CN117025829B - KASP molecular marker related to salt tolerance of soybean in germination period and application thereof - Google Patents
KASP molecular marker related to salt tolerance of soybean in germination period and application thereof Download PDFInfo
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Abstract
The invention discloses a KASP molecular marker related to salt tolerance of soybean in germination period and application thereof, and belongs to the technical field of molecular markers. The molecular marker comprises at least one of a sequence shown as SEQ ID NO.1 and a sequence shown as SEQ ID NO. 2. And (3) designing and detecting a KASP primer group according to the molecular marker, performing fluorescent quantitative PCR amplification on DNA of the soybean sample to be detected, and judging whether the soybean shows salt tolerance in the germination period by using an amplification result. The method provides powerful technical support for the breeding of the salt-tolerant soybean variety, is beneficial to the molecular breeding improvement of the salt-tolerant soybean, and compared with the traditional breeding screening, the method can rapidly screen and identify the soybean material with salt tolerance in the germination period, has remarkable effects on reducing soybean breeding workload and accelerating soybean breeding progress, and has great significance on accelerating the soybean salt-tolerant molecular breeding progress.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to a KASP molecular marker related to salt tolerance of soybean in germination period and application thereof.
Background
Soil salinization is an important factor that jeopardizes crop growth and is also a global problem that restricts grain production.
The soybean (Glycine max) belongs to the diploids of leguminous plants, butterfly flower subfamilies and soybean, is an important economic crop, oil crop and edible plant protein source in China, and is also an important industrial raw material. The planting area is enlarged by multiple ways in China, on one hand, salt-tolerant soybeans are developed to produce soybeans in saline-alkali soil; on the other hand, the soybean and corn banded compound planting is applied to improve the total soybean yield in China.
Genome-wide association analysis (GWAS) is an effective method for researching quantitative traits, and in recent years, with the continuous development of modern molecular biology techniques, molecular marker techniques have been deeply developed in the molecular breeding fields of wheat, rice, corn, sorghum, rape and the like. The majority of soybean traits are complex quantitative traits controlled by multiple genes, under the combined actions of genotype and environment. Liang Tengyue and the like, 9 SNPs closely related to the single plant weight under low phosphorus treatment are obtained by carrying out whole genome association analysis on 395 soybean germplasm resources by utilizing GAPIT, 227 soybean varieties or resources in Chuan Yuan area are taken as materials by Yang Hao and the like, 135 SSR markers and 107,081 effective SNPs markers are utilized for genotyping, and 51 and 70 sites obviously related to the growth-period characters are detected by whole genome association analysis. Compared with the seedling stage, the research on the salt tolerance correlation analysis of the soybean germination stage is just started, and the research on the salt tolerance correlation analysis of the soybean germination stage is recently reported.
KASP (kompetitive allele specific PCR) molecular marker is a novel SNP typing method based on allele specificity amplification and high-sensitivity fluorescence detection, has the characteristics of low cost and high flux, can accurately conduct double allele typing on SNP and InDel loci through specific matching of primer terminal bases, and is widely applied to molecular marker-assisted selection of crops such as rice, wheat and soybean. Therefore, the soybean salt tolerance major site is discovered by utilizing the whole genome association analysis (GWAS) in a high throughput way, and the KASP mark based on the obvious association SNP is developed for early selection of soybean salt tolerance breeding, so that the method has obvious effects on reducing soybean breeding workload and accelerating soybean breeding progress, and has great significance on accelerating soybean salt tolerance molecular breeding progress.
Disclosure of Invention
The invention aims to provide a KASP molecular marker related to salt tolerance of soybean in germination period and application thereof, so as to solve the problems in the prior art. The KASP molecular marker provided by the invention can be used for rapidly identifying the salt tolerance of soybeans in a batch manner, provides powerful technical support for the breeding of salt-tolerant varieties of soybeans, and is beneficial to the molecular breeding improvement of salt-tolerant soybeans.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a molecular marker related to salt tolerance in soybean germination, which comprises at least one of a sequence shown as SEQ ID NO.1 and a sequence shown as SEQ ID NO. 2.
Further, the sequence shown as SEQ ID NO.1 has an A/C mutation at the 54 th base; A/G mutation exists at the 76 th base of the sequence shown in SEQ ID NO. 2.
The invention also provides a KASP primer group for detecting the molecular marker, when the molecular marker is a sequence shown as SEQ ID NO.1, the KASP primer group comprises an upstream primer F1 with a nucleotide sequence shown as SEQ ID NO.3, an upstream primer F2 with a nucleotide sequence shown as SEQ ID NO.4 and a downstream primer R with a nucleotide sequence shown as SEQ ID NO. 5;
when the molecular marker is a sequence shown as SEQ ID NO.2, the KASP primer group comprises an upstream primer F1 with a nucleotide sequence shown as SEQ ID NO.6, an upstream primer F2 with a nucleotide sequence shown as SEQ ID NO.7 and a downstream primer R with a nucleotide sequence shown as SEQ ID NO. 8.
The invention also provides a reagent or a kit for detecting the molecular marker, which comprises the KASP primer group.
The invention also provides an application of the molecular marker, the KASP primer group or the reagent or the kit, which is used in any one of the following applications:
identifying the salt tolerance of soybean in germination period;
screening salt-tolerant soybean varieties or strains in germination period;
auxiliary breeding of soybean molecular markers;
improving the salt-resistant germplasm resource of soybean.
The invention also provides a method for identifying the salt tolerance of soybean in germination period, which comprises the following steps:
taking genomic DNA of a soybean sample to be detected as a template, carrying out fluorescent quantitative PCR amplification on the template by using the KASP primer group or the reagent or the kit, and judging whether the soybean shows salt tolerance in a germination period by using an amplification result;
if the amplification result shows that blue fluorescence is released, judging that the soybean sample to be detected is salt-tolerant in the germination period; and if the amplification result shows that red fluorescence is released, judging that the soybean sample to be detected is in a non-salt tolerance state in the germination period.
Further, the fluorescent quantitative PCR amplification procedure is as follows: 94 ℃ for 15min;94 ℃ for 20sec and 61-55 ℃ for 1min, wherein the annealing temperature is reduced by 0.6 ℃ for 10 cycles; 94 ℃ for 20sec and 55 ℃ for 1min, 26 cycles in total; 1min at 37 ℃.
Further, the fluorescent quantitative PCR amplification system comprises: 4. Mu.L of DNA template, 5. Mu.L of 2 XSKASP Master Mix, 0.14. Mu.L of primer Mix KASP Assay Mix, ddH 2 O2.0. Mu.L; wherein the primer mixture KASP Assay Mix consists of 6. Mu.L each of the upstream primers F1 and F2 and 15. Mu.L each of the downstream primers R and H 2 O23. Mu.L.
The invention discloses the following technical effects:
according to the invention, a representative 283 soybean germplasm resource is taken as a research material, salt tolerance identification is carried out in a soybean germination period, the phenotype of the germination rate, the germination potential, the germination index and the relative values of all indexes under salt stress treatment is explored and analyzed, two SNP loci S05-41921861 and S02-6088007 related to the salt tolerance of the soybean in the germination period are obtained through screening, KASP mark development is carried out on the SNP loci, SNP typing is obtained through KASP technology, the salt tolerance of the soybean in the germination period can be rapidly identified in batches, powerful technical support is provided for the breeding of the salt tolerance variety of the soybean, the molecular breeding improvement of the salt tolerance soybean is facilitated, and compared with the traditional breeding screening, the method can rapidly screen and identify the soybean material with the salt tolerance in the germination period, has remarkable effects on reducing soybean breeding workload and accelerating soybean breeding progress, and has great significance on accelerating soybean salt tolerance molecular breeding.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a differential analysis of RGR (A), RGE (B), RGI (C) for soybean germplasm at different years;
FIG. 2 is a graph showing the frequency distribution of RGR (A), RGE (B), RGI (C) and RGR (D), RGE (E) and RGI (F) in 2022 and 2023 germination period;
FIG. 3 is a diagram showing significant correlation of salt tolerance related traits in soybean natural populations with SNP haplotype analysis;
FIG. 4 is a genotyping of the KASP markers, wherein A is the genotyping result of S05_ 41921861; b is the genotyping result of S02_ 6088007.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
1 materials and methods
1.1 test materials
283 parts of a representative soybean germplasm resource, comprising 52 parts of local species, 212 parts of cultivars and 19 parts of wild soybeans, are provided by the soybean subject group of the institute of economic crops, academy of agricultural sciences, jiangsu province.
1.2 test methods and phenotypic data analysis
In order to find stress concentration suitable for salt tolerance evaluation indexes of soybean materials to be tested in germination period, 8 varieties are randomly selected from all the materials to be tested, germination pre-tests are repeatedly carried out for three times on each variety, concentration gradients of 0, 30, 60, 90, 120, 150 and 180mmol/LNaCl are respectively set for the pre-tests, and test results show that when the concentration of NaCl reaches 150mmol/L, various indexes such as germination rate of the materials are inhibited, and when the concentration is increased to 180mmol/L, various indexes such as germination rate of the materials and germination potential germination index are obviously different. The experiment determines 180mmol/L as stress concentration.
The germination test is carried out in a germination room, 25g of vermiculite is covered on each lattice, 20 healthy, full and pest-free seeds with the same size are selected from each material, 90mL of 0mM and 150mM NaCl solution are used for stress treatment after the vermiculite is paved, then the seeds are paved on the vermiculite which is watered with treatment liquid, 3-4 layers of filter paper which is soaked with the treatment liquid are covered on the vermiculite, the germination number is counted every 24 hours, and 7-8 days are counted. And calculating the relative salt damage rate of each index of the germination rate, the germination potential and the germination index of each material according to a formula. The calculation formula is as follows:
germination Rate (GR)% = (N) t /N)×100，N t The germination number of each grid of seeds on the t day is set, and N is the number of the seeds to be tested;
germination Index (GI) = Σgt/D t ，G t Number of germination per grid of seeds on day t, D t Day t of germination test;
germination Potential (GP)% =n 3 /N×100，N 3 The germination number of each grid of seeds on day 3 is N, and the number of the seeds to be tested is N;
relative salt damage index (ST) =s/C, C is control germination rate, germination index and germination vigor, S is germination rate, germination index and germination vigor under salt treatment.
The 3 relative salt damage indices for linkage analysis were calculated and expressed as ST-GR, ST-GI, ST-GP, respectively.
1.3 Whole genome correlation analysis
The inventors previously resequenced 283 parts of material with an average sequencing depth of 12.4×, resulting in a high density physical map containing 2597425 SNPs in total (thorappl Genet,2021,134 (5): 1329-1341.). The whole genome association analysis is calculated by using a GAPIT algorithm package based on R software, and is carried out by using a General Linear Model (GLM). takes-LogP not less than 5 as a significant threshold, and is considered as a significant association site when the threshold of SNP is in-LogP not less than 5.
1.4 haplotype and candidate Gene analysis
And determining the interval on the chromosome according to the target gene, and generating a target interval SNP annotation file and genotype data. Genotypes of different classes in the target region are classified into 5 major classes, such as gene-related region (exon, stock, splicing etc.), intron and UTR region (intron and UTR), upstream region (upstream), downstream region (downstream), and intergenic region (inter). And respectively carrying out haplotype analysis on SNP loci of different categories, and constructing a haplotype network by using PopART v1.7 software. Haplotype analysis was accomplished using the R program.
After SNP (single nucleotide polymorphism) which is obviously related to salt tolerance in soybean germination is obtained through whole genome association analysis, genes which are related to soybean plant height in 120kb intervals before and after SNP which is obviously related to salt tolerance in soybean germination are searched by utilizing soybean genome information (https:// Phytozome-next.jgi.doe/info/Gmax_Wm82_a2_v1) in an online database and Blastp comparison is carried out on the genes in an Arabidopsis genome database, so that candidate genes are determined.
1.5KASP marker development
KASP-PCR amplification primers were designed according to SNP sites s05_41921861 (a/C), s02_6088007 (a/G) significantly associated with germination rate, germination vigor, germination index of soybean, respectively, using the Primer-BLAST function of NCBI (https:// www.ncbi.nlm.nih.gov /), each pair of primers comprising two specific forward primers F1, F2 and one universal reverse Primer R. Wherein F1 and F2 comprise 6-carboxyfluorescein (FAM) and hexachloro-6-methylfluorescein (HEX) fluorescent linker sequences, respectively. Primer sequences were synthesized by the family of the organisms (Nanjing) and are shown in Table 4.
2. Results
2.1 analysis of Soybean germination stage phenotypes
According to the investigation test of salt tolerance related characters in the germination period of 283 parts of soybean material, 3 germination related characters (GR, GE and GI) are obtained through statistics, three basic indexes of the soybean material are subjected to statistical analysis, and relative indexes (RGR, RGE, RGI) of the soybean material are obtained, and specific results are shown in Table 1.
Table 1 descriptive statistics of three germination-related traits of soybean populations under NaCl conditions
From the data in table 1, the relative germination traits of 283 parts of soybean material showed large phenotypic variation in both years (2022, 2023). In two years, the change range of RGR, RGE, RGI is respectively between 0.05 and 1.00, 0.00 and 1.00 and 0.04 and 1.00. 3 the personality Coefficient of Variation (CV) ranges from 31.81% to 50.60%, the generalized genetic rate (h 2 ) In the range of 95.75% to 99.64%, soybean germplasm is significantly affected by strain, environment and strain-to-environment interactions as seen by the broad genetic rates of the phenotypes. The quantitative traits are controlled by multiple genes, and meanwhile, the true genetic difference of the population growth-period traits is indicated, so that the association analysis can be further carried out.
2.2 analysis of salt tolerance box diagram and frequency distribution in germination period
Drawing a box diagram (figure 1) of the relative germination rate, relative germination vigor and relative germination index obtained by calculation under the two-year stress treatment, and obtaining that the two-year variation of the relative indexes has obvious difference. The relative germination rate, relative germination potential, and relative germination index frequency of 283 soybean germplasm for two years were calculated by using Microsoft excel 2016, and the frequency distribution chart and the density curve were plotted (FIG. 2). It can be seen that several indicators are inhibited to varying degrees under salt stress. The histograms of the phenotype data all show the characteristic of approximate normal distribution, which indicates that the soybean natural population of 283 materials studied by the invention has rich genetic variation and is suitable for the subsequent whole genome association analysis.
2.3 analysis of salt tolerance related traits in soybean germination period GWAS
The invention uses GAPIT in R to carry out whole genome association analysis by combining phenotypic results of 283 tested materials based on natural population under salt treatment in germination period, germination vigor, germination index and relative index (RGR, RGE, RGI) with sequencing data and using GLM model, and draws Manhattan diagram and QQ diagram of corresponding indexes. In 2022, 447 SNPs (-log 10P > 5) closely related to soybean germination were detected, wherein the SNPs associated with relative germination vigor were the most, 269, and the most, and the least, of the SNPs associated with relative germination indexes were distributed on chromosomes 2, 5 and 20. In 2023, there were 1841 SNPs closely related to soybean germination, the most SNP sites related to the relative germination rate, 1512 and the most distribution on chromosome 5. The sites associated with the relative germination index are minimal and are predominantly distributed on chromosomes 9 and 20. Table 2 shows the SNPs significantly correlated with each trait in germination period, as counted by GWAS analysis results.
TABLE 2 germination-related trait GWAS analysis result statistics
2.4 haplotype and candidate Gene analysis
In order to study the phenotypic effect of allelic variation of the obviously-associated SNP locus, haplotype analysis is carried out on the obviously-associated SNP locus with the highest detection threshold value of the salt tolerance related trait in 2022 germination period.
The allelic variation of the SNP locus S05_41921861 is found to be A/C, the average value of the relative germination rate with S05_41921861-A is 0.52, and the average value of the relative germination rate with S05_41921861-C is significantly lower than 0.71. The S05_41921861 locus is positioned at the 54 th base of the sequence shown in SEQ ID NO. 1;
SEQ ID NO.1：
GGAGGGCCTCAGTCAAGTAACCAGAAAAATAAAATGTATAAAGTTGAGGACTGACGTGTTATTGTATTTGTAGGCACTAAATTTGAAATTAGAAGTTAAACAAAGCCCCATCATGGCCTTTAAAGGTCAGCCTGACAAAATTATTGACAGTTTAAATGTATATTACTATCTTTCAATCCCATCGAAAAGAATGAGAAAATAATTAAATGAATGATATTATATTAGATGATAGAAGCTGAGCACCCTGCATGATTGGATTAAGCGGGTGCAATGGAGACTTTTTCAACCTCTAGAATGTATTCAAGTGTTGCAAGTGGAGGAATTTGCACACCAGTGCCTAAGTCAGCACCATTCTCTCCAAACCCCAATTTAGGAGGGACTATTACCTTCCTCTTGCC, (underlined bold indicates SNP site, where A/C mutation is present).
The allelic variation of the SNP locus S02_6088007 is A/G, the average value of the relative germination potential carrying S02_6088007-A is 0.42, and the relative germination potential carrying S02_6088007-G is significantly lower than that of 0.66. The S02_6088007 locus is positioned at the 76 th base of the sequence shown in SEQ ID NO. 2;
SEQ ID NO.2：
TTTTATTAGATCATATCATTAATCTCAGTTGTACTTTTTTAAAGACTAATATTAGTTATTAATTTATTATTTTTTTTAATGAGAGAGGTATATTCTAAATCACATGACCTCTTCTTTTTGTTTCTTTTATAATTAAGTTAATTTTATAACTTATTTTTTCTTATCCCTTTAAGGCAGATAATCATTTTTCCTATTCCGTGTTTTCCTTTCTGAAATATGGGCTAGTTTGCTTGCACTTGATTTGTTGACACGTATTAGCTAGCATGGATTTTTAATCTTACAATTCACATCGTAACACATTGAATCACTTCTTTTTTTATTCTTACATGCTTTCATGTCACAATCATGACCTCTACCTGTTTGAACTACGTTGGTACGTGGGAATGAGGTGCATGCCATTGCTATAAATTAAAACACAACCACTTTATCAACAAAACCACAAGCACACAAACACT, (underlined bold indicates SNP site, where A/G mutation is present).
The allelic variation of SNP site S09_3907313 was T/C, the average value of the relative germination index with S09_3907313-C was 0.40, and the average value of the relative germination index with S09_3907313-T was 0.53 (FIG. 3).
Screening candidate genes and predicting functions within 120kb range on the upstream and downstream of the SNP locus which is obviously related to salt tolerance in soybean germination (log 10 (P) is more than or equal to 5). Referring to the genetic functional annotation information of soybean genome, 12 candidate genes (table 3) which are obviously related to soybean salt tolerance in germination period are identified, and are respectively related to coordinating cell reaction, regulating osmotic stress, weakening oxidative stress and scavenging of Reactive Oxygen Species (ROS) and heavy metal ion transport protein, and play a vital role in plant development and stress tolerance so as to realize normal growth and development of plants, immune response and response to abiotic stress to resist biotic stress.
TABLE 3 functional annotation of salt tolerance related candidate genes for soybean germination
Wherein the mutation at the s05_41921861 (a/C) site is associated with the glyma.05g244600 gene, and annotation information shows that the gene coordinates cellular responses to achieve normal growth and development of plants, immune responses, and responses to abiotic stress against biotic stress. The mutation at the s02_6088007 (a/G) site was correlated with the increased expression of the glyma.02g067600 gene as shown by the information on release of interest. Under salt stress, it induces GAOX20 expression, which encodes a adC7-GA inhibitor.
2.5 application of salt-tolerant KASP (KASP) mark in soybean germination period
KASP markers were developed for SNP sites s05_41921861 (a/C) and s02_6088007 (a/G) that are significantly associated with soybean germination salt tolerance, and the KASP primer sequences are shown in table 4.
TABLE 4 specific primers for KASP
Note that: the underlined part is the linker sequence, and the bolded part is the base corresponding to the SNP locus.
Extracting genomic DNA of the selected soybean germplasm, taking the genomic DNA as a reaction template, and adopting KASP primers designed for the SNP loci to carry out PCR reaction to obtain PCR amplification products.
The reaction system is as follows: 4. Mu.L of DNA template, 5. Mu.L of 2 XSKASP Master Mix, 0.14. Mu.L of primer Mix KASP Assay Mix, ddH 2 O2.0. Mu.L; the preparation of the primer Mix KASP Assay Mix was: F1:6.mu.L, F2:6.mu.L, R:15.mu.L, H 2 O:23 μl, total volume: 50. Mu.L of the system was used for the PCR reaction system by taking 0.14. Mu.L of the system of 50. Mu.L.
The reaction procedure is: 94 ℃ for 15min;94 ℃ for 20sec and 61-55 ℃ for 1min, wherein the annealing temperature is reduced by 0.6 ℃ for 10 cycles; 94 ℃ for 20sec and 55 ℃ for 1min, 26 cycles in total; 1min at 37 ℃.
And after the reaction is finished, directly reading the generated fluorescence data result on a real-time fluorescence quantitative PCR system. Genotyping was performed on 24 selected soybean germplasm using the KASP-labeled primer pair designed for s05_41921861 (a/C) and s02_6088007 (a/G) loci (known salt tolerance quality (relative germination rate and relative germination potential index) of soybean germplasm are shown in table 5), and the results are shown in fig. 4, in which two different genotypes can be clearly separated after PCR with 2 different molecular-labeled primers designed.
When the primer of S05_41921861 is used for genotyping to detect the salt tolerance of soybean in germination, when the alleles where SNP loci are located are all C, the detection sample is combined with a specific FAM detection primer and releases a blue fluorescent group, and the blue fluorescent signal is enhanced along with the increase of the number of PCR reaction cycles, so that the soybean can be judged to be the salt tolerance genotype of soybean in germination through fluorescent color (the genotyping result of KASP mark is defined as CC). When the allelic bases of the locus are all A, the detection sample is combined with a specific HEX detection primer and releases a red fluorescent group, and the red fluorescent signal is enhanced along with the increase of the PCR reaction cycle number, so that the locus can be judged to be a non-salt tolerant genotype of soybean germination period through fluorescent color (the genotyping result of KASP mark is defined as AA).
When the primer of S02_6088007 is used for genotyping to detect the salt tolerance of soybean in germination, when the alleles of SNP loci are G, the detection sample is combined with a specific FAM detection primer and releases a blue fluorescent group, and the blue fluorescent signal is enhanced along with the increase of the PCR reaction cycle number, so that the soybean can be judged to be the salt tolerance genotype of soybean in germination through fluorescent color (the genotyping result of KASP mark is defined as GG). When the alleles of the locus are all A, the detection sample is combined with a specific HEX detection primer and releases a red fluorescent group, and the red fluorescent signal is enhanced along with the increase of the PCR reaction cycle number, so that the locus can be judged to be a non-salt tolerant genotype of soybean germination period through fluorescent color (the genotyping result of KASP mark is defined as AA).
Table 524 genotyping results for soybean germplasm
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (4)
1. Use of a set of KASP primers for detecting a molecular marker associated with salt tolerance in soybean germination, for use in any one of the following applications:
identifying the salt tolerance of soybean in germination period;
screening salt-tolerant soybean varieties or strains in germination period;
auxiliary breeding of soybean molecular markers;
improving soybean salt-tolerant germplasm resources;
the molecular marker comprises at least one of a sequence shown as SEQ ID NO.1 and a sequence shown as SEQ ID NO. 2; an A/C mutation exists at the 54 th base of the sequence shown in SEQ ID NO. 1; the A/G mutation exists at the 76 th base of the sequence shown in SEQ ID NO. 2;
when the molecular marker is a sequence shown as SEQ ID NO.1, the KASP primer group comprises an upstream primer F1 with a nucleotide sequence shown as SEQ ID NO.3, an upstream primer F2 with a nucleotide sequence shown as SEQ ID NO.4 and a downstream primer R with a nucleotide sequence shown as SEQ ID NO. 5;
when the molecular marker is a sequence shown as SEQ ID NO.2, the KASP primer group comprises an upstream primer F1 with a nucleotide sequence shown as SEQ ID NO.6, an upstream primer F2 with a nucleotide sequence shown as SEQ ID NO.7 and a downstream primer R with a nucleotide sequence shown as SEQ ID NO. 8.
2. A method for identifying salt tolerance of soybeans in germination, comprising the steps of:
taking genomic DNA of a soybean sample to be detected as a template, carrying out fluorescent quantitative PCR amplification on the template by using a KASP primer group for detecting a molecular marker related to the salt tolerance of the soybean in the germination period, and judging whether the soybean shows the salt tolerance in the germination period by using an amplification result;
the molecular marker comprises at least one of a sequence shown as SEQ ID NO.1 and a sequence shown as SEQ ID NO. 2; an A/C mutation exists at the 54 th base of the sequence shown in SEQ ID NO. 1; the A/G mutation exists at the 76 th base of the sequence shown in SEQ ID NO. 2;
the KASP primer group comprises an upstream primer F1, an upstream primer F2 and a downstream primer R; the upstream primer F1 is marked with a FAM fluorescent group, and the upstream primer F2 is marked with a HEX fluorescent group;
when the molecular marker is a sequence shown as SEQ ID NO.1, the KASP primer group comprises an upstream primer F1 with a nucleotide sequence shown as SEQ ID NO.3, an upstream primer F2 with a nucleotide sequence shown as SEQ ID NO.4 and a downstream primer R with a nucleotide sequence shown as SEQ ID NO. 5;
when the molecular marker is a sequence shown as SEQ ID NO.2, the KASP primer group comprises an upstream primer F1 with a nucleotide sequence shown as SEQ ID NO.6, an upstream primer F2 with a nucleotide sequence shown as SEQ ID NO.7 and a downstream primer R with a nucleotide sequence shown as SEQ ID NO. 8;
if the amplification result shows that blue fluorescence is released, judging that the soybean sample to be detected is salt-tolerant in the germination period; and if the amplification result shows that red fluorescence is released, judging that the soybean sample to be detected is in a non-salt tolerance state in the germination period.
3. The method of claim 2, wherein the fluorescent quantitative PCR amplification procedure is: 94 ℃ for 15min;94 ℃ for 20sec and 61-55 ℃ for 1min, wherein the annealing temperature is reduced by 0.6 ℃ for 10 cycles; 94 ℃ for 20sec and 55 ℃ for 1min, 26 cycles in total; 1min at 37 ℃.
4. The method of claim 2, wherein the fluorescent quantitative PCR amplification system is: 4. Mu.L of DNA template, 5. Mu.L of 2 XSKASP Master Mix, 0.14. Mu.L of primer Mix KASP Assay Mix, ddH 2 O2.0. Mu.L; wherein the primer mixture KASP Assay Mix consists of 6. Mu.L each of the upstream primers F1 and F2 and 15. Mu.L each of the downstream primers R and H 2 O23. Mu.L.
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