KASP molecular marker of wheat stripe rust resistant gene and application of KASP molecular marker

Abstract

The invention relates to the technical field of molecular breeding and phytopathology, in particular to a KASP molecular marker of a wheat stripe rust resistant gene and application of the KASP molecular marker. The invention particularly relates to a functional SNP (Single Nucleotide Polymorphism) marker of a wheat stripe rust resistance major gene Chr6B excavated on the basis of Meta-GWAS (Complete Genome Association Analysis), and application of a KASP (Kinase Amplified Sequence Protein) molecular marker system developed on the basis of an SNP locus in wheat disease-resistant germplasm resource screening, molecular marker-assisted breeding and gene pyramiding breeding. The major effect of the MQTL6B.2 site on the Chr6B chromosome in resistance phenotypic variation is disclosed for the first time, and the contribution rate is greater than or equal to 28.54%. The primer can distinguish a disease-resistant material from a susceptible material, and provides an effective molecular marking tool for subsequent disease-resistant breeding. A molecular marker detection tool can be provided for identification of stripe rust-resistant germplasm resources and breeding of disease-resistant varieties, the selection efficiency is improved, and the breeding process is accelerated.

Description

Technical Field

[0001] The present invention relates to the technical fields of molecular breeding and plant pathology, and particularly relates to a KASP molecular marker for wheat stripe rust resistance genes and its application. Background Art

[0002] Wheat is one of the most important food crops in the world. However, stripe rust is a disease that seriously threatens the yield and quality of wheat.

[0003] In the context of the rapid development of molecular biology technologies, the KASP marker technology has emerged. Traditional wheat stripe rust resistance research mainly relies on phenotypic identification, which is time-consuming, vulnerable to environmental influences, and has limited accuracy. From a genetic perspective, the stripe rust resistance trait in wheat is controlled by multiple genes, and accurately identifying and tracking these disease-resistant genes is crucial for wheat disease-resistant breeding. The KASP marker technology has the characteristics of high accuracy, high throughput, and low cost, and can rapidly detect single nucleotide polymorphisms (SNPs) in the wheat genome. Plant pathology research shows that the stripe rust pathogen is constantly evolving, and new physiological races are constantly emerging, making the resistance of wheat disease-resistant varieties prone to loss. Using the KASP marker technology for marker-assisted selection of wheat stripe rust resistance genes can more efficiently screen wheat varieties or materials with durable resistance, which is of great significance in accelerating the process of wheat disease-resistant breeding, improving wheat resistance to stripe rust, and ensuring the stable global wheat yield. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a KASP molecular marker for wheat stripe rust resistance genes in view of the deficiencies of the above-mentioned prior art. By using the genome-wide association analysis technology, a gene Chr6B that affects wheat disease resistance is excavated, and competitive allele-specific PCR (KASP) primers are designed based on a single-base variation of this gene.

[0005] The present invention provides a KASP molecular marker for wheat stripe rust resistance genes. The KASP molecular marker includes three primers, namely forward specific primer R34F1, forward specific primer R34F2, and reverse primer R34R. Among them, the nucleotide sequence of the forward specific primer R34F1 is as shown in SEQ ID NO:1, the nucleotide sequence of the forward specific primer R34F2 is as shown in SEQ ID NO:2, and the nucleotide sequence of the reverse primer R34R is as shown in SEQ ID NO:3.

[0006] According to the KASP molecular marker of the wheat stripe rust resistance gene provided by the present invention, the PCR amplification reaction system of the KASP molecular marker is 5 μL, including 2.5 μL of 2×KASP Master mix, 0.056 μL of the mixed primer, 100 ng of genomic DNA, and made up to 5 μL with ultrapure water.

[0007] According to the KASP molecular marker of the wheat stripe rust resistance gene provided by the present invention, the concentrations of the forward specific primer R34F1 and the forward specific primer R34F2 are both 12 mmol / L, and the concentration of the shared reverse primer R34R is 30 mmol / L.

[0008] According to the KASP molecular marker of the wheat stripe rust resistance gene provided by the present invention, the PCR amplification program of the KASP molecular marker is: pre-denaturation at 94 °C for 15 min, denaturation at 94 °C for 20 s; annealing at 62 °C for 60 s, decreasing by 0.6 °C for each cycle, for 10 cycles; denaturation at 94 °C for 20 s, annealing at 55 °C for 60 s, for 32 cycles.

[0009] The present invention also provides an application of the above KASP molecular marker of the wheat stripe rust resistance gene, and the KASP molecular marker of the wheat stripe rust resistance gene can be used for disease-resistant molecular marker-assisted breeding.

[0010] The present invention has the following advantages compared with the prior art:

[0011] The present invention specifically relates to a functional SNP marker for the major wheat stripe rust resistance gene Chr6B mined based on genome-wide association meta-analysis (Meta-GWAS), and the application of the KASP molecular marker system developed based on this SNP locus in the screening of wheat disease-resistant germplasm resources, molecular marker-assisted breeding, and gene pyramiding breeding. Traditional disease-resistant phenotype identification relies on artificial inoculation identification, which has problems such as a long cycle, large environmental interference, and low accuracy during the entire growth period; conventional molecular markers such as SSR and RFLP have low throughput and high costs, making it difficult to meet the needs of large-scale germplasm screening; the reported stripe rust resistance QTL / genes mostly have environmental specificity or minor genetic characteristics, making it difficult to directly apply them to breeding practice. Through cross-population meta-analysis integration, a total of 110 linkage-based mapping studies were used, among which different types of mapping populations were used for mapping, including recombinant inbred lines RIL, double haploid DH, and F2:3 or F3 populations. At the same time, part of the stripe rust resistance data of GWAS published between 2014 and 2023 was collected, and these data were used to verify the effectiveness of MQTL. For the first time, it was revealed that the MQTL6B.2 locus on chromosome Chr6B plays a major role in resistance phenotype variation, with a contribution rate ≥ 28.54%. Such primers can distinguish disease-resistant materials from susceptible materials, providing an effective molecular marker tool for subsequent disease-resistant breeding. The present invention can provide a molecular marker detection tool for the identification of stripe rust-resistant germplasm resources and the breeding of disease-resistant varieties, improve the selection efficiency, and accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, after processing the published genetic map to further densify the map distance and developing markers to prove their effectiveness, the purpose is to make them objective and evidentiary. Other drawings can also be obtained based on these drawings.

[0013] Figure 1 is a meta-analysis mapping diagram;

[0014] Figure 2 is a genotyping diagram. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0015] Example 1

[0016] This example provides a KASP molecular marker for the wheat stripe rust resistance gene. The molecular marker contains three primers, namely the forward specific primer R34F1, the forward specific primer R34F2, and the reverse primer R34R;

[0017] Among them, the nucleotide sequence of the forward specific primer R34F1 is shown in SEQ ID NO:1, the nucleotide sequence of the forward specific primer R34F2 is shown in SEQ ID NO:2, and the nucleotide sequence of the reverse primer R34R is shown in SEQ ID NO:3;

[0018] The reaction system for molecular marker PCR amplification is 5 μL, including 2.5 μL of 2×KASP Master mix, 0.056 μL of the mixed primers, 100 ng of genomic DNA, and made up to 5 μL with ultrapure water;

[0019] The mixed primers include two forward specific primers and one common reverse primer. The concentration of each forward specific primer is 12 mmol / L, and the concentration of the common reverse primer R34R is 30 mmol / L;

[0020] The PCR amplification program is as follows: pre-denaturation at 94 °C for 15 min, denaturation at 94 °C for 20 s; annealing at 62 °C for 60 s, with a decrease of 0.6 °C per cycle for 10 cycles; denaturation at 94 °C for 20 s, annealing at 55 °C for 60 s for 32 cycles;

[0021] Extract genomic DNA from samples

[0022] According to the plant genomic DNA extraction kit provided by TIANGEN Company (Catalog number: DP-305), extract the genomic DNA of the tested wheat samples according to the kit instructions. The specific steps are as follows:

[0023] S1. Grind 100 mg of fresh or 20 °C frozen sample materials in liquid nitrogen;

[0024] The sample materials are selected from the GWAS population composed of 376 domestic and foreign wheat varieties (lines), as shown in Table 1;

[0025] S2. Quickly transfer the ground powder to a centrifuge tube pre-filled with 700 μL of buffer GP1 preheated at 65 °C. The buffer GP1 contains 2-mercaptoethanol with a final concentration of 0.1 wt%. After quickly inverting and mixing evenly, place the centrifuge tube in a water bath at 65 °C for 20 min. During the water bath process, invert the centrifuge tube several times to mix the samples;

[0026] S3. Add 700 μL of chloroform, mix well, and centrifuge at 12000 rpm for 5 min; if extracting plant tissues rich in polyphenols or starch, equal volume extraction can be carried out in advance with a 1:1 mixture of phenol and chloroform;

[0027] S4. Transfer the upper aqueous phase obtained after centrifugation in S3 to a new centrifuge tube, and add 700 μL of buffer GP2, and mix well;

[0028] S5. Transfer the mixed liquid into adsorption column CB3, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid.

[0029] S6. Add 500 μL of buffer GD (check whether absolute ethanol has been added in a 1:1 ratio before use) to adsorption column CB3, centrifuge at 12,000 rpm for 30 s to allow the liquid to pass through the adsorption column, and discard the waste liquid. The waste liquid contains impurities, and DNA will bind to the adsorption column.

[0030] In DNA extraction experiments, absolute ethanol usually needs to be added to buffer GD in advance to ensure its ability to effectively bind DNA. Absolute ethanol is a key component of buffer GD, which is used to adjust the polarity of the solution so that DNA can effectively bind to the adsorption column. If buffer GD is premixed (i.e., absolute ethanol has been added), there is no need to add it additionally. If it is not premixed, absolute ethanol needs to be added and mixed according to the ratio in the instruction manual.

[0031] Ensure that the centrifuge speed is 12,000 rpm and the time is 30 seconds to ensure that the liquid completely passes through the adsorption.

[0032] S7. Add 600 μL of wash buffer PW (check whether absolute ethanol has been added before use) to adsorption column CB3, centrifuge at 12,000 rpm for 30 s, pour out the waste liquid, and then rinse once more.

[0033] S8. Place adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, pour out the waste liquid, and leave adsorption column CB3 at room temperature to thoroughly dry the residual wash buffer in the adsorption material.

[0034] S9. Place adsorption column CB3 into a clean centrifuge tube, suspend and add 50 - 200 μL of elution buffer TE with a pH value between 7.0 and 8.5 dropwise to the middle position of the adsorption membrane, let it stand at room temperature for 2 - 5 min, and centrifuge at 12,000 rpm for 2 min to collect the DNA solution.

[0035] S10. Use an ultraviolet spectrophotometer to detect the content and purity of DNA. The results show that the measured A260 / 280 of the sample is between 1.8 and 2.0, indicating that the extracted DNA has a high purity.

[0036] PCR amplification:

[0037] Using genomic DNA as a template, DNA dilution and transfer to plates were carried out on a TECAN liquid automation workstation. The entire PCR process was completed on a Douglas Scenfitic Array Tape platform. DNA and PCR mix were added to a 384-well PCR reaction Array Tape on a Nexar workstation, and the PCR reaction was completed in a Soellex water bath. Fluorescence intensity was detected on an Araya, and data were read. Program settings and data analysis were completed in an Intellics management system.

[0038] The PCR reaction system was 3 μL, and the components were as follows: 0.4 μL of 2×KASP PCR mix, 0.16 μM of Allele-specific primer 1, 0.16 μM of Allele-specific primer 2, 0.41 μM of universal primer, 12 ng of DNA, and ddH 2 O was added to a total volume of 0.8 μL.

[0039] The nucleotide sequence of Allele-specific primer 1 was as shown in SEQ ID NO:4, the nucleotide sequence of Allele-specific primer 2 was as shown in SEQ ID NO:5, and the nucleotide sequence of the universal primer was as shown in SEQ ID NO:6.

[0040] The PCR amplification reaction conditions were as follows: pre-denaturation at 94 °C for 15 min; the first-step amplification reaction, pre-denaturation at 94 °C for 20 s, annealing at 65 - 55 °C for 60 s, for 10 cycles with a 1 °C decrease in each cycle; the second-step amplification reaction, pre-denaturation at 94 °C for 20 s, annealing at 55 °C for 60 s, for 35 cycles.

[0041] Fluorescence signals were read on an Araya fluorescence reader. Data reading was performed using the Intellics software of Douglas Scenfitic Company, and result analysis was performed using the Intellics software of Douglas Scenfitic Company for genotyping. The results are shown in Table 1 and Table 2 below. Among a total of 276 wheat varieties, 245 varieties showed the genotype AA, 14 varieties showed the genotype GG, and the stripe rust resistance of AA-genotype wheat was higher than that of GG-genotype wheat.

[0042] The SNP molecular marker was designated as AX-109072850, and its nucleotide sequence was as shown in SEQ ID NO:7.

[0043] Table 1 Detection results of genotypes and stripe rust resistance of 376 wheat materials

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] In the above table,? represents the absence of phenotypic or genotypic data;

[0060] Table 2 Analysis of Molecular Markers and Disease Resistance of 376 Wheat Varieties

[0061]

[0062] The SNP molecular markers identified by the present invention, the primers for detecting the SNP molecular markers, and the corresponding detection and genotyping methods have the characteristics of high throughput, low cost, and high accuracy, providing a new technical means for molecular marker-assisted selection of wheat offspring resistant to different wheat rusts, and can be used for molecular identification of wheat rust resistance.

[0063] The genotyping results of the SNP locus AX-109072850 in wheat varieties are shown in Table 1, and the average value of MDS for stripe rust is shown in the column of "Stripe Rust MDS BLUP" in Table 1. According to the genotyping results, the BLUP average value of MDS for stripe rust of each genotype was calculated, as shown in Table 2. The results showed that the BLUP of MDS for stripe rust in wheat varieties with the AX-109072850 genotype of AA was significantly higher than that in wheat varieties with the AX-109072850 genotype of GG, indicating that the stripe rust resistance of wheat with the AX-109072850 genotype was higher than that of wheat with the AA genotype at the SNP locus AX-109072850.

[0064] This gene is located on wheat chromosome 6B, at position 159299163 on chromosome 6B. Using this base difference, primers were designed. The last base of the forward primer fell at the mutated base, so the last bases of the two forward primer sequences were different, but they shared a common reverse primer.

[0065] In this example, the detection refers to reading the terminal fluorescence readings with a microplate reader after PCR amplification, and then importing the data into software for genotyping. The model of the microplate reader used is FLUOstar Omega, and the data processing software for genotyping is KlusterCaller software. The processing results are as Figure 2 shown.

[0066] The present invention can provide a molecular marker detection tool for the identification of stripe rust-resistant germplasm resources and the breeding of disease-resistant varieties, increase the selection efficiency, and accelerate the breeding progress.

[0067] The present invention specifically relates to a functional SNP marker for the major wheat stripe rust resistance gene Chr6B mined based on genome-wide association meta-analysis (Meta-GWAS), and the application of a KASP molecular marker system developed based on this SNP locus in the screening of wheat disease-resistant germplasm resources, molecular marker-assisted breeding, and gene pyramiding breeding. Traditional disease-resistant phenotype identification relies on artificial inoculation identification, which has problems such as a long cycle (requiring a complete growth period), large environmental interference, and low accuracy; conventional molecular markers (such as SSR, RFLP) have low throughput and high costs, making it difficult to meet the needs of large-scale germplasm screening; the reported stripe rust resistance QTL / genes mostly have environmental specificity or minor genetic characteristics, making it difficult to be directly used in breeding practice. Through cross-population meta-analysis integration, a total of 110 linkage-based mapping studies were used, among which different types of mapping populations were used for mapping, including recombinant inbred lines (RIL), double haploids (DH), and F2:3 or F3 populations. At the same time, part of the stripe rust resistance data of GWAS published between 2014 and 2023 was collected, and these data were used to verify the effectiveness of MQTL. For the first time, it was revealed that the MQTL6B.2 locus on chromosome Chr6B plays a major role in resistance phenotype variation (contribution rate ≥ 28.54%). Such primers can distinguish disease-resistant materials from susceptible materials, providing an effective molecular marker tool for subsequent disease-resistant breeding. The present invention can provide a molecular marker detection tool for the identification of stripe rust-resistant germplasm resources and the breeding of disease-resistant varieties, improve the selection efficiency, and accelerate the breeding process.

[0068] As described above, it is only a preferred embodiment of the present invention and does not impose any limitation on the present invention. Any simple modification, change, and equivalent change made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A KASP molecular marker for wheat stripe rust resistance gene, characterized in that: The KASP molecular marker comprises three primers, namely a forward specific primer R34F1, a forward specific primer R34F2 and a reverse primer R 3 4R; wherein the nucleotide sequence of the forward specific primer R34F1 is shown in SEQ ID NO: 1, the nucleotide sequence of the forward specific primer R34F2 is shown in SEQ ID NO: 2, and the nucleotide sequence of the reverse primer R 3 4R is shown in SEQ ID NO:

3.

2. The KASP molecular marker of wheat stripe rust resistance gene according to claim 1, characterized in that: The PCR amplification reaction system of the KASP molecular marker is 5 μL, including 2.5 μL of 2×KASP Master mix, 0.056 μL of mixed primers, 100 ng of genomic DNA, and ultrapure water to make up to 5 μL.

3. The KASP molecular marker of wheat stripe rust resistance gene according to claim 1, characterized in that: The concentrations of the forward specific primer R34F1 and the forward specific primer R34F2 are both 12 mmol / L, and the concentration of the shared reverse primer R34R is 30 mmol / L.

4. The KASP molecular marker of wheat stripe rust resistance gene according to claim 1, characterized in that: The PCR amplification procedure of the KASP molecular marker is: pre-denaturation at 94°C for 15 min, denaturation at 94°C for 20 s, annealing at 62°C for 60 s, decreasing 0.6°C per cycle, 10 cycles; denaturation at 94°C for 20 s, annealing at 55°C for 60 s, 32 cycles.

5. An application of the KASP molecular marker of wheat stripe rust resistance gene as claimed in any one of claims 1 to 4, characterized in that: The KASP molecular marker of the wheat stripe rust resistance gene can be used for disease resistance molecular marker assisted breeding.

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