KASP molecular markers related to soybean alkali tolerance and application thereof

Abstract

The present application relates to the technical field of plant molecular breeding, and particularly relates to a KASP molecular marker related to soybean alkali tolerance and application thereof. The KASP molecular marker corresponds to a Chr20_25666202 site in a soybean genome, the Chr20_25666202 site is located in a gene Glyma.20G072500 interior, and a C to T nucleotide mutation exists; the alkali tolerance of a soybean with a CC genotype of the Chr20_25666202 site is significantly higher than that of a soybean with a TT genotype, and the application of the KASP molecular marker in soybean alkali tolerance identification or molecular marker assisted breeding. Advantages lie in: solving the pain points of phenotype identification being easily interfered by environment, existing genetic markers being scarce, and breeding selection efficiency being low in alkali tolerance breeding; through an allele specific fluorescent PCR technology, rapid and accurate determination of alkali tolerance genotype is realized, and individual screening of alkali tolerance can be completed at the seedling stage without long-term field stress identification.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding technology, and in particular to a KASP molecular marker related to soybean alkali tolerance and its application. Background Technology

[0002] Soybeans are an important oilseed crop and protein source globally, playing a vital role in agricultural production and the national economy. However, salinity stress is one of the major abiotic stress factors limiting soybean growth, development, and yield. Approximately 20% of global arable land is affected by salinization, and this area continues to expand due to environmental changes and improper irrigation, seriously threatening the safe production of soybeans.

[0003] Alkaline stress, a significant type of saline-alkali stress, is primarily caused by alkaline salts such as sodium bicarbonate and sodium carbonate. It leads to increased soil pH, disrupts the ion balance and pH homeostasis of plant cells, inhibits physiological processes such as root growth and photosynthesis, and ultimately results in reduced crop yield or even death. Therefore, developing new alkaline-tolerant soybean varieties is an effective way to address alkaline stress, expand soybean planting area, and increase yield.

[0004] Traditional soybean alkali tolerance breeding mainly relies on phenotypic identification. However, soybean alkali tolerance is a complex quantitative trait controlled by multiple genes, and phenotypic identification is easily affected by environmental conditions and the timing of identification, resulting in problems such as long identification cycles, low efficiency, and poor accuracy, which seriously limits the progress of alkali-tolerant soybean breeding. Molecular marker-assisted breeding (MAS) technology can directly screen based on genotypes, is not affected by environmental interference, and can significantly shorten the breeding cycle and improve selection efficiency, providing a new technical means for alkali-tolerant soybean breeding.

[0005] Genome-wide association analysis (GWAS) is an effective method for identifying genetic loci associated with complex traits. Through GWAS analysis, several quantitative trait loci (QTLs) and candidate genes associated with soybean alkali tolerance have been identified. Among them, [the following gene is missing from the original text]. Glyma.20G072500 The homologous gene of this gene is annotated as L-type lectin receptor kinase S.7 in Arabidopsis thaliana, belonging to the stress-related protein family, whose function is closely related to plant stress tolerance. Further research revealed a C-to-T single nucleotide polymorphism (SNP) at the Chr20_25666202 site within this gene, and this SNP is significantly associated with alkali tolerance in soybean, making it an important target for developing molecular markers.

[0006] Competitive allele-specific PCR (KASP) is a fluorescent signal-based SNP genotyping technique with advantages such as high specificity, high accuracy, low cost, and high throughput. It has been widely used in marker-assisted breeding of crops. However, a specific KASP marker for the Chr20_25666202 locus that can be directly applied to marker-assisted breeding for soybean alkali tolerance has not yet been developed. Therefore, developing a KASP molecular marker closely related to soybean alkali tolerance and with accurate and efficient genotyping is a pressing technical problem to be solved in the field of soybean alkali tolerance breeding. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a KASP molecular marker related to soybean alkali tolerance and its application.

[0008] The primary objective of this invention is to provide a KASP molecular marker associated with alkali tolerance in soybeans. This molecular marker corresponds to the Chr20_25666202 locus in the soybean genome, which is located in the gene... Glyma.20G072500 Internally, there is a C-to-T nucleotide mutation.

[0009] Preferably, the alkali tolerance of CC genotype soybeans at the Chr20_25666202 locus is significantly higher than that of TT genotype soybeans.

[0010] The second objective of this invention is to provide a specific primer set for detecting KASP molecular markers associated with soybean alkali tolerance. The primer set includes two upstream specific primers and one universal downstream primer, specifically recognizing the C and T alleles at the Chr20_25666202 locus. The two upstream specific primers and one universal downstream primer are as follows:

[0011] 5'-AAAGTGGCAATTCGAGTTCGTC-3'; 5'-AAAGTGGCAATTCGAGTTCGTT-3'; 5'-GATAATTGCTGGCCAAACATTC-3'.

[0012] Preferably, the 5' end of upstream specific primer 1 is labeled with a FAM fluorescent tag; the 5' end of upstream specific primer 2 is labeled with a HEX fluorescent tag.

[0013] The third objective of this invention is to provide a kit for detecting alkali tolerance in soybeans, comprising the aforementioned specific primer set; the reaction system is 20 μL, comprising: 2×KASP Master Mix, 10μL; 0.3 μL of primer mixture containing the specific primer set described above; Genomic DNA template, 2 μL; Make up the rest with sterile water to a total of 20 μL.

[0014] Preferably, the final concentration of the two upstream specific primers is 0.15 μmol / L, and the final concentration of the one universal downstream primer is 0.3 μmol / L.

[0015] The fourth objective of this invention is to provide an application of KASP molecular markers related to soybean alkali tolerance in soybean alkali tolerance identification or molecular marker-assisted breeding.

[0016] Preferably, the application includes: screening soybean plants, lines or varieties with CC homozygous genotypes by detecting the genotype of the Chr20_25666202 locus in soybean materials; In the soybean alkali resistance identification, the CC homozygous genotype corresponds to soybean materials with strong alkali resistance; In the aforementioned marker-assisted breeding, alkali-tolerant single plants from the early stages of hybridization breeding are screened, and individuals with the CC homozygous genotype are retained for breeding.

[0017] The fifth objective of this invention is to provide a rapid method for identifying the alkali tolerance of soybeans, specifically including the following steps: S1. Extract genomic DNA from the soybean material to be identified; S2. Using genomic DNA as a template, KASP-PCR amplification was performed using the specific primer set described above; S3. The fluorescence signal of the amplified product was detected using a real-time PCR instrument, and the genotype was determined according to the fluorescence signal type: only FAM fluorescence signal was detected, indicating CC homozygous genotype, corresponding to soybean material with strong alkali resistance; only HEX fluorescence signal was detected, indicating TT homozygous genotype, corresponding to soybean material with weak alkali resistance; both fluorescence signals were detected at the same time, indicating CT heterozygous genotype.

[0018] Preferably, the reaction program for KASP-PCR amplification in step S2 is as follows: pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 20 s, annealing and extension at 61~55℃ for 60 s, for a total of 10 cycles, with the annealing temperature of each cycle decreasing by 0.6℃ compared to the previous cycle; denaturation at 94℃ for 20 s, annealing and extension at 55℃ for 60 s, for a total of 30 cycles; and holding at 10℃ for 5 min.

[0019] Compared with the prior art, the present invention can achieve the following beneficial effects: (1) High specificity and high accuracy: The marker of this invention directly corresponds to the alkali tolerance-related gene. Glyma.20G072500 The functional variation sites can accurately distinguish soybean materials with different alkali tolerance genotypes, avoiding the subjectivity of phenotypic identification and environmental interference.

[0020] (2) Rapid and efficient detection: Using KASP technology, the entire process from DNA extraction to genotype identification can be completed within one day, and high-throughput detection can be achieved, making it suitable for large-scale screening of breeding materials.

[0021] (3) Simple operation and low cost: KASP reaction does not require complicated steps such as electrophoresis, and the fluorescence signal can be read directly, making the operation simple; at the same time, the primer design is highly targeted, and the reagent consumption is low, which reduces the detection cost.

[0022] (4) High application value: It can be used for early identification of soybean alkali tolerance, and alkali-tolerant materials can be screened out at the seedling stage, which significantly shortens the breeding cycle. At the same time, it can be used for molecular marker-assisted breeding of new alkali-tolerant soybean varieties, which improves the efficiency and accuracy of breeding selection and provides important technical support for the breeding of new alkali-tolerant soybean varieties.

[0023] In summary, the soybean-based [material] provided by this invention... Glyma.20G072500 The KASP molecular marker for soybean alkali tolerance, along with its specific primer set, detection methods, and applications, can directly address the core challenges in soybean alkali tolerance breeding: phenotypic identification is susceptible to environmental interference, existing genetic markers are scarce, and breeding selection efficiency is low. This molecular marker targets a functional SNP site (Chr20_25666202) validated in 326 natural soybean populations and rigorous alkaline stress experiments. It enables rapid and accurate determination of alkali tolerance genotypes using allele-specific fluorescent PCR technology, allowing for screening of alkali-tolerant individuals at the seedling stage without the need for long-term field stress assessment. It not only possesses the technical advantages of high specificity, high stability, and high throughput, but also fills the application gap for efficient molecular markers of soybean alkali tolerance traits. It provides a key tool for the precise breeding of alkali-tolerant soybean varieties, rapid evaluation of germplasm resources suitable for saline-alkali land, and molecular marker-assisted breeding, possessing broad industrial application value and significant implications for promoting agricultural development in saline-alkali land and increasing soybean production capacity. Attached Figure Description

[0024] Figure 1 This is a Manhattan plot of genome-wide association analysis (GWAS) for soybean alkali tolerance; the horizontal axis represents the chromosome numbers of soybean 1-20, and the vertical axis represents the -log10(P) value of the association test p-value. The higher the value, the stronger the association between the SNP and the alkali tolerance trait; different colored scatter points in the plot represent the associated SNP loci of different types of alkali tolerance-related traits. Among them, the specific locus on chromosome 20 that is significantly above the threshold line is the target SNP locus Chr20_25666202 (C / T mutation), which is highly significantly associated with the soybean alkali tolerance trait.

[0025] Figure 2The figure shows the QQ plot of the MLMM model GWAS under the alkali tolerance trait of soybean; the horizontal axis is the theoretically expected -log10(P) value, and the vertical axis is the actual observed -log10(P) value; the points in the figure that deviate from the theoretical line correspond to SNP sites that are significantly associated with alkali tolerance.

[0026] Figure 3 A schematic diagram of the flanking sequence and primer design for the SNP site Chr20_25666202.

[0027] Figure 4 This is a comparison of the aboveground phenotypes of soybean sensitive variety A033 and alkali-tolerant variety A052 under control (CK) and 150mM mixed alkali stress (AT). The leaves of A033 withered under stress, while A052 still grew well after stress. The scale bar on the right is 5cm. Detailed Implementation

[0028] In the following description, embodiments of the invention will be described with reference to the accompanying drawings. In the description below, the same modules are denoted by the same reference numerals. Where the same reference numerals are used, their names and functions are also the same. Therefore, their detailed description will not be repeated.

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not constitute a limitation thereof.

[0030] This invention provides a KASP molecular marker associated with alkali tolerance in soybeans. This KASP molecular marker corresponds to the Chr20_25666202 locus in the soybean genome, which is located in the gene... Glyma.20G072500 Internally, the nucleotide sequence contains a C-to-T mutation. Association analysis results show that this SNP site is significantly associated with soybean alkali tolerance, with the CC genotype soybean material exhibiting significantly higher alkali tolerance than the TT genotype material.

[0031] Provide a specific primer set for detecting this KASP molecular marker; design a specific primer set based on the flanking sequence and SNP variant type of the Chr20_25666202 site, including: (1) Upstream specific primer 1 (corresponding to C allele): 5'-FAM-AAAGTGGCAATTCGAGTTCGTC-3' (SEQ ID NO.1), with a FAM fluorescent tag at the 5' end; (2) Upstream specific primer 2 (corresponding to T allele): 5'-HEX-AAAGTGGCAATTCGAGTTCGTT-3' (SEQ ID NO.2), with a HEX fluorescent tag at the 5' end; (3) Universal downstream primer: Designed by combining conserved sequences downstream of the SNP site to ensure specific amplification of the target fragment, 5'-GATAATTGCTGGCCAAACATTC-3' (SEQ ID NO.3).

[0032] A kit containing the above-mentioned specific primer set is provided. In addition to the above-mentioned specific primer set, the kit also includes conventional reagents required for the KASP reaction, such as 2×KASP Master Mix (containing fluorescent probes, dNTPs, hot-start Taq enzyme, etc.), sterile water, etc., which can be directly used for KASP-PCR amplification reaction and are convenient for practical applications.

[0033] This document provides the application of the KASP molecular marker, primer set, and kit. The KASP molecular marker can be used for the identification of alkali tolerance in soybeans and for marker-assisted breeding of alkali tolerance soybeans. In specific applications, genomic DNA is extracted from soybean materials, and KASP-PCR amplification is performed using the aforementioned specific primer set. The genotype is determined based on the fluorescence signal type, thereby predicting the alkali tolerance of soybean materials and enabling early screening and targeted breeding of alkali-tolerant soybean materials.

[0034] A rapid method for identifying soybean alkali tolerance is provided, which specifically includes the following steps: S1. Genomic DNA extraction: Genomic DNA was extracted from the soybean material to be identified using the conventional CTAB method or a commercial DNA extraction kit, ensuring that the DNA purity and concentration met the requirements for PCR reaction (OD260 / OD280 of 1.8-2.0, concentration of approximately 50 ng / μL).

[0035] S2. KASP-PCR amplification: Using extracted genomic DNA as a template, amplification was performed using the specific primer set and kit described above. The reaction volume was 20 μL, including: 2×KASP Master Mix, 10μL; Primer mixture (final concentrations of upstream specific primer 1, upstream specific primer 2, and downstream universal primer were 0.15 μmol / L, 0.15 μmol / L, and 0.3 μmol / L, respectively), 0.3 μL; Genomic DNA template, 2 μL; Make up the rest with sterile water to a total of 20 μL.

[0036] The reaction program was as follows: pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 20 s, annealing and extension at 61-55℃ for 60 s, for a total of 10 cycles (the annealing temperature decreased by 0.6℃ in each cycle); denaturation at 94℃ for 20 s, annealing and extension at 55℃ for 60 s, for a total of 30 cycles; and holding at 10℃ for 5 min.

[0037] S3. Fluorescence Signal Detection and Genotype Determination: After amplification, the fluorescence signal of the amplified products is detected using a quantitative real-time PCR instrument. If only FAM fluorescence signal is detected, the genotype is CC homozygous, corresponding to materials with strong alkali resistance; if only HEX fluorescence signal is detected, the genotype is TT homozygous, corresponding to materials with weak alkali resistance; if both fluorescence signals are detected simultaneously, the genotype is CT heterozygous.

[0038] Example 1 The identification of KASP molecular markers associated with soybean alkali tolerance specifically includes: The experimental materials consisted of 326 soybean cultivars from different ecological regions in China. Using the Illumina HiSeq sequencing platform and MS medium, the aim was to identify functional SNPs related to soybean alkali tolerance through GWAS analysis and to develop targeted therapies. Glyma.20G072500 KASP molecular markers for genes; The experimental methods include: (1) Soybean seedlings were subjected to alkaline stress for 7 days using 150mM mixed alkaline solution (NaHCO3:Na2CO3=5:1, pH9.0±0.1), while the control group was treated with standard MS medium (pH7.0±0.1). (2) Trait determination and genome sequencing: Eight alkali-tolerant related traits, such as seedling fresh weight, dry weight, and chlorophyll content, were determined and the AT / CK ratio was calculated. At the same time, genomic DNA was extracted for whole-genome resequencing, and 3,311,166 high-quality SNPs were screened out. (3) Significantly associated SNPs were screened through GWAS analysis of 7 models, and QTLs were located based on LD attenuation distance (±71.6kb). Figure 1 , Figure 2 As shown), the final selection was made. Glyma.20G072500 As candidate genes and target SNP sites; (4) Based on the significant association between the target SNP site Chr20_25666202 (C / T mutation) and alkali tolerance, a specific primer set (SEQ ID NO.1-3) was designed for this target SNP. Figure 3 ) and perform PCR amplification and fluorescence signal detection.

[0039] The results showed that the successfully developed KASP molecular marker could accurately distinguish between the three genotypes CC, CT, and TT. Furthermore, the alkali tolerance traits of the CC genotype soybean, such as chlorophyll content and biomass, were significantly better than those of other genotypes, indicating that the marker was strongly associated with the alkali tolerance of soybean.

[0040] Example 2 The application of KASP molecular markers in the screening of alkali tolerance in soybean germplasm aims to verify the screening effect of KASP molecular markers developed based on the Chr20_25666202 locus of the Glyma.20G072500 gene on alkali tolerance of soybean germplasm resources. Details are as follows: The experimental materials consisted of 16 soybean germplasms from different sources, including alkali-tolerant and alkali-sensitive materials; The experimental methods include: (1) DNA extraction: DNA was extracted from soybean seedling leaves using the CTAB method and the concentration was adjusted to 50 ng / μL; (2) Genotype identification: KASP-PCR amplification was performed using the specific primer set developed in Example 1, and the genotypes of various types were determined by a real-time PCR instrument. (3) Phenotypic verification: All germplasm was subjected to alkaline stress treatment with 150mM mixed alkaline solution, and the alkalinity-related traits such as fresh weight, dry weight, and chlorophyll content of seedlings were measured to verify the correlation between genotype and phenotype.

[0041] The results are shown in Table 1. The KASP marker achieved an accuracy of 91.67% in screening alkali-tolerant (CC genotype) germplasm and 100% in screening sensitive (TT genotype) germplasm, indicating that it can be efficiently used for rapid evaluation of alkali tolerance in soybean germplasm resources. In Table 1, a_SPAD represents the chlorophyll content of soybean materials.

[0042] Table 1. Correspondence between KASP phenotypes and alkali tolerance phenotypes of 16 soybean varieties

[0043] Example 3 Early screening for soybean hybrid breeding is detailed below: The experimental material was the F2 generation population (46 plants) obtained by crossing the alkali-tolerant variety A052 (CC genotype) with the sensitive variety A033 (TT genotype). The experimental method is as follows: (1) Early genotype screening: DNA was extracted from the leaves of F2 generation seedlings and genotypes were identified using the KASP molecular markers of this invention to screen out CC genotype seedlings; (2) Alkali tolerance verification: Alkali stress was applied to the selected CC genotype plants, and alkali tolerance-related traits such as seedling fresh weight, dry weight, and chlorophyll content were measured. (3) Tracking genetic stability: Tracking the stability of alkali tolerance traits in F3 generation lines.

[0044] The experimental results showed that the number of plants with the CC, CT, and TT genotypes in the F2 generation was 12, 24, and 10, respectively, and the segregation ratio conformed to Mendelian inheritance law of 1:2:1. Among them, the alkali tolerance trait of individual plants with the CC genotype was not significantly different from that of the maternal parent A052. The alkali tolerance trait of the F3 generation line remained stable, indicating that this molecular marker can be effectively applied to the screening of alkali tolerance in the early generations of soybean breeding, and can shorten the breeding cycle by 2-3 generations.

[0045] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0046] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A KASP molecular marker associated with soybean alkali tolerance, characterized in that: The molecular marker corresponds to the Chr20_25666202 locus in the soybean genome, and the Chr20_25666202 locus is located in the gene... Glyma.20G072500 Internally, there is a C-to-T nucleotide mutation.

2. The KASP molecular marker related to soybean alkali tolerance according to claim 1, characterized in that: The alkali tolerance of CC genotype soybeans at the Chr20_25666202 locus is significantly higher than that of TT genotype soybeans.

3. A specific primer set for detecting the KASP molecular marker related to soybean alkali tolerance as described in claim 1, characterized in that: The primer set includes two upstream-specific primers and one universal downstream primer, specifically recognizing the C and T alleles at the Chr20_25666202 locus; the two upstream-specific primers and one universal downstream primer are as follows: 5'-AAAGTGGCAATTCGAGTTCGTC-3'; 5'-AAAGTGGCAATTCGAGTTCGTT-3'; 5'-GATAATTGCTGGCCAAACATTC-3'.

4. A specific primer set according to claim 3, characterized in that: The 5' end of upstream specific primer 1 is labeled with a FAM fluorescent tag; the 5' end of upstream specific primer 2 is labeled with a HEX fluorescent tag.

5. A reagent kit for detecting alkali tolerance in soybeans, characterized in that: Includes the specific primer set as described in claim 3; the reaction system is 20 μL, comprising: 2×KASP Master Mix, 10μL; 0.3 μL of primer mixture containing the specific primer set described above; Genomic DNA template, 2 μL; Make up the rest with sterile water to a total of 20 μL.

6. The reagent kit for detecting soybean alkali tolerance according to claim 5, characterized in that: The final concentration of the two upstream specific primers was 0.15 μmol / L, and the final concentration of the one universal downstream primer was 0.3 μmol / L.

7. The application of the KASP molecular marker related to soybean alkali tolerance as described in claim 1 in the identification of soybean alkali tolerance or molecular marker-assisted breeding.

8. The application according to claim 7, characterized in that: The application includes: screening soybean plants, lines or varieties with CC homozygous genotypes by detecting the genotype of the Chr20_25666202 locus in soybean materials; In the soybean alkali resistance identification, the CC homozygous genotype corresponds to soybean materials with strong alkali resistance; In the aforementioned marker-assisted breeding, alkali-tolerant single plants from the early stages of hybridization breeding are screened, and individuals with the CC homozygous genotype are retained for breeding.

9. A rapid method for identifying the alkali tolerance of soybeans, characterized in that: Specifically, the following steps are included: S1. Extract genomic DNA from the soybean material to be identified; S2. Using genomic DNA as a template, KASP-PCR amplification was performed using the specific primer set described in claim 3; S3. The fluorescence signal of the amplified product was detected using a real-time PCR instrument, and the genotype was determined according to the fluorescence signal type: only FAM fluorescence signal was detected, indicating CC homozygous genotype, corresponding to soybean material with strong alkali resistance; only HEX fluorescence signal was detected, indicating TT homozygous genotype, corresponding to soybean material with weak alkali resistance; both fluorescence signals were detected at the same time, indicating CT heterozygous genotype.

10. A rapid identification method for soybean alkali tolerance according to claim 9, characterized in that: The reaction program for KASP-PCR amplification in step S2 is as follows: pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 20 s, annealing and extension at 61~55℃ for 60 s, for a total of 10 cycles, with the annealing temperature of each cycle decreasing by 0.6℃ compared to the previous cycle; denaturation at 94℃ for 20 s, annealing and extension at 55℃ for 60 s, for a total of 30 cycles; and holding at 10℃ for 5 min.

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