A SNP molecular marker related to soybean 100-grain weight trait, characterized in that: The SNP molecular marker is located at the 40068386bp position of chromosome 15 of the soybean genome, and the polymorphism is C or T. The phenotype corresponding to the CC genotype is a soybean variety with a large 100-grain weight, and the phenotype corresponding to the TT genotype is a soybean variety with a small 100-grain weight.

2. A KASP-specific primer for the SNP molecular marker associated with soybean 100-grain weight trait according to claim 1, characterized in that: The primers include: Upstream primer F1, nucleotide sequence is shown in SEQ ID NO.1: 5'-GAAGGTGACCAAGTTCATGCTAAAATGCAAGCAGAACCAAACCAC-3'; Upstream primer F2, nucleotide sequence is shown in SEQ ID NO.2: 5'-GAAGGTCGGAGTCAACGGATTAAAATGCAAGCAGAACCAAACCAT-3'; The nucleotide sequence of the downstream primer R is shown in SEQ ID NO.3: 5'-AACAGTGACTCAAACCAAACCTTG-3'.

3. Use of the SNP molecular marker associated with soybean 100-grain weight trait according to claim 1 in identifying soybean 100-grain weight trait.

4. The use according to claim 3, characterized in that: The application is specifically: The base type of the SNP molecular marker related to the soybean 100-grain weight trait in the soybean sample to be tested is detected. If the test result shows that the base type here is T, it is determined that the soybean variety has a smaller 100-grain weight; if the test result is C, it is determined that the soybean variety has a larger 100-grain weight.

5. Use of the SNP molecular marker related to soybean 100-grain weight trait according to claim 1 in genetic breeding.

6. Use of the KASP specific primers according to claim 2 in identifying the soybean 100-grain weight trait.

7. The use according to claim 6, characterized in that: The following steps are involved: (1) extracting genomic DNA from the soybean sample to be tested; (2) using the genomic DNA of the soybean sample to be tested as a template, and using KASP-specific primers of the SNP molecular marker related to the soybean 100-grain weight trait to perform PCR amplification reaction to obtain an amplified product fragment; (3) Perform KASP genotyping test on the PCR amplification product fragment. If the test result shows that the base type here is T, it is judged that the 100-grain weight of the soybean variety is small; if the test result is C, it is judged that the 100-grain weight of the soybean variety is large.

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