KASP molecular marker related to soybean multi-branch character and application of KASP molecular marker

Abstract

The invention belongs to the technical field of plant molecular breeding, and particularly relates to a KASP molecular marker related to soybean multi-branch traits and application of the KASP molecular marker. The KASP molecular marker provided by the invention corresponds to two core SNP (Single Nucleotide Polymorphism) loci (Chr166095339 and Chr166096184, and the variation types of the two core SNP loci are both A / G) associated with a soybean Glyma. 16G062400 gene. Through KASP-PCR amplification and fluorescence signal detection, early accurate determination of soybean branch genotypes can be realized, and the multi-environment stability is strong. According to the method, the breeding period can be greatly shortened, the breeding cost is reduced, a key technical support is provided for breeding of a new variety of multi-branch soybeans, and the method has important industrial application value.

Description

A KASP molecular marker associated with the multibranching trait in soybean and its application Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to a KASP molecular marker related to the multibranching trait of soybean and its application. Background Technology

[0002] Branching number is a core agronomic trait affecting soybean yield. High-branching varieties can increase yield by increasing the number of pod-setting sites and are suitable for low-density, simplified cultivation, reducing planting costs, thus playing a significant role in high-yield soybean breeding. Traditional soybean branching trait breeding relies on field phenotypic identification, which has drawbacks such as a long cycle (requiring 3-5 years), significant environmental influence, and low accuracy, severely limiting breeding efficiency.

[0003] Molecular marker-assisted breeding technology can achieve early and precise screening of target genotypes, effectively overcoming the limitations of traditional breeding. Although a study has located a stable QTL (qBN16) related to branch number on soybean chromosome 16, existing molecular markers related to branch number often suffer from low resolution, unstable linkage with target traits, cumbersome detection steps, and low throughput, making it difficult to meet the needs of large-scale breeding.

[0004] KASP technology has become a preferred technology for crop molecular breeding due to its advantages of high throughput, low cost, speed and accuracy. However, there are currently no reports on KASP molecular markers developed based on the Glyma.16G062400 gene that can accurately screen multi-branched soybeans. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a KASP molecular marker, a dedicated detection primer set, and a screening method that are closely related to the multi-branching trait of soybean, so as to achieve early, rapid, and accurate identification of multi-branching soybean genotypes, solve the problems of long breeding cycles and environmental interference in phenotypic identification in traditional breeding, provide an efficient tool for soybean molecular marker-assisted breeding, and accelerate the breeding process of new multi-branching soybean varieties.

[0006] To achieve the above objectives, the present invention is implemented through the following solution:

[0007] This invention provides a KASP molecular marker associated with the multibranching trait in soybean, wherein the KASP molecular marker targets Chr16_6095339 and Chr16_6096184;

[0008] The Chr16_6095339 and Chr16_6096184 are located within the qBN16QTL region associated with the soybean Glyma.16G062400 gene;

[0009] The Chr16_6095339 is located at 6095339bp on soybean chromosome 16, and the mutation type is A / G.

[0010] The Chr16_6096184 variant is located at 6096184bp on soybean chromosome 16 and has an A / G variant type.

[0011] In this invention, the core gene Glyma.16G062400 (based on the Williams 82 reference genome Wm82.a2.v1, physical interval: 6,094,000~6,097,000 bp) is located within the stable QTL (qBN16) interval for soybean branch number, and its associated haplotype is significantly correlated with the branch number phenotype. This invention found that when both SNP loci (Chr16_6095339 and Chr16_6096184) are both G homozygous (Hap_2 haplotype), the average number of branches in soybean is ≥1.35, i.e., a multi-branched genotype; when both are A homozygous (Hap_1 haplotype), the average number of branches is ≤0.55, i.e., a low-branched genotype.

[0012] This invention provides primers for detecting the above-mentioned KASP molecular markers:

[0013] (1) First set of primers (targeting SNP1 site, 6095339bp):

[0014] The allele-specific downstream primer K-16BN-SNP1-FAM (corresponding to the A allele of SNP1) has the sequence shown in SEQ ID NO.1:

[0015] 5'-GAAGGTGACCAAGTTCATGCTAAGTAAATGTTTTGTAGTAAAATAACATAT-3',5' fluorescently labeled FAM (F1);

[0016] The allele-specific downstream primer K-16BN-SNP1-HEX (corresponding to the G allele of SNP1) has the sequence shown in SEQ ID NO.2:

[0017] 5'-GAAGGTCGGAGTCAACGGATTAAGTAAATGTTTTGTAGTAAAATAACATAC-3', 5' fluorescently labeled HEX (F2);

[0018] The universal upstream primer K-16BN-SNP1-Common (targeting the SNP1 site) has the sequence shown in SEQ ID NO.3:

[0019] 5'-AAGTAAATGTTTTTGTAGTAAAATAACATAT-3' (R1);

[0020] (2) Second set of primers (targeting SNP2 site, 6096184bp):

[0021] The allele-specific upstream primer K-16BN-SNP2-FAM (corresponding to the SNP2-A allele) has the sequence shown in SEQ ID NO.4:

[0022] 5'-GAAGGTGACCAAGTTCATGCTGAAAATGAAACGGAGGGGAAAAA-3', 5' fluorescently labeled FAM (F3);

[0023] The allele-specific upstream primer K-16BN-SNP2-HEX (corresponding to the SNP2-G allele) has the sequence shown in SEQ ID NO. 5:

[0024] 5'-GAAGGTCGGAGTCAACGGATTGAAAATGAAACGGAGGGGAAAAG-3', 5' fluorescently labeled HEX (F4);

[0025] The universal downstream primer K-16BN-SNP2-Common (targeting the SNP2 site) has the sequence shown in SEQ ID NO.6:

[0026] 5'-TACTAGTGGTGGCCCCAGGCACGTCCACT-3' (R2);

[0027] The primers were verified by BLAST comparison with the soybean genome database, and there was no risk of primer dimers or non-specific binding.

[0028] This invention provides a method for screening multi-branched soybeans, the method comprising the following steps:

[0029] (1) Fresh leaves of soybean seedlings to be tested were taken, and genomic DNA was extracted using the CTAB method. The purity and integrity of the DNA were tested to ensure OD. 260 / OD 280 The value is between 1.8 and 2.0;

[0030] (2) PCR amplification: Using the extracted genomic DNA as a template, the KASP-PCR amplification system was 2 μl: soybean sample DNA template (5 ng / μl) 1 μl, 2×Master Mix for ASPCR V1 1 μl, KASP Assay Mix (F1:F2:R1=F3:F4:R2=1:1:3) 0.02 μl;

[0031] The reaction conditions included: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 sec, annealing at 61–55℃ for 40 sec, with a decrease of 0.6℃ per cycle, for 10 cycles; and denaturation at 95℃ for 20 sec, annealing at 55℃ for 40 sec, for 30 cycles.

[0032] (3) Use a real-time PCR instrument to detect the fluorescence signal of the amplification products and screen multi-branched soybeans according to the fluorescence type;

[0033] The determination method is as follows:

[0034] When only HEX fluorescence signals are detected at Chr16_6095339 and Chr16_6096184, it indicates that both SNP sites are G homozygous, the soybean sample corresponds to a multi-branched phenotype, and the average number of branches is ≥1.35.

[0035] When only FAM fluorescence signals are detected at Chr16_6095339 and Chr16_6096184, it indicates that both SNP sites are A homozygous, and the soybean sample corresponds to a low-branching phenotype with an average number of branches ≤0.55.

[0036] When two fluorescent signals are detected simultaneously, the sample is considered to carry a heterozygous genotype and is not recommended for breeding.

[0037] The present invention provides a kit for detecting the above-mentioned KASP molecular marker, the kit comprising the above-mentioned primer set, as well as KASP Master Mix, ddH2O and positive control DNA.

[0038] This invention also provides the application of the above-mentioned KASP molecular marker, the above-mentioned primer set, any of the above-mentioned screening methods, or the above-mentioned kit in screening multi-branched soybean germplasm resources.

[0039] This invention also provides the application of the above-mentioned KASP molecular marker, the above-mentioned primer set, any of the above-mentioned screening methods, or the above-mentioned kit in marker-assisted breeding of soybean.

[0040] This invention also provides the application of the above-mentioned KASP molecular marker, the above-mentioned primer set, any of the above-mentioned screening methods, or the above-mentioned kit in the breeding of new multi-branched soybean varieties.

[0041] This invention also provides the application of the above-mentioned KASP molecular marker, the above-mentioned primer set, any of the above-mentioned screening methods, or the above-mentioned kit in the screening of multibranched traits in soybeans.

[0042] Two hundred core soybean germplasm resources, representing diverse genetic regions across my country, were selected as validation materials to ensure that the samples could represent the genetic variation characteristics of the soybean germplasm population. The branching phenotype was investigated according to the standard that "lateral branches (excluding secondary branches) have more than two nodes and more than one mature pod." Ten plants with consistent growth were selected from each material, and the average number of branches was calculated. The results showed that the average number of branches in HEX fluorescent positive materials (i.e., Hap_2 genotype with both SNP loci homozygous for G, totaling 13 accessions) was 1.35±0.12, while the average number of branches in FAM fluorescent positive materials (i.e., Hap_1 genotype with both SNP loci homozygous for A, totaling 187 accessions) was 0.55±0.08. There was a highly significant difference in branching numbers between the two genotypes (P<0.01). In summary, this molecular marker demonstrates extremely high screening accuracy. Attached Figure Description

[0043] Figure 1 is a schematic diagram of the linkage disequilibrium (LD) block of the SNP site associated with the Glyma.16G062400 gene on soybean Chr.16;

[0044] In the diagram, "Block0" represents the target LD block, and the red area within the block represents the region with high chain strength.

[0045] The labeled “Chr16_6095339” and “Chr16_6096184” are two core SNP sites that are significantly associated with soybean branch number. They are located in the same LD block, indicating that there is a strong linkage relationship between these two sites, which together constitute the haplotype block that regulates soybean branch number. This figure intuitively shows the genomic location and linkage characteristics of the target SNP sites, which is one of the core bases for the development of KASP molecular markers in this invention.

[0046] Figure 2 shows the gene locus and haplotype locus information of soybean gene Glyma.16G062400;

[0047] The genotypes of this gene at two loci, 6095339 (intergenic region) and 6096184 (intergenic region), and the base composition of the corresponding two haplotypes, Hap_1 and Hap_2, are shown.

[0048] Figure 3 is a box plot of the number of branches corresponding to the Hap_1 and Hap_2 haplotypes associated with the soybean Glyma.16G062400 gene;

[0049] The horizontal axis represents the two haplotypes (Hap_1, Hap_2), and the vertical axis represents the average number of branches in soybean.

[0050] The box bars in the figure represent the distribution range of the number of branches, the horizontal line inside the box is the median, the error bar is the range of data fluctuation, and the "a" and "b" indicate that there is a highly significant difference in the number of branches between the two haplotypes (P<0.01). The results show that the average number of branches of the Hap_1 haplotype (corresponding to the A homozygous sites Chr16_6095339 and Chr16_6096184) is about 0.55, and the average number of branches of the Hap_2 haplotype (corresponding to the G homozygous sites) is about 1.35. This intuitively reflects the significant association between the target haplotype of this invention and the soybean multi-branched phenotype, and is the phenotypic basis for the development of KASP molecular markers. Detailed Implementation

[0051] This invention aims to provide a KASP molecular marker associated with the multibranching trait in soybean and its applications. The technical solution of this invention is described in detail below with reference to the accompanying drawings, primer sequences, and specific embodiments.

[0052] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions.

[0053] Example 1

[0054] Mining and identification of target SNP sites

[0055] Twenty-one core soybean germplasm resources (including high-branching and low-branching phenotypes) covering five accumulated temperature zones in Northeast my country were selected and planted in three environments: Jiamusi, Heilongjiang (JMS17 in 2017, JMS18 in 2018) and Changchun, Jilin (JL20 in 2020). A randomized complete block design with three replicates was used. The number of branches at maturity was counted according to standard. Joint environmental analysis showed that the variances of branch number genotypes, environmental variances, and G×E interaction variances were all highly significant (P<0.0001), and the broad-sense heritability (h²) was 0.61, suitable for association analysis. Genomic DNA was extracted from soybean leaves at the three-leaf stage using the CTAB method, and resequencing (sequencing depth ≥16×) and quality control filtering were performed on the Illumina HiSeq platform to obtain 2,715,610 high-quality SNPs.

[0056] Association analysis was performed using five GWAS models with GAPIT3 software, using -log 10With a threshold of P>7.73, the SNP site Chr16_6096184 (physical location 6,096,184 bp) on Chr.16 was screened. This site was detected in both the MLMM and SUPER models, located within the stable QTL interval qBN16, and showed strong linkage (r²=0.98) with Chr16_6095339 (A / G variant) 845 bp upstream, together forming the Hap16 haplotype block. The average number of branches corresponding to the Hap_2 haplotype, where both SNPs are G homozygous, was 1.35±0.12, which was significantly higher than that of the A homozygous haplotype (Hap_1, 0.55±0.08) (P<0.01). These two were identified as the core target SNP sites (Figures 1 and 2).

[0057] Example 2

[0058] KASP primer set design and optimization based on target sites

[0059] Based on the flanking sequences of the target SNP site (referencing the Williams 82 genome), KASP primers were designed using Primer 5.0 software according to primer design principles. The specific primer set information is as follows:

[0060] (1) First set of primers (targeting SNP1 site, 6095339bp):

[0061] The allele-specific downstream primer K-16BN-SNP1-FAM (corresponding to the A allele of SNP1) has the sequence shown in SEQ ID NO.1:

[0062] 5'-GAAGGTGACCAAGTTCATGCTAAGTAAATGTTTTGTAGTAAAATAACATAT-3' (Fluorescently labeled FAM);

[0063] The allele-specific downstream primer K-16BN-SNP1-HEX (corresponding to the G allele of SNP1) has the sequence shown in SEQ ID NO.2:

[0064] 5'-GAAGGTCGGAGTCAACGGATTAAGTAAATGTTTTGTAGTAAAATAACATAC-3' (fluorescently labeled HEX);

[0065] The universal upstream primer K-16BN-SNP1-Common (targeting the SNP1 site) has the sequence shown in SEQ ID NO.3:

[0066] 5'-AAGTAAATGTTTTTGTAGTAAAATAACATAT-3';

[0067] (2) Second set of primers (targeting SNP2 site, 6096184bp):

[0068] The allele-specific upstream primer K-16BN-SNP2-FAM (corresponding to the SNP2-A allele) has the sequence shown in SEQ ID NO.4:

[0069] 5'-GAAGGTGACCAAGTTCATGCTGAAAATGAAACGGAGGGGAAAAA-3' (Fluorescently labeled FAM);

[0070] The allele-specific upstream primer K-16BN-SNP2-HEX (corresponding to the SNP2-G allele) has the sequence shown in SEQ ID NO. 5:

[0071] 5'-GAAGGTCGGAGTCAACGGATTGAAAATGAAACGGAGGGGAAAAG-3' (fluorescently labeled HEX);

[0072] The universal downstream primer K-16BN-SNP2-Common (targeting the SNP2 site) has the sequence shown in SEQ ID NO.6:

[0073] 5'-TACTAGTGGTGGCCCCAGGCACGTCCACT-3'.

[0074] The KASP-PCR amplification system suitable for the above KASP primer set optimization is a 2μl reaction system: 1μl soybean sample DNA template (5ng / μl), 1μl 2×Master Mix for ASPCR V1, and 0.02μl KASP Assay Mix (F1:F2:R1=F3:F4:R2=1:1:3).

[0075] The reaction conditions included: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 sec, annealing at 61–55°C for 40 sec, with a decrease of 0.6°C per cycle, for 10 cycles; and denaturation at 95°C for 20 sec, annealing at 55°C for 40 sec, for 30 cycles.

[0076] Example 3

[0077] Validation of the accuracy and stability of KASP molecular markers

[0078] Accuracy verification was performed on 200 core soybean germplasm resources. Detection using optimized KASP primers showed that the average number of branches in 187 FAM fluorescent positive materials (both SNPs were homozygous for A) was 0.55±0.08 (low branching), and the average number of branches in 13 HEX fluorescent positive materials (both SNPs were homozygous for G) was 1.35±0.12 (high branching). The marker and phenotype showed high consistency (Figure 3).

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A KASP molecular marker associated with the multibranching trait in soybean, characterized in that, The KASP molecular marker targets Chr16_6095339 and Chr16_6096184; Chr16_6095339 and Chr16_6096184 are located within the qBN16QTL region associated with the soybean Glyma.16G062400 gene; Chr16_6095339 is located at 6095339 bp on soybean chromosome 16, with a variant type of A / G; Chr16_6096184 is located at 6096184 bp on soybean chromosome 16, with a variant type of A / G.

2. Primers for detecting the KASP molecular marker of claim 1, characterized in that, The primers for detecting Chr16_6095339 include: upstream specific primer 1 with the nucleotide sequence shown in SEQ ID NO. 1; upstream specific primer 2 with the nucleotide sequence shown in SEQ ID NO. 2; and downstream universal primer 1 with the nucleotide sequence shown in SEQ ID NO.

3. The primers for detecting Chr16_6096184 include: upstream specific primer 3 with the nucleotide sequence shown in SEQ ID NO. 4; upstream specific primer 4 with the nucleotide sequence shown in SEQ ID NO. 5; and downstream universal primer 2 with the nucleotide sequence shown in SEQ ID NO.

6.

3. A method for screening multi-branched soybeans, characterized in that, The method includes the following steps: (1) extracting genomic DNA from the soybean sample to be tested; (2) using the genomic DNA as a template, performing KASP-PCR amplification using the primer set described in claim 2; (3) using a fluorescence quantitative PCR instrument to detect the fluorescence signal of the amplification product, and screening multi-branched soybeans according to the fluorescence type.

4. The screening method according to claim 3, characterized in that, The reaction program for KASP-PCR amplification in step (2) is as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s, annealing at 61℃~55℃ for 40 s, decreasing by 0.6℃ for each cycle, for a total of 10 cycles; denaturation at 95℃ for 20 s, annealing at 55℃ for 40 s, for a total of 30 cycles.

5. The screening method according to claim 3, characterized in that, The judgment method described in step (3) is as follows: when only HEX fluorescence signal is detected at Chr16_6095339 and Chr16_6096184, it indicates that both SNP sites are G homozygous, the soybean sample corresponds to a multi-branched phenotype, and the average number of branches is ≥1.35; when only FAM fluorescence signal is detected at Chr16_6095339 and Chr16_6096184, it indicates that both SNP sites are A homozygous, the soybean sample corresponds to a low-branched phenotype, and the average number of branches is ≤0.

55.

6. A kit for detecting the KASP molecular marker of claim 1, characterized in that, The kit includes the primer set as described in claim 2.

7. The application of the KASP molecular marker of claim 1, the primer set of claim 2, the screening method of any one of claims 3 to 5, or the kit of claim 6 in screening multi-branched soybean germplasm resources.

8. The application of the KASP molecular marker of claim 1, the primer set of claim 2, the screening method of any one of claims 3 to 5, or the kit of claim 6 in soybean molecular marker-assisted breeding.

9. The application of the KASP molecular marker of claim 1, the primer set of claim 2, the screening method of any one of claims 3 to 5, or the kit of claim 6 in the breeding of new multi-branched soybean varieties.

10. The application of the KASP molecular marker of claim 1, the primer set of claim 2, the screening method of any one of claims 3 to 5, or the kit of claim 6 in the screening of soybean multibranching traits.

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