Single nucleotide mutation site snp, kasp marker significantly associated with soybean plant height and application thereof

Abstract

The application discloses a single nucleotide mutation site SNP significantly related to soybean plant height, a KASP marker and application thereof. The SNP molecular marker is located at the position of 36108741 bp of the 5th chromosome of soybean, and is significantly related to the soybean plant height phenotype, and the plant height of the soybean variety with the TT genotype is significantly lower than that of the soybean variety with the CC genotype. 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. The primers are used for PCR amplification and genotyping of the soybean to be tested. If the detection result shows that the base type at the marker position is T, it is determined that the soybean variety has a short plant height. If the detection result is C, it is determined that the plant height is high. The SNP molecular marker can be used as an auxiliary selection marker of the plant height trait in the soybean breeding process, improves the selection accuracy, and speeds up the breeding process related to the soybean plant height trait.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics breeding and provides a single nucleotide mutation site (SNP) and KASP marker that are significantly associated with soybean plant height and their application. These markers can be used for early molecular-assisted selection of soybean plant height traits to improve breeding efficiency. Background Technology

[0002] Soybeans are widely cultivated globally as a key oilseed crop, food protein source, and industrial material. China is both the origin of soybeans and a major consumer market; however, its soybean yield per unit area is lower than that of some major producing countries. Therefore, increasing soybean production is of great significance.

[0003] Traditional soybean breeding relies on individual plant selection based on the height of the offspring. This method is not only time-consuming and labor-intensive but also susceptible to environmental interference, leading to low accuracy. Developing specific molecular markers to assist selection by utilizing base differences in target genes is the best way to improve the efficiency of soybean plant height selection. Molecular markers have advantages in crop breeding, including early selection, immunity to environmental influences, accuracy, speed, and efficiency, making them an accurate and efficient tool. Among these, Kompetitive Allele-Specific PCR (KASP) is a novel SNP genotyping method based on Amplification Refractory Mutation System (ARMS) and highly sensitive fluorescence detection. Its principle involves designing two forward primers and one universal reverse primer targeting allele SNP sites. Each forward primer has a specific sequence that can bind to different fluorescent markers. PCR amplification of the sample DNA using forward primers with different fluorescent binding sequences and the universal reverse primer reveals allelic variations reflected by different fluorescence signals.

[0004] Studies have shown that plant height is a crucial factor in soybean yield trait formation and is a complex trait controlled by multiple quantitative trait loci (QTLs). The SoyBase (https: / / soybase.org / ) database of soybean genomics and molecular biology has reported over 250 plant height-related QTLs, covering 20 chromosomes of the soybean genome. These QTLs were primarily obtained through linkage analysis or GWAS methods. Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. Genome-wide association studies (GWAS), as an effective gene mapping tool, can rapidly and accurately identify SNPs significantly associated with soybean plant height. Therefore, based on the identified SNPs significantly associated with soybean plant height, developing KASP markers closely linked to soybean plant height for early (lower generations) selection has a significant effect on reducing breeding workload and accelerating breeding progress, while also yielding significant economic benefits. Based on the discovery of SNPs that are significantly associated with soybean plant height, KASP molecular markers were developed for assisted breeding to achieve early molecular-assisted selection of target traits, which is of great importance to improve breeding efficiency. Summary of the Invention

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

[0006] The objective of this invention can be achieved through the following technical solutions:

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

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

[0009] The specific application involves 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 is T, the soybean variety is determined to have a shorter plant height; if the detection result is C, the soybean variety is determined to have a taller plant height.

[0010] Thirdly, the application of the aforementioned SNP molecular markers related to soybean plant height in genetic breeding is provided.

[0011] As a fourth aspect, a KASP-specific primer for the soybean plant height-related SNP molecular marker is provided, comprising 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 one 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 specific FAM and HEX fluorescent linker sequences of the KASP reaction reagent from Chengdu Hanchen Guangyi Biotechnology Co., Ltd.

[0012] KASP tags the upstream primer F1 sequence as follows:

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

[0014] KASP tags the upstream primer F2 sequence as follows:

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

[0016] The KASP-tagged downstream primer R sequence is as follows:

[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 tagged with a fluorescent signal tag of carboxyfluorescein FAM (first 21 positions of the primer sequence); the 5' end of the forward primer F2 is tagged with a fluorescent signal tag of hexachlorofluorescein aminophosphate HEX (first 21 positions of the primer sequence).

[0019] Fifthly, the application of the aforementioned KASP-specific primers in identifying soybean plant height is provided. The KASP molecular marker targeting nucleotide mutation sites (SNPs) significantly associated with soybean plant height is used for identification or screening by detecting whether the genotype of the deoxyribonucleotide at position 36108741 bp on soybean chromosome 5 is TT or CC. The TT genotype indicates shorter plant height, while the CC genotype indicates taller plant height. Molecular markers assist in the genetic improvement of soybean plant height.

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

[0021] The specific steps are as follows:

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

[0023] (2) The genomic DNA of the biological sample was amplified by PCR using the PCR-specific primers described above to obtain the amplified product fragment; the PCR amplified product fragment was subjected to KASP genotyping detection. If the detection result showed that the base type was T, the soybean variety was determined to be short; if the detection result was C, the soybean variety was determined to be tall.

[0024] The molecular marker primers were added to the same PCR reaction system, and a blank control was set up with ultrapure water instead of sample template DNA. The DNA of soybean germplasm resources was amplified on the Gene Matrix high-throughput genotyping system.

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

[0026] 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 molecular marker primers could clearly separate the two genotypes. The red dots near the X-axis indicated the T allele variation site, with the genotype being TT; the blue dots near the Y-axis indicated the C allele variation site, with the genotype being CC.

[0027] (3) Select the desired soybean plant height or lineage based on the genotype in different segregating generations.

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

[0029] (1) The SNP significantly associated with soybean plant height in this invention, S05_36108741, is derived from the whole-genome resequencing information and related GWAS loci of 270 cultivated soybean varieties. The SNP locus S05_36108741, significantly associated with soybean plant height, can be detected, located at 36108741 bp on chromosome 5 of the soybean genome (version number Glycine max Wm82.a2.v1). Based on the desired selection of TT or CC genotype soybean low-generation breeding materials, this provides technical support for marker-assisted breeding of soybean plant height.

[0030] (2) This invention identifies a SNP site on soybean chromosome 5 that controls soybean plant height. The developed KASP molecular marker can directly distinguish and detect the T or C bases 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) Using KASP molecular marker primers, 185 soybean accessions were amplified and genotyped 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 indicated the presence of the T allele, corresponding to the TT genotype (160 accessions), with an average plant height of 82.64 cm. The blue dots near the Y-axis indicated the presence of the C allele, corresponding to the CC genotype (25 accessions), with an average plant height of 88.82 cm. The dots near the origin of the XY axes served as the blank control. The plant height of the two genotypes of soybean materials was statistically significant, indicating that the markers described in this invention are practical. Attached Figure Description

[0032] Figure 1 These are Manhattan and QQ-plots showing the association analysis results between genotypes and plant height phenotypes at 207 SNP loci in soybean; (a) is the Manhattan plot of the association analysis results, and (b) is the QQ-plot of the association analysis results.

[0033] Figure 2 This is a graph showing the genotyping results of different soybean varieties using KASP markers; (a) shows the genotyping results of soybean materials SPBX001-SPBX093 (excluding SPBX052), and (b) shows the genotyping results of soybean materials SPBX097-SPBX189; the black squares near the origin represent blank controls without template DNA; the blue dots near the Y-axis and the red dots near the X-axis represent soybean varieties carrying C allele variants and soybean varieties carrying T allele variants, respectively. Detailed Implementation

[0034] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of methods consistent with some aspects of this application as detailed in the appended claims.

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

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

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

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

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

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

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

[0042] (2) SNP detection: Young soybean leaves at stage V4 were collected, and high-quality soybean genomic DNA was extracted using the CTAB method. This DNA was used for genome resequencing, and high-quality clean data of 8100Gb was obtained, with an average of 30G per sample and a sequencing depth of approximately 30-fold. The sequencing data were 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) Genome-wide association analysis: Genome-wide association analysis was performed on the obtained SNP marker sites and the measured plant height phenotype information. The analysis software was TASSEL, and a mixed linear model was used for the analysis.

[0044] (4) Acquisition of 20K sites: The requirements for acquiring 20K marker sites are that in 270 cultivated soybeans, the genotype deletion 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 sites are preferred, followed by markers in the gene coding region. One marker is selected for every 25kb. If there are no markers of the above two types for 75kb consecutively, a marker in the non-coding region is selected. Finally, 20,648 SNP marker sites were obtained, of which 17,588 markers are located on functional genes, covering 31% of soybean coding genes. The SNP molecular markers are evenly distributed, and the average marker spacing is 46kb.

[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, with a physical distance of about 5 Mbp between sites.

[0046] (6) Gene-phenotype association analysis: The genotypes of 207 SNP loci were associated with the plant height phenotype. The analysis software was TASSEL, and a mixed linear model was used for the analysis. The SNP locus S05_36108741, which is significantly associated with soybean plant height, was detected. It is located at 36108741 bp on chromosome 5 of the soybean genome (version number Glycine max Wm82.a2.v1).

[0047] Example 2: Development of KASP-labeled 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: upstream primer F1 (SEQ ID NO.1), upstream primer F2 (SEQ ID NO.2), and downstream primer R (SEQ ID NO.3). F1 and F2 contain FAM and HEX fluorescent linker sequences (the first 21 positions of the sequence), respectively, as shown below:

[0049] KASP tags the upstream primer F1 sequence as follows:

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

[0051] KASP tags the upstream primer F2 sequence as follows:

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

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

[0054] Example 3: Genotyping of SNP loci in 185 different soybean varieties and its application

[0055] Genomic DNA was extracted from 185 soybean samples of different varieties. Using the genomic DNA as a template, the DNA from the soybean samples was amplified using KASP-labeled primers on the Gene Matrix high-throughput genotyping system. The amplification system consisted of 2 μl reaction volumes: soybean sample DNA template, 5 ng / μl, 1 μl; 2x Master Mix for ASPCR V, 1 μl; KASPassay Mix, F1:F2:R = 1:1:3, 0.02 μl. Reaction conditions included: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 sec, annealing at 61–55℃ for 40 sec, decreasing by 0.6℃ per cycle, for 10 cycles; and 95℃ denaturation for 20 sec, annealing at 55℃ for 40 sec, for 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 as follows: Figure 2185 soybean accessions were amplified and genotyped using KASP molecular marker primers. The results showed that the primers clearly separated the two genotypes. The blue dots near the Y-axis indicated C alleles, corresponding to the CC genotype (25 accessions, average plant height 88.82 cm); the red dots near the X-axis indicated T alleles, corresponding to the TT genotype (160 accessions, average plant height 82.64 cm) (Tables 1 & 2); the dots near the origin of the X and Y axes represented the blank control. Figure 2 The plant height of the two soybean genotypes was statistically significant (Table 2), indicating that the markers described in this 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 variety groups

[0064]

[0065] The various embodiments of this disclosure have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

[0066] The above are merely optional embodiments of this disclosure and are not intended to limit this disclosure. Various modifications and variations can be made to this disclosure by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure.

Claims

1. The use of a SNP molecular marker related to soybean plant height trait in identifying soybean plant height trait, characterized in that, The SNP molecular marker is located at the position of 36108741bp of chromosome 5 of Glycine max Wm82.a2.v1, the polymorphism is T or C, the phenotype corresponding to the TT genotype is a soybean variety with lower plant height, and the phenotype corresponding to the CC genotype is a soybean variety with higher plant height.

2. Use according to claim 1, characterized in that, The application is specifically: The base type of the SNP molecular marker related to the soybean plant height trait in the to-be-tested soybean sample is detected, if the detection result shows that the base type at this position is T, it is determined that the soybean variety has lower plant height, and if the detection result is C, it is determined that the soybean variety has higher plant height.

3. The use of a SNP molecular marker related to soybean plant height trait in the genetic breeding of soybean plant height trait, characterized in that, The SNP molecular marker is located at the position of 36108741bp of chromosome 5 of Glycine max Wm82.a2.v1, the polymorphism is T or C, the phenotype corresponding to the TT genotype is a soybean variety with lower plant height, and the phenotype corresponding to the CC genotype is a soybean variety with higher plant height.

4. Use of KASP specific primers of a SNP molecular marker associated with soybean plant height trait in identifying soybean plant height trait, characterized in that, The primer comprises: The upstream primer F1 has the nucleotide sequence shown in SEQ ID NO. 1: 5'-GAAGGTGACCAAGTTCATGCTAGCATATTATGTCAACCCCATCCT-3'; The upstream primer F2 has the nucleotide sequence shown in SEQ ID NO. 2: 5'-GAAGGTCGGAGTCAACGGATTAGCATATTATGTCAACCCCATCCC-3'; The downstream primer R has the nucleotide sequence shown in SEQ ID NO. 3: 5'-CCAATTTTACATCTATGGCATGGCT-3'.

5. Use according to claim 4, characterized in that, The method comprises the following steps: (1) extracting the genomic DNA of the to-be-tested soybean sample; (2) using the genomic DNA of the to-be-tested soybean sample as a template, performing a PCR amplification reaction by using the KASP-specific primer of the SNP molecular marker related to the soybean plant height trait to obtain an amplified product fragment; (3) performing KASP genotyping detection on the PCR amplified product fragment, if the detection result shows that the base type at this position is T, it is determined that the soybean variety has lower plant height, and if the detection result is C, it is determined that the soybean variety has higher plant height.

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