Single nucleotide mutation site SNP and KASP markers significantly associated with soybean plant height and application thereof

Abstract

The invention discloses a single nucleotide mutation site SNP (Single Nucleotide Polymorphism) and KASP (Kinase Associated Specific Protein) marker remarkably associated with soybean plant height and application thereof. The SNP molecular marker is located at the 36108741bp position of the fifth chromosome of the soybean, the basic group is T or C, the SNP molecular marker is significantly related to the plant height phenotype of the soybean, and the plant height of the soybean variety with the locus genotype of TT is significantly lower than that of the soybean variety with the genotype of CC; three KASP primers are developed according to the SNP site and are respectively SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, PCR amplification and genetic typing are carried out 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, it is judged that the plant height of the soybean variety is relatively low; if the detection result is C, it is judged that the plant height is high. The SNP molecular marker disclosed by the invention can be used as an auxiliary selection marker for the plant height character in the soybean breeding process, so that the selection accuracy is improved, and the soybean plant height character related breeding process 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 significantly associated with soybean plant height, a KASP marker and an application thereof, which can be used for early molecular assisted selection of soybean plant height traits to improve breeding efficiency. Background Art

[0002] As a key oil crop, food protein source and industrial material, soybean is widely cultivated around the world. China is both the origin of soybean and a major consumer market, but its soybean yield per unit area is lower than that of some major producing countries. Therefore, it is of great significance to increase soybean yield.

[0003] Traditional soybean breeding is based on the selection of individual plants according to the height of the offspring. This method is not only time-consuming and labor-intensive, but also susceptible to environmental interference, resulting in low accuracy. Using the base differences in the target gene to develop specific molecular markers for auxiliary selection is the best way to improve the efficiency of soybean plant height selection. Molecular markers have the advantages of early selection, no environmental influence, and accuracy, rapidity, and efficiency in crop breeding, and have become an accurate and efficient tool. Among them, competitive allele-specific PCR (Kompetitive Allele-Specific PCR, KASP) molecular markers are a new SNP typing method based on allele-specific amplification (Amplification Refractory Mutation System, ARMS) and highly sensitive fluorescence detection. The principle is to design two forward primers and a universal reverse primer for the allele SNP site. Each forward primer has a specific sequence that can bind to different fluorescent markers. The forward primer with different fluorescent binding sequences and the universal reverse primer PCR amplify the DNA of the sample, and its allelic variation can be reflected by different fluorescent signals.

[0004] Studies have shown that plant height is an important factor in the formation of soybean yield traits and is a complex trait controlled by multiple quantitative trait loci (QTL). In the Soybean Genomics and Molecular Biology Database (SoyBase, https: / / soybase.org / ), more than 250 QTLs related to plant height have been reported, covering 20 chromosomes of the soybean genome. These QTLs are mainly obtained through linkage analysis or GWAS methods. Single nucleotide polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. As an effective gene positioning tool, genome-wide association study (GWAS) can quickly and accurately mine SNPs that are significantly associated with soybean plant height. Therefore, based on the identified SNPs that are significantly associated with soybean plant height, the development of KASP markers that are closely linked to soybean plant height for early breeding (low generation) selection has a significant effect on reducing breeding workload and accelerating breeding progress, and has obvious economic benefits. Based on the discovery of SNPs that are significantly associated with soybean plant height, it is particularly important to develop KASP molecular markers for assisted breeding and achieve early molecular-assisted selection of target traits to improve breeding efficiency. Summary of the invention

[0005] The purpose of the present invention is to identify single nucleotide mutation sites (SNPs) significantly associated with soybean plant height, and develop KASP molecular markers and primer pairs based on the SNP site information to provide molecular assisted selection technology support for early identification and screening of this trait.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] As a first aspect, a SNP molecular marker related to soybean plant height traits is provided. The SNP S05_36108741 significantly associated with soybean plant height is located at the 36108741bp position of chromosome 5 of the soybean genome (version number is Glycine max Wm82.a2.v1), and a substitution of base T to C occurs. The phenotype corresponding to the TT genotype is a soybean variety with shorter plant height, and the phenotype corresponding to the CC genotype is a soybean variety with taller plant height.

[0008] As a second aspect, the application of the above-mentioned SNP molecular markers related to soybean plant height traits in identifying soybean plant height traits is provided.

[0009] The application is specifically: detecting the base type of the SNP molecular marker related to the soybean plant height trait in the soybean sample to be tested. If the detection result shows that the base type here is T, it is determined that the soybean variety has a shorter plant height; if the detection result is C, it is determined that the soybean variety has a taller plant height.

[0010] As a third aspect, the application of the above-mentioned SNP molecular markers related to soybean plant height traits in genetic breeding is provided.

[0011] As a fourth aspect, a KASP-specific primer for the SNP molecular marker related to soybean plant height trait is provided, which respectively comprises two specific primers designed for base differences at key sites, namely upstream primer F1 (SEQ ID NO.1) and upstream primer F2 (SEQ ID NO.2), and a universal primer, namely downstream primer R (SEQ ID NO.3). The 3' ends of the two specific primers are allelic variant bases, and the 5' ends are connected to the FAM and HEX fluorescent linker sequences specific to the KASP reaction reagent of Chengdu Hanchen Guangyi Bioengineering Co., Ltd.

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

[0013] 5'-GAAGGTGACCAAGTTCATGCTAGCATATTATGTCAACCCCATCCT-3' (SEQ ID NO. 1);

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

[0015] 5'-GAAGGTCGGAGTCAACGGATTAGCATATTATGTCAACCCCATCCC-3' (SEQ ID NO. 2);

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

[0017] 5'-CCAATTTTACATCTATGGCATGGCT-3' (SEQ ID NO. 3).

[0018] When synthesizing the KASP molecular marker primers, the 5' end of the forward primer F1 is added with a fluorescent signal label of carboxyfluorescein FAM (the first 21 positions of the primer sequence); the 5' end of the forward primer F2 is added with a fluorescent signal label of hexachlorofluorescein phosphoramidate HEX (the first 21 positions of the primer sequence).

[0019] As a fifth aspect, the application of the above-mentioned KASP specific primers in identifying soybean plant height traits is provided. The KASP molecular marker for the nucleotide mutation site SNP significantly associated with soybean plant height is used in the identification or auxiliary screening method to detect whether the genotype of the deoxyribonucleotide at the 36108741bp position of soybean chromosome 5 is TT or CC, the TT genotype indicates a shorter plant height, and the CC genotype indicates a taller plant height, and the molecular marker assists the genetic improvement of the soybean plant height trait.

[0020] In the above method, the KASP primer set consists of upstream primer F1, upstream primer F2 and downstream primer R. A 384-well plate PCR system was constructed using a Matrix Arrayer 3250 reaction plate preparation instrument, and PCR amplification was performed using a Matrix Cycler 2010 high-throughput water bath thermal cycler. After the reaction was completed, fluorescence scanning was performed on a Matrix Scanner 2100 high-speed fluorescence scanner, and genotyping was performed using the matching Matrix Master software; if the typing was insufficient, amplification was continued, and the typing was checked every 5 cycles until the typing was complete.

[0021] The specific steps are as follows:

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

[0023] (2) using the PCR specific amplification primers to perform PCR amplification on the genomic DNA of the biological sample to obtain an amplified product fragment; performing KASP genotyping detection on the PCR amplified product fragment; if the detection result shows that the base type here is T, it is determined that the soybean variety has a shorter plant height; if the detection result is C, it is determined that the soybean variety has a taller plant height:

[0024] The molecular marker primers are added into the same PCR reaction system, and a blank control is set up in which ultrapure water is used instead of sample template DNA, and the DNA of soybean germplasm resources is amplified on the Gene Matrix high-throughput genotyping system;

[0025] 2μl reaction system: soybean sample DNA template, 5ng / μl, 1μl; 2x Master Mix for ASPCR V1 1μl; KASPAssay Mix, F1:F2:R=1:1:3, 0.02μl. Reaction conditions include 95℃ pre-denaturation for 10min; 95℃ denaturation for 20sec, 61~55℃ annealing for 40sec, 0.6℃ lowering for each cycle, 10 cycles; 95℃ denaturation for 20sec, 55℃ annealing for 40sec, 30 cycles.

[0026] After the reaction was completed, fluorescence scanning was performed on the Matrix Scanner 2100 high-speed fluorescence scanner, and genotyping was performed using the accompanying Matrix Master software. The molecular marker primers can clearly separate the two genotypes, where the red dots close to the X-axis are T allele variant sites with a genotype of TT; the blue dots close to the Y-axis are C allele variant sites with a genotype of CC.

[0027] (3) Selecting soybean plants or strains of desired plant height in different separation generations based on genotype.

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

[0029] (1) The SNP significantly associated with soybean plant height, S05_36108741, of the present invention is derived from the whole genome resequencing information and related GWAS sites of 270 cultivated soybean varieties. The SNP site S05_36108741 significantly associated with soybean plant height can be detected, located at the 36108741bp position of chromosome 5 of the soybean genome (version number is Glycine max Wm82.a2.v1). Select TT or CC genotype soybean low-generation breeding materials as needed to provide technical support for molecular marker-assisted breeding of soybean plant height traits.

[0030] (2) The present invention identifies a SNP site on soybean chromosome 5 that controls soybean plant height. The developed KASP molecular marker can directly and specifically distinguish and detect the T or C base of the SNP mutation site. The KASP molecular marker has good application value and can realize the pre-selection of soybean plant height traits and molecular-assisted breeding.

[0031] (3) KASP molecular marker primers were used to amplify and genotype 185 soybean materials on the Gene Matrix high-throughput genotyping system. The results showed that the molecular marker primers could clearly separate the two genotypes. The red dots near the X-axis were T allele variant sites, genotype TT, 160 of which had an average plant height of 82.64 cm; the blue dots near the Y-axis were C allele variant sites, genotype CC, 25 of which had an average plant height of 88.82 cm; the dots near the origin of the XY axis were blank controls. The plant heights of the two genotypes of soybean materials were statistically significant, indicating that the markers described in the present invention are practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The Manhattan and QQ-plot diagrams of the association analysis results between 207 SNP loci genotypes and plant height phenotypes in soybean; (a) is the Manhattan diagram of the association analysis results, and (b) is the QQ-plot diagram of the association analysis results;

[0033] Figure 2 The figures are the genotyping results of different soybean varieties using KASP markers; (a) is the genotyping result of soybean materials SPBX001-SPBX093 (excluding SPBX052), and (b) is the genotyping result of soybean materials SPBX097-SPBX189; the black squares close to the origin represent blank controls without template DNA; the blue dots close to the Y-axis and the red dots close to the X-axis represent soybean varieties carrying C allele variation sites and soybean varieties carrying T allele variation sites, respectively. DETAILED DESCRIPTION

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

[0035] The terminology used in the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0036] As used in this application and the appended claims, the singular forms "a", "an", "said", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and encompasses 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 the present application to describe various information, these information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present 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 "at the time of" or "when" or "in response to determining".

[0038] The present invention is further described below with reference to the accompanying drawings and embodiments. The methods used are conventional methods unless otherwise specified.

[0039] Example 1: Obtaining nucleotide mutation sites (SNPs) significantly associated with soybean plant height

[0040] The SNP significantly associated with soybean plant height in the present invention, S05_36108741, is derived from whole genome resequencing information and related GWAS sites of 270 cultivated soybean varieties, and the method for obtaining the SNP includes the following steps:

[0041] (1) Sample collection and acquisition: 270 core soybean germplasm resources were sown in the experimental field of Northeast Institute of Geography and Agricultural Ecology, Chinese Academy of Sciences in 2018 and managed normally; and the plant height values ​​were obtained. Plant height measurement: The length from the ground to the top growth point of the main stem was measured.

[0042] (2) SNP detection: Young soybean leaves at the V4 stage were taken and high-quality soybean genomic DNA was extracted using the CTAB method. The DNA was used for genome resequencing. The amount of high-quality clean data obtained by sequencing was 8100Gb, with an average of 30G per sample and a sequencing depth of about 30 times. The sequencing data was aligned to the soybean reference genome (version number: Glycinemax Wm82.a2.v1) using BWA software, duplicates were removed using PICARD software, and high-quality SNPs were obtained using GATK software. The SNP detection results were annotated using ANNORVAR software.

[0043] (3) Whole-genome association analysis: A whole-genome association analysis was performed on the obtained SNP marker loci and the measured plant height phenotype information. The analysis software was TASSEL, and a mixed linear model was used for analysis.

[0044] (4) Acquisition of 20K loci: The acquisition of 20K marker loci requires that among 270 cultivated soybeans, the genotype missing rate is less than 20%, the heterozygous genotype rate is less than 30%, the maximum allele frequency is less than 95%, and the minimum allele frequency is greater than 5%. GWAS marker loci are selected first, followed by markers in the gene coding region, and one marker is selected every 25 kb. If there are no two markers in a continuous 75 kb, non-coding region markers are selected. Finally, the 20,648 SNP marker loci were obtained, of which 17,588 markers were located on functional genes, covering 31% of soybean coding genes. The SNP molecular markers were evenly distributed, and the average marker spacing was 46 kb.

[0045] (5) Obtaining 207 SNP sites evenly distributed on soybean chromosomes: 61 SNP sites were selected from published literature or patents, and 146 SNP sites were selected from 20K sites. A total of 207 SNP sites can be evenly distributed on soybean chromosomes, and the physical position distance between the sites is about 5 Mbp.

[0046] (6) Gene-phenotype association analysis: The genotypes of 207 SNP loci were analyzed for association with plant height phenotypes using TASSEL software and a mixed linear model. The SNP locus S05_36108741, which was significantly associated with soybean plant height, was detected and located at 36108741 bp on chromosome 5 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 S05_36108741 site, namely, upstream primer F1 (SEQ ID NO.1), upstream primer F2 (SEQ ID NO.2) and downstream primer R (SEQ ID NO.3), wherein F1 and F2 respectively contain FAM and HEX fluorescent linker sequences (the first 21 positions of the sequence), and the sequences are as follows:

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

[0050] 5'-GAAGGTGACCAAGTTCATGCTAGCATATTATGTCAACCCCATCCT-3' (SEQ ID NO. 1);

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

[0052] 5'-GAAGGTCGGAGTCAACGGATTAGCATATTATGTCAACCCCATCCC-3' (SEQ ID NO. 2);

[0053] KASP marker downstream primer R 5'-CCAATTTTACATCTATGGCATGGCT-3' (SEQ ID NO. 3).

[0054] Example 3: Detection of genotypes of SNP sites of 185 soybean varieties and their application

[0055] Genomic DNA of 185 soybean samples of different varieties were extracted respectively, and the genomic DNA was used as a template and KASP marker special primers were used to amplify the DNA of soybean samples on the Gene Matrix high-throughput genotyping system. The amplification system was a 2μl reaction system: soybean sample DNA template, 5ng / μl, 1μl; 2x Master Mix for ASPCR V11μl; KASPAssay Mix, F1:F2:R=1:1:3, 0.02μl. The reaction conditions included 95℃ pre-denaturation for 10min; 95℃ denaturation for 20sec, 61~55℃ annealing for 40sec, reducing 0.6℃ for each cycle, 10 cycles; 95℃ denaturation for 20sec, 55℃ annealing for 40sec, 30 cycles.

[0056] After the reaction was completed, fluorescence scanning was performed on a Matrix Scanner 2100 high-speed fluorescence scanner, and genotyping was performed using the accompanying Matrix Master software. The results are shown in Figure 2. KASP molecular marker primers were used to amplify and genotype 185 soybean materials. The results showed that the molecular marker primers could clearly separate the two genotypes. The blue dots close to the Y axis were C allele variant sites, with 25 samples of genotype CC and an average plant height of 88.82 cm. The red dots close to the X axis were T allele variant sites, with 160 samples of genotype TT and an average plant height of 82.64 cm (Tables 1 and 2). The dots close to the origin of the XY axis were blank controls ( Figure 2 ). The plant heights of the two genotypes of soybean materials were statistically significant (Table 2), indicating that the markers of the present invention are practical.

[0057] Table 1. Plant height of 185 different soybean varieties

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] Table 2. Comparison of mean values ​​among 185 soybean varieties

[0064]

[0065] The embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent 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 technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

[0066] The above are only optional embodiments of the present disclosure and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A SNP molecular marker related to soybean plant height trait, characterized in that: The SNP molecular marker is located at the 36108741bp position of chromosome 5 of the soybean genome, and the polymorphism is T or C. The phenotype corresponding to the TT genotype is a soybean variety with a shorter plant height, and the phenotype corresponding to the CC genotype is a soybean variety with a taller plant height.

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

3. Use of the SNP molecular marker associated with soybean plant height trait according to claim 1 in identifying soybean plant height 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 plant height 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 shorter plant height; if the test result is C, it is determined that the soybean variety has a taller plant height.

5. Use of the SNP molecular marker associated with soybean plant height trait according to claim 1 in genetic breeding.

6. Use of the KASP specific primers according to claim 2 in identifying soybean plant height traits.

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 plant height 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 soybean variety has a short plant height; if the test result is C, it is judged that the soybean variety has a tall plant height.

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