KASP marker of soybean shade-tolerant gene Glyma. 06g213100 and application of KASP marker

Abstract

The invention provides a KASP marker of a soybean shade-tolerant gene Glyma.06g213100 and application of the KASP marker, the KASP marker is four SNP variation sites of the soybean shade-tolerant gene Glyma.06g213100, soySNP-1 is subjected to base T-A substitution at 21, 485 and 858 bp, soySNP-2 is subjected to base A-G substitution at 21, 486 and 271 bp, soySNP-3 is subjected to base A-G substitution at 21, 485 and 456 bp, and soySNP-4 is subjected to base T-G substitution at 21, 486 and 286 bp. The SNP and KASP markers provided by the invention can be used for molecular marker-assisted selective breeding of the soybean shade-tolerant character, and have important theoretical and practical guiding significance for accelerating the genetic improvement process of soybean shade-tolerant breeding and improving the breeding selection efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of molecular genetic breeding, and provides a KASP marker related to the soybean shade tolerance gene Glyma.06g213100 and its application, which can be used for early molecular assisted selection of soybean shade tolerance traits to improve breeding efficiency. Background Art

[0002] In recent years, the demand for soybeans in China has been increasing year by year. The production capacity of soybeans in China is seriously insufficient, and the external dependence is as high as over 80%. At present, if China wants to fully achieve self-sufficiency in soybeans, it needs to occupy 700-800 million mu of cultivated land for planting, which is difficult to achieve under the existing overall cultivated land conditions. Intercropping soybeans has the advantages of intensive utilization of land, space and time, improving light energy utilization rate, and improving the crop population structure. Without affecting the yield of other crops, an additional crop of soybeans can be harvested. It can be seen that expanding the planting area of soybeans and increasing soybean yield through intercropping can alleviate the contradiction between soybean supply and demand in China to a certain extent. Therefore, developing the soybean intercropping planting mode is an important way to improve China's soybean production capacity and effectively utilize the planting space, which can break through the cultivated land constraint and solve China's soybean self-sufficiency rate.

[0003] However, when soybeans are intercropped with tall crops such as corn, sugarcane, and cassava, they will be subjected to shade stress, which is extremely unfavorable to soybean growth and defense (Liu, et al., 2019). When soybeans are subjected to shade stress, the plant height becomes taller, the internodes of the main stem, hypocotyls and petioles over-elongate, the leaf angle becomes smaller, the stem diameter becomes thinner, the root-shoot ratio decreases, the branches decrease, and flowering is advanced, ultimately leading to lodging, excessive growth, premature senescence, and a decline in the ability to resist diseases and pests, resulting in difficulty in improving yield and quality (Raza, et al., 2020; Khalid, et al., 2019; Gholamhoseini, et al., 2018; Wu, et al., 2017; Liu, et al., 2016; Su, et al., 2014). Shade has become a limiting factor restricting the potential of soybean intercropping and is the biggest obstacle to promoting the soybean intercropping mode. With the vigorous development of soybean intercropping production, the problem of shade tolerance will become more prominent. Screening shade-tolerant soybean resources and cultivating shade-tolerant, high-yielding and suitable intercropping soybean varieties are effective ways to solve the above problems. However, there are technical problems such as a lack of shade-tolerant soybean germplasm resources, a shortage of shade-tolerant, high-yielding and high-quality varieties suitable for intercropping, and low efficiency of traditional breeding techniques, which greatly restrict the sustainable development of the soybean intercropping planting mode. Cultivating high-quality, shade-tolerant and high-yielding new varieties has become the key to the development and high-yield and stable production of intercropping soybeans, and is an urgent need for the development of intercropping soybean production.

[0004] Traditional identification of soybean shade tolerance involves data collection and analysis at multiple growth stages, including the seedling stage, branching stage, flowering stage, pod-setting stage, and maturity stage, etc., which results in a relatively long identification cycle; during the identification process, multiple indicators such as leaf area index, specific leaf weight, and chlorophyll content need to be measured regularly, and the data collection and processing process is relatively complex; when conducting large-scale identification in the field, the relative error of some traits such as stem diameter, number of branches, fresh weight of stems and leaves, and dry weight of stems and leaves is relatively large, which may lead to inaccurate identification results; the variation of some traits is small, such as leaf shape index, petiole length, leaf length, and leaf width, etc., and the differences in the relative values of these traits among varieties are not significant after shading, so their sensitivity is insufficient and they are not suitable as the main indicators for shade tolerance identification; the identification results of soybean shade tolerance are easily affected by environmental conditions, such as meteorological factors like continuous rainy weather may interfere with the test results; the environmental conditions in different ecological regions vary greatly, which may lead to differences in the shade tolerance performance of the same variety when conducting identification in different regions. There is an urgent need to develop more molecular markers closely linked to shade tolerance traits to shorten the breeding cycle and improve the breeding efficiency of shade-tolerant varieties.

[0005] Molecular marker technology has the advantages of high efficiency, accuracy, being unaffected by the environment, and rapidity in crop breeding, and is an important part of modern breeding technology. KASP (kompetitive allele specific PCR) is a new type of genotyping technology with the characteristics of high throughput, low cost, high precision, and wide applicability. It accurately performs dual-allele genotyping of SNP and InDel loci through specific matching of the terminal bases of primers, and has been widely used in molecular marker-assisted selection of soybeans. Given the advantages of KASP marker detection technology such as good stability, high accuracy, low detection cost, and high throughput, with the help of KASP markers, a large number of samples can be accurately subjected to dual-allele genotyping, achieving the effect of high-throughput verification and detection of target genes.

[0006] This patent designs and develops a simple and high-throughput KASP marker for the differential sequences of the shade tolerance gene Glyma.06g213100 in different soybean resources, which can perform molecular detection of the shade tolerance of soybean germplasm resources, provides molecular markers that can be applied to molecular marker-assisted selection for soybean breeders, and will accelerate the process of molecular marker-assisted polymerization of excellent shade tolerance genes and cultivation of shade-tolerant new varieties. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the purpose of the present invention is to identify single nucleotide variant sites (SNPs) closely associated with the soybean shade tolerance gene Glyma.06g213100, and develop KASP molecular markers and their primers based on the SNP site information, so as to provide molecular-assisted selection technical support for the early large-scale identification and screening of shade tolerance traits.

[0008] The object of the present invention can be achieved by the following technical solutions:

[0009] In the first aspect, the present invention provides a nucleotide variation site SNP closely associated with the soybean shade tolerance gene Glyma.06g213100. The 4 SNP variation sites of the soybean shade tolerance gene Glyma.06g213100 are located at the position of 21376611_21490108 bp on chromosome 6 of the soybean genome Wm82.a2.v2.0. Among them, soySNP-1 has a base substitution of T to A at 21,485,858 bp, soySNP-2 has a base substitution of A to G at 21,486,271 bp, soySNP-3 has a base substitution of A to G at 21,485,456 bp, and soySNP-4 has a base substitution of T to G at 21,486,286 bp. The phenotypic variation explanation rate reaches 15.25%. The nucleotide sequence where the SNP is located is as described in Seq ID NO:1.

[0010] In the second aspect, the present invention provides a KASP marker primer set for detecting the shade tolerance performance of soybeans. Each KASP marker contains three primers, including two specific primers designed for the base differences at the key sites, namely the upstream primer F1 and the upstream primer F2, and one universal primer, i.e., the downstream primer R. The 3'-ends of the two specific primers are allelic variant bases, and the 5'-ends are connected with the specific FAM and HEX fluorescence adapter sequences of the KASP reaction reagent of LGC (Laboratory of the Government Chemist) Company in the UK.

[0011] The sequence of the upstream primer F1 of the soySNP-1KASP marker is:

[0012] 5'-GAAGGTGACCAAGTTCATGCTAGGGAGCGGCTCGAGAAA-3', as shown in SEQ ID NO:2;

[0013] The sequence of the upstream primer F2 of the soySNP-1KASP marker is:

[0014] 5'-GAAGGTCGGAGTCAACGGATTAGGGAGCGGCTCGAGAAT-3', as shown in SEQ ID NO:3;

[0015] The sequence of the downstream primer R of the soySNP-1KASP marker is: 5'-CTTGTACGCGTTTGACCCCAAAT-3', as shown in SEQID NO:4;

[0016] The sequence of the upstream primer F1 of the soySNP-2KASP marker is:

[0017] 5’-GAAGGTGACCAAGTTCATGCTCAATTTTACAGACGCGACTCTGG-3’, as shown in SEQ ID NO:5;

[0018] The upstream primer F2 sequence of the soySNP-2KASP marker is:

[0019] 5’-GAAGGTCGGAGTCAACGGATTCAATTTTACAGACGCGACTCTGA-3’, as shown in SEQ ID NO:6;

[0020] The downstream primer R sequence of the soySNP-2KASP marker is: 5’-TAAACAAAAAGAGAAAACCTGCCG-3’, as shown in SEQ ID NO:7;

[0021] The upstream primer F1 sequence of the soySNP-3KASP marker is:

[0022] 5’-GAAGGTGACCAAGTTCATGCTCGGCTTCACGTCTTCGAGC-3’, as shown in SEQ ID NO:8;

[0023] The upstream primer F2 sequence of the soySNP-3KASP marker is:

[0024] 5’-GAAGGTCGGAGTCAACGGATTCGGCTTCACGTCTTCGAGT-3’, as shown in SEQ ID NO:9;

[0025] The downstream primer R sequence of the soySNP-3KASP marker is: 5’-CGGTCGGTTAATGTTAGATTTGCA-3’, as shown in SEQ ID NO:10;

[0026] The upstream primer F1 sequence of the soySNP-4KASP marker is:

[0027] 5’-GAAGGTGACCAAGTTCATGCTTGGTTGGGGGTTTCGCTG-3’, as shown in SEQ ID NO:11;

[0028] The upstream primer F2 sequence of the soySNP-4KASP marker is:

[0029] 5’-GAAGGTCGGAGTCAACGGATTTGGTTGGGGGTTTCGCTT-3’, as shown in SEQ ID NO:12;

[0030] The R sequence of the downstream primer of the soySNP-4KASP marker is: 5’-CCCGTCACTTTCATTCGGTTTTAA-3’, as shown in SEQ ID NO:13.

[0031] When synthesizing the KASP molecular marker primers described above, a fluorescence signal tag of carboxyfluorescein FAM is added to the 5’ end of the forward primer F1); a fluorescence signal tag of hexachlorofluorescein phosphoramidite HEX is added to the 5’ end of the forward primer F2.

[0032] The application of the KASP molecular marker for the 4 nucleotide variant sites SNP of the soybean shade tolerance gene Glyma.06g213100 in the identification or assisted screening method is to detect whether the genotypes of the 4 deoxyribonucleotides at the position of 21376611_21490108bp on chromosome 6 of soybean are AGCG, TACG or TATT, and select the soybean shade tolerance germplasm resources carrying the allelic variant sites of AGCG or TACG genotypes for selecting strong shade tolerance soybean germplasm resources with AGCG genotype or medium shade tolerance soybean germplasm resources with TACG genotype, and molecular marker assisted genetic improvement of soybean shade tolerance traits.

[0033] In the third aspect, the present invention protects a reagent of KASP markers for detecting soybean shade tolerance performance, and the reagent contains the primer group described above.

[0034] In the fourth aspect, the present invention also protects a kit of KASP markers for detecting soybean shade tolerance performance, and the kit contains the primer group described above or contains the reagent described above.

[0035] In the fifth aspect, the present invention also protects the primer group described above, the reagent described above, or the kit described above in any of the following applications:

[0036] (A1) Assisted breeding of shade tolerance soybean;

[0037] (A2) Preparation of products for assisted breeding of shade tolerance soybean;

[0038] (A3) Identification or assisted identification of soybean shade tolerance;

[0039] (A4) Preparation of products for identification or assisted identification of soybean shade tolerance;

[0040] (A5) Breeding or assisted breeding of shade tolerance soybean;

[0041] (A6) Preparation of products for breeding or assisted breeding of shade tolerance soybean.

[0042] Sixth aspect, the present invention protects a method for breeding or assisting in breeding shade-tolerant soybeans, which involves extracting genomic DNA of soybeans, performing PCR amplification on the extracted genomic DNA using the primer pairs described above, and performing genotyping through the fluorescence signals of the PCR amplification products.

[0043] Seventh aspect, the present invention protects a method for identifying or assisting in identifying the shade tolerance of soybeans, which involves extracting genomic DNA of soybeans, performing PCR amplification on the extracted genomic DNA using the primer pairs described above, and performing genotyping through the fluorescence signals of the PCR amplification products.

[0044] In the above method, the KASP primer set consists of upstream primer F1, upstream primer F2, and downstream primer R. PCR amplification is carried out in an ABI7500 real-time fluorescence quantitative PCR instrument, and after PCR, this instrument can perform genotyping according to the fluorescence signals. After the reaction is completed, the ABI7500 real-time fluorescence quantitative PCR instrument will directly read the fluorescence data of the PCR reaction products, and the results of the fluorescence scan will be automatically converted into graphs; if the genotyping is insufficient, continue the amplification, check the genotyping situation every 3 cycles until the genotyping is complete.

[0045] The specific operation steps are as follows:

[0046] (1) Extraction of genomic DNA of soybean plants;

[0047] (2) Performing PCR amplification on the genomic DNA of biological samples using the above-mentioned PCR specific amplification primer pairs and detecting the SNP genotypes:

[0048] Adding the above-mentioned molecular marker primers to the same PCR reaction system, setting 2 blank controls with ultrapure water instead of sample template DNA, and amplifying the DNA of soybean germplasm resources on a fluorescence quantitative PCR instrument;

[0049] 5.0 μL reaction system: 5 - 100 ng of soybean sample DNA template; 2.5 μL of FLu-Arms 2x PCR Mix; 0.075 μL of KASP upstream genotyping primer F1 (10 μM), 0.075 μL of KASP upstream genotyping primer F2 (10 μM), 0.2 μL of KASP downstream universal primer R (10 μM); make up the volume to 5 μL with water.

[0050] The reaction conditions include pre-denaturation at 95°C for 3 min, 1 cycle; denaturation at 95°C for 15 s, annealing at 63.4 - 57°C for 45 s, with a decrease of 0.8°C for each cycle, 9 cycles; denaturation at 94°C for 15 s, annealing at 57.5°C for 45 s, 45 cycles; after the cycles are completed, extend at 30°C for 30 s.

[0051] After the reaction is completed, the fluorescence is detected using a multifunctional microplate reader. According to the fluorescence read, the ratios of FAM / ROX and HEX / ROX are calculated respectively, and a coordinate graph is drawn using software to obtain the genotype of the sample.

[0052] (3) Select shade-tolerant soybean germplasm resources from different soybean germplasm resources based on their genotypes.

[0053] Among them, in step (2), "ROX" refers to the fluorescence intensity of the internal reference ROX in the PCR Mix. The applicant reads the fluorescence intensity of FAM, HEX and the internal reference ROX respectively, and then calculates the ratios of FAM / ROX and HEX / ROX, and uses software to draw a coordinate graph to obtain the genotype of the sample.

[0054] Among them, in step (2), in response to the problem of "the same PCR reaction system", the applicant prepared PCR reaction systems for the different SNP molecular markers mentioned above respectively to distinguish the genotype of each site respectively, instead of distinguishing the genotype of the four SNP sites at the same time.

[0055] Beneficial Effects

[0056] (1) The four SNP variation sites closely related to the soybean shade tolerance gene Glyma.06g213100 of the present invention were selected from 394 representative soybean germplasm resources in southern China, and 5× resequencing was performed. After elimination and filtering, a high-density SNP molecular marker map covering the whole genome was obtained, and then the shade tolerance index data of soybeans in three environments (spring 2014, summer 2014 and spring 2015) were detected by field phenotypic identification method. The soybean shade tolerance phenotypic data were subjected to genome-wide association analysis. Four SNP variation sites of the Glyma.06g213100 gene significantly associated with soybean shade tolerance were detected in all three environments, and the phenotypic variation explanation rate reached 15.25%, which were located at the 21376611_21490108bp position of chromosome 6 of the soybean genome v2.0. Select shade-tolerant soybean germplasm resources of the AGCG or TACG genotype carrying allelic variation sites to select strong shade-tolerant soybean germplasm resources of the AGCG genotype or medium shade-tolerant soybean germplasm resources of the TACG genotype, providing technical support for molecular marker-assisted breeding of soybean shade tolerance traits.

[0057] (2) The present invention identified four SNP sites on soybean chromosome 6 that control soybean shade tolerance. The developed KASP molecular marker can directly distinguish and detect whether the bases of the four SNP variation sites are AGCG, TATT or TACG. The KASP molecular marker has good application value and can realize the pre-selection of soybean shade tolerance and molecular-assisted breeding.

[0058] (3) Using KASP molecular marker primers, 33 soybean materials (including 5 strongly shade-tolerant materials, 23 moderately shade-tolerant materials, and 5 extremely shade-intolerant materials) were amplified and genotyped on a real-time fluorescence quantitative PCR instrument (Table 1). The results showed that this molecular marker primer could clearly separate the three genotypes. Among them, 5 strongly shade-tolerant materials had mutations at the soySNP-1, soySNP-2, soySNP-3, and soySNP-4 loci, carried the AGCG allelic variant locus, and had the genotype AGCG, accounting for 100%; 3 out of 5 extremely shade-intolerant materials did not carry variant loci, accounting for 60.0%, with the genotype TATT, and the other 2 had mutations at the soySNP-3 and soySNP-4 loci and carried the CG allelic variant locus; among 23 moderately shade-tolerant materials, mutations occurred at both the soySNP-3 and soySNP-4 loci, carried the CG allelic variant locus, and had the genotype TACG (the TATT type was the reference genome); the ratio of the genotypes of the 33 materials that matched the phenotypes was 90.91%. The comparison between the genotypes and the shade-tolerance phenotype results was relatively consistent, and it could be safely used in molecular marker-assisted selection breeding for soybean seedling shade tolerance. Specific implementation manner

[0059] The following further describes the present invention in conjunction with embodiments. Unless otherwise specified, the methods used are all conventional methods. If the production manufacturers of the reagents or instrument devices are not indicated, they are all regarded as conventional products that can be purchased on the market.

[0060] The 4 SNP variant loci of the soybean shade-tolerance gene Glyma.06g213100 in the present invention were obtained through the following steps:

[0061] (1) 394 representative ones were selected from 1498 soybean germplasm resources in southern China for 5× resequencing. After filtering, a high-density SNP molecular marker map covering the whole genome was obtained. (2) Using the field phenotypic identification method, the shade-tolerance index data of soybeans in 3 environments (spring 2014, summer 2014, and spring 2015) were detected. Genome-wide association analysis was performed on the soybean shade-tolerance phenotypic data, and 4 SNP loci of the Glyma.06g213100 gene that were significantly associated with soybean shade tolerance were detected in all 3 environments. The phenotypic variation interpretation rate reached 15.25%, and it was located at the position of 21376611_21490108 bp on chromosome 6 of soybean.

[0062] Example 1: Development of KASP marker specific primers

[0063] Using the Primer-BLAST function of NCBI (https: / / www.ncbi.nlm.nih.gov / ), according to the sequence of the Glyma.06g213100 gene on the genome, which corresponds one by one to the reverse complement of the sequence of this gene in the phytozome database, primers for 4 SNP mutation sites were designed, including upstream primer F1, upstream primer F2 and downstream primer R, where F1 and F2 contain FAM and HEX fluorescent linker sequences respectively, and the sequences are as follows:

[0064] The sequence of the upstream primer F1 of the soySNP-1KASP marker is:

[0065] 5’-GAAGGTGACCAAGTTCATGCTAGGGAGCGGCTCGAGAAA-3’, as shown in SEQ ID NO:2;

[0066] The sequence of the upstream primer F2 of the soySNP-1KASP marker is:

[0067] 5’-GAAGGTCGGAGTCAACGGATTAGGGAGCGGCTCGAGAAT-3’, as shown in SEQ ID NO:3;

[0068] The sequence of the downstream primer R of the soySNP-1KASP marker is: 5’-CTTGTACGCGTTTGACCCCAAAT-3’, as shown in SEQID NO:4.

[0069] The sequence of the upstream primer F1 of the soySNP-2KASP marker is:

[0070] 5’-GAAGGTGACCAAGTTCATGCTCAATTTTACAGACGCGACTCTGG-3’, as shown in SEQ ID NO:5;

[0071] The sequence of the upstream primer F2 of the soySNP-2KASP marker is:

[0072] 5’-GAAGGTCGGAGTCAACGGATTCAATTTTACAGACGCGACTCTGA-3’, as shown in SEQ ID NO:6;

[0073] The sequence of the downstream primer R of the soySNP-2KASP marker is: 5’-TAAACAAAAAGAGAAAACCTGCCG-3’, as shown in SEQID NO:7.

[0074] The sequence of the upstream primer F1 of the soySNP-3KASP marker is:

[0075] 5’-GAAGGTGACCAAGTTCATGCTCGGCTTCACGTCTTCGAGC-3’, as shown in SEQ ID NO:8;

[0076] The upstream primer F2 sequence of the soySNP-3KASP marker is:

[0077] 5’-GAAGGTCGGAGTCAACGGATTCGGCTTCACGTCTTCGAGT-3’, as shown in SEQ ID NO:9;

[0078] The downstream primer R sequence of the soySNP-3KASP marker is: 5’-CGGTCGGTTAATGTTAGATTTGCA-3’, as shown in SEQ ID NO:10.

[0079] The upstream primer F1 sequence of the soySNP-4KASP marker is:

[0080] 5’-GAAGGTGACCAAGTTCATGCTTGGTTGGGGGTTTCGCTG-3’, as shown in SEQ ID NO:11;

[0081] The upstream primer F2 sequence of the soySNP-4KASP marker is:

[0082] 5’-GAAGGTCGGAGTCAACGGATTTGGTTGGGGGTTTCGCTT-3’, as shown in SEQ ID NO:12;

[0083] The downstream primer R sequence of the soySNP-4KASP marker is: 5’-CCCGTCACTTTCATTCGGTTTTAA-3’, as shown in SEQ ID NO:13.

[0084] When synthesizing the KASP molecular marker primers, a fluorescence signal tag of carboxyfluorescein FAM is added to the 5’ end of the forward primer F1); a fluorescence signal tag of hexachlorofluorescein phosphoramidite HEX is added to the 5’ end of the forward primer F2.

[0085] Example 2: Detecting the genotypes of SNP loci in different soybean varieties and their applications

[0086] Genomic DNA of different soybean samples was extracted separately. Using the genomic DNA as a template, PCR amplification was performed with the KASP marker-specific primers to obtain PCR amplification products. The PCR amplification was carried out in an ABI7500 real-time fluorescence quantitative PCR instrument, and after PCR, the instrument could perform genotyping based on the fluorescence signals.

[0087] The amplification system described above is a 5.0 μL reaction system: 5 - 100 ng of soybean sample DNA template; 2.5 μL of FLu-Arms 2xPCR Mix; 0.075 μL of KASP upstream genotyping primer F1 (10 μM), 0.075 μL of KASP upstream genotyping primer F2 (10 μM), 0.2 μL of KASP downstream universal primer R (10 μM); make up the volume to 5 μL with water.

[0088] The PCR reaction conditions include: pre-denaturation at 95°C for 3 min, 1 cycle; denaturation at 95°C for 15 s, annealing at 63.4 - 57°C for 45 s, with a decrease of 0.8°C for each cycle, 9 cycles; denaturation at 94°C for 15 s, annealing at 57.5°C for 45 s, 45 cycles; after the cycles are completed, extend at 30°C for 30 s. After the reaction is completed, the ABI7500 real-time fluorescence quantitative PCR instrument directly reads the fluorescence data of the PCR reaction products.

[0089] Using the KASP molecular marker primers, 33 soybean materials (including 5 strongly shade-tolerant materials, 23 moderately shade-tolerant materials, and 5 extremely shade-intolerant materials) that have been identified for shade tolerance over multiple years and multiple locations were amplified and genotyped on a real-time fluorescence quantitative PCR instrument (Table 1). The results showed that: this molecular marker primer could clearly separate the three genotypes. Among them, 5 strongly shade-tolerant materials had mutations at the soySNP-1, soySNP-2, soySNP-3, and soySNP-4 loci, carried the AGCG allele variant sites, and the genotype was AGCG, accounting for 100%; among the 5 extremely shade-intolerant materials, 3 did not carry variant sites, accounting for 60.0%, with the genotype of TATT, and the other 2 had mutations at the soySNP-3 and soySNP-4 loci (TATT type was the reference genome); among the 23 moderately shade-tolerant materials, mutations occurred at both the soySNP-3 and soySNP-4 loci, carried the CG allele variant sites, and the genotype was TACG (TATT type was the reference genome); the ratio of the genotypes of the 33 materials that matched the phenotypes was 90.91%. The comparison between the genotypes and the shade tolerance phenotypes was relatively consistent, and it could be safely used in molecular marker-assisted selection breeding for soybean seedling shade tolerance.

[0090] Table 1. Names and shade tolerance of soybeans used for KASP molecular markers

[0091]

[0092]

[0093] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, the changes and advantages that those skilled in the art can think of are included in the present invention, and the appended claims are used as the protection scope.

Claims

1. A KASP marker primer set for detecting the shade tolerance of soybeans, characterized in that, The KASP markers are 4 SNP variant sites closely related to the soybean shade tolerance gene Glyma.06g213100. The 4 SNP variant sites are located at the position of 21376611_21490108 bp on chromosome 6 of the soybean genome Wm82.a2.v2.

0. Among them, at soySNP-1, a base substitution of T to A occurred at 21,485,858 bp; at soySNP-2, a base substitution of A to G occurred at 21,486,271 bp; at soySNP-3, a base substitution of A to G occurred at 21,485,456 bp; at soySNP-4, a base substitution of T to G occurred at 21,486,286 bp. The sequences of the KASP marker primer sets are as follows: The upstream primer F1 sequence of the soySNP-1 KASP marker is: 5’-GAAGGTGACCAAGTTCATGCTAGGGAGCGGCTCGAGAAA-3’, as shown in SEQ ID NO:2; The upstream primer F2 sequence of the soySNP-1 KASP marker is: 5’-GAAGGTCGGAGTCAACGGATTAGGGAGCGGCTCGAGAAT-3’, as shown in SEQ ID NO:3; The downstream primer R sequence of the soySNP-1 KASP marker is: 5’-CTTGTACGCGTTTGACCCCAAAT-3’, as shown in SEQ IDNO:

4. The upstream primer F1 sequence of the soySNP-2 KASP marker is: 5’-GAAGGTGACCAAGTTCATGCTCAATTTTACAGACGCGACTCTGG-3’, as shown in SEQ ID NO:5; The upstream primer F2 sequence of the soySNP-2 KASP marker is: 5’-GAAGGTCGGAGTCAACGGATTCAATTTTACAGACGCGACTCTGA-3’, as shown in SEQ ID NO:6; The downstream primer R sequence of the soySNP-2 KASP marker is: 5’-TAAACAAAAAGAGAAAACCTGCCG-3’, as shown in SEQ IDNO:7; The upstream primer F1 sequence of the soySNP-3 KASP marker is: 5’-GAAGGTGACCAAGTTCATGCTCGGCTTCACGTCTTCGAGC-3’, as shown in SEQ ID NO:8; The upstream primer F2 sequence of the soySNP-3 KASP marker is: 5’-GAAGGTCGGAGTCAACGGATTCGGCTTCACGTCTTCGAGT-3’, as shown in SEQ ID NO:9; The downstream primer R sequence of the soySNP-3 KASP marker is: 5’-CGGTCGGTTAATGTTAGATTTGCA-3’, as shown in SEQ IDNO:10; The upstream primer F1 sequence of the soySNP-4KASP marker is: 5’-GAAGGTGACCAAGTTCATGCTTGGTTGGGGGTTTCGCTG-3’, as shown in SEQ ID NO:11; the upstream primer F2 sequence of the soySNP-4KASP marker is: 5’-GAAGGTCGGAGTCAACGGATTTGGTTGGGGGTTTCGCTT-3’, as shown in SEQ ID NO:12; The downstream primer R sequence of the soySNP-4KASP marker is: 5’-CCCGTCACTTTCATTCGGTTTTAA-3’, as shown in SEQ ID NO:

13.

2. The primer set according to claim 1, characterized in that When the genotypes of the 4 SNP mutation sites are AGCG, the test sample is a strongly shade-tolerant soybean germplasm resource; when the genotype is TACG, the test sample is a moderately shade-tolerant soybean germplasm resource; when the test sample is TATT, the test sample is a shade-intolerant soybean germplasm resource.

3. A reagent of KASP markers for detecting the shade tolerance of soybeans, characterized in that, The reagent contains the primer set described in claim 1.

4. A kit of KASP markers for detecting the shade tolerance of soybeans, characterized in that, The kit contains the primer set described in claim 1 or contains the reagent described in claim 3.

5. Use of the primer set according to any one of claims 1 or 2, or the reagent according to claim 3, or the kit according to claim 4 in any of the following: (A1) Assisted breeding of shade-tolerant soybeans; (A2) Preparation of products for assisted breeding of shade-tolerant soybeans; (A3) Identification or assisted identification of soybean shade tolerance; (A4) Preparation of products for identification or assisted identification of soybean shade tolerance; (A5) Selection or assisted selection of shade-tolerant soybeans; (A6) Preparation of products for selection or assisted selection of shade-tolerant soybeans.

6. A method for selecting or assisting in the selection of shade-tolerant soybeans, which comprises extracting genomic DNA of soybeans, performing PCR amplification on the extracted genomic DNA using the primer set described in claim 1, and performing genotyping through the fluorescence signal of the PCR amplification product; when the genotypes of the 4 SNP mutation sites are AGCG, the test sample is a strongly shade-tolerant soybean germplasm resource; when the genotype is TACG, the test sample is a moderately shade-tolerant soybean germplasm resource; when the test sample is TATT, the test sample is a shade-intolerant soybean germplasm resource.

7. A method for identifying or assisting in the identification of soybean shade tolerance, which comprises extracting genomic DNA of soybeans, performing PCR amplification on the extracted genomic DNA using the primer set described in claim 1, and performing genotyping through the fluorescence signal of the PCR amplification product; when the genotypes of the 4 SNP mutation sites are AGCG, the test sample is a strongly shade-tolerant soybean germplasm resource; when the genotype is TACG, the test sample is a moderately shade-tolerant soybean germplasm resource; when the test sample is TATT, the test sample is a shade-intolerant soybean germplasm resource.

8. The application according to claim 5 or the method according to claim 6 or 7, characterized in that Comprising the following specific steps: (1) Extraction of genomic DNA of soybean plants; (2) Performing PCR amplification on the genomic DNA of biological samples using the primer set described in claim 1 and detecting the SNP genotypes described in claim 1: Add the primer described in Claim 1 into the same PCR reaction system, and set up 2 blank controls with ultrapure water replacing the sample template DNA, and amplify the DNA of soybean germplasm resources on a real-time fluorescence quantitative PCR instrument; After the reaction is completed, use a multifunctional microplate reader to detect the fluorescence. According to the read fluorescence, calculate the ratios of FAM / ROX and HEX / ROX respectively, and use software to draw a coordinate graph to obtain the genotype of the sample. (3) Select shade-tolerant soybean germplasm resources from different soybean germplasm resources according to the genotype.

9. The application or method according to Claim 8, characterized in that In the step (2), the 5.0 μL reaction system: 5 - 100 ng of soybean sample DNA template; 2.50 μL of FLu-Arms 2x PCR Mix; 0.075 μL of 10 μM KASP upstream genotyping primer F1, 0.075 μL of 10 μM KASP upstream genotyping primer F2, 0.20 μL of 10 μM KASP downstream universal primer R1; make up the volume to 5.0 μL with water.

10. The application or method according to Claim 8, characterized in that In the step (2), the PCR reaction conditions are as follows: pre-denaturation at 95 °C for 3 min, 1 cycle; denaturation at 95 °C for 15 s; annealing at 63.4 - 57 °C for 45 s, with a decrease of 0.8 °C for each cycle, 9 cycles; denaturation at 94 °C for 15 s; annealing at 57.5 °C for 45 s, 45 cycles; after the cycles are completed, extend at 30 °C for 30 s, 1 cycle.

Images