KASP marker primer group linked with wheat stalk rot resistance gene and application thereof

Abstract

The invention discloses a KASP marker primer group linked with a wheat stem rot resistance gene and application thereof, and relates to the technical field of biology. The KASP labeled primer group comprises an upstream primer F1 with a nucleotide sequence as shown in SEQ ID NO.1, an upstream primer F2 with a nucleotide sequence as shown in SEQ ID NO.2 and a universal downstream primer R with a nucleotide sequence as shown in SEQ ID NO.3. Through whole genome association analysis, a QTL site closely associated with disease resistance of basal stem rot is found on a long arm of a 5D chromosome of wheat, and a disease-resistant gene is found and named as TaLRK-D1. And a KASP molecular marker capable of being used for large-scale material screening is further developed aiming at the SNP of the TaLRK-D1 in an anti-infection material. Through verification, the molecular marker is closely related to the disease resistance of wheat basal stem rot and can be applied to screening of high-disease-resistance wheat germplasm.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a KASP marker primer set linked to a wheat stem base rot resistance gene and application thereof. BACKGROUND

[0002] Wheat is an important food crop, and in recent years, stem base rot (Fusarium crown rot, FCR) has become an important new disease in wheat production. Wheat stem base rot is a soil-borne disease caused by Fusarium spp., which has strong saprophytic characteristics, a wide host range, and a main disease site at the stem base, and can occur throughout the growth period.

[0003] Due to the characteristics of the pathogen of stem base rot, which is a semi-saprophytic fungus, a wide host range, a main disease site at the stem base, and can occur throughout the growth period, it is difficult to effectively eradicate using traditional methods such as fungicides, and planting disease-resistant varieties is the most economical and effective measure to prevent and control the occurrence and damage of stem base rot. Exploring disease-resistant sites in wheat materials and developing linked molecular markers are necessary prerequisites for disease-resistant variety breeding. The existing genes related to wheat stem base rot resistance include TaCWI-B1, TaDIR-B1, TaRLK-6A, TaOPR3, etc., and the disease-resistant sites include Qcrs.cpi-3B, Qfcr.cau.3D-3, etc.

[0004] Currently, there are few disease-resistant genes and sites found, and some of the genes and sites have not developed linked molecular markers, making them unable to be applied. In order to cope with the increasingly serious wheat stem base rot, it is urgent to find more disease-resistant sites and develop linked high-throughput molecular markers. SUMMARY

[0005] The purpose of the present application is to provide a KASP marker primer set linked to a wheat stem base rot resistance gene and application thereof, in order to solve the problems existing in the prior art. The KASP marker primer set can effectively identify the stem base rot resistance of wheat.

[0006] To achieve the above purpose, the present application provides the following solutions:

[0007] The present application provides a KASP marker primer set linked to a wheat stem base rot resistance gene, which comprises an upstream primer F1 with a nucleotide sequence as shown in SEQ ID NO. 1, an upstream primer F2 with a nucleotide sequence as shown in SEQ ID NO. 2, and a universal downstream primer R with a nucleotide sequence as shown in SEQ ID NO. 3.

[0008] Further, the upstream primer F1 and the upstream primer F2 label different fluorescent groups.

[0009] Further, the upstream primer F1 is labeled with a FAM fluorescent group; and the upstream primer F2 is labeled with a HEX fluorescent group.

[0010] The application also provides application of the KASP marker primer set in identifying resistance of wheat to stem base rot, and the wheat variety with the genotype GG has higher resistance to stem base rot than the wheat variety with the genotype AA.

[0011] The application also provides application of the KASP marker primer set in preparing a product for identifying resistance of wheat to stem base rot.

[0012] Further, the product is a kit.

[0013] The application also provides a product for identifying resistance of wheat to stem base rot, comprising the KASP marker primer set.

[0014] Further, the product is a kit.

[0015] The application also provides application of the product in identifying resistance of wheat to stem base rot, and the wheat variety with the genotype GG has higher resistance to stem base rot than the wheat variety with the genotype AA.

[0016] The application also provides a method for identifying resistance of wheat to stem base rot, comprising the following steps:

[0017] KASP amplification is performed on the genomic DNA of the wheat to be tested by using the KASP marker primer set, and the wheat to be tested is genotyped according to the amplification result; and the resistance of the wheat to stem base rot is determined according to the genotyping result, and the wheat variety with the genotype GG has higher resistance to stem base rot than the wheat variety with the genotype AA.

[0018] The application discloses the following technical effects:

[0019] The application utilizes a natural population of 223 hexaploid wheat germplasm resources to perform whole genome association analysis, and finds a QTL site (Qfcr.cau.5D) closely related to stem base rot disease resistance on the long arm of the 5D chromosome of wheat. The transcriptome sequencing of the resistant control (04zhong36) and the susceptible control (Xinmai26) materials inoculated for 72h is performed, the up-regulated expression genes are analyzed, and an anti-disease gene TraesCS5D02G525400.1 located in the Qfcr.cau.5D is found and named as TaLRK-D1. Further, the KASP molecular marker KASP5DTaLRK which can be used for large-scale material screening is developed for the SNP of TaLRK-D1 in the resistant and susceptible materials. It is verified that the molecular marker is closely related to the wheat stem base rot disease resistance and can be applied to screening of high-disease-resistant wheat germplasm. The KASP molecular marker developed in the application provides a very valuable molecular tool for screening of wheat FCR-resistant germplasm resources. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0021] Figure 1 Figure 1 is a disease index distribution diagram of 223 hexaploid wheat germplasm resources;

[0022] Figure 2 Figure 2 is a transcriptome up-regulated differential expression gene Wayne diagram; wherein, A / B represents the differential expression genes before and after injection inoculation; C / D represents the differential expression genes before and after seed soaking inoculation;

[0023] Figure 3 Figure 3 is a schematic diagram of the anti-disease gene TaLRK-D1 structure;

[0024] Figure 4 Figure 4 is a result diagram of genotyping detection using KASP5DTaLRK marker in Example 2; wherein, 04z36 represents 04zhong36 genotype; XM26 represents Xinmai26 genotype;

[0025] Figure 5 Figure 5 is a statistical diagram of disease index after grouping according to genotype in Example 2; wherein, A is the genotyping result of the first batch of samples; B is the genotyping result of the second batch of samples; C is the genotyping result of the third batch of samples; 04z36 represents 04zhong36 genotype; XM26 represents Xinmai26 genotype. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be non-limiting examples of the present application, and are understood to be a description of certain aspects, features and embodiments of the present application.

[0027] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise stated, the inclusion of either a lower or an upper limit of a range of values is specifically asserted. The exclusion of either a lower or an upper limit of a range of values is specifically asserted. Where a range of values is stated, it is specifically asserted that any intermediate value of that range is also disclosed. Any smaller range that falls within a disclosed range is also specifically disclosed. The upper and lower limits of any smaller range can each independently be included or excluded in the smaller range.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials that are related to the present application. In the case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification controls.

[0029] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0030] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to mean including, but not limited to.

[0031] Example 1

[0032] 1. Identification of disease resistance loci for wheat foot rot

[0033] Strain WZ-8A was cultured in CMC medium at 25°C, 180 r / min for 4 days after PDA medium. The bacterial solution was collected after 4 days and adjusted to 1 x 10 7Concentration of spores. After germination, wheat seeds were sowed in 5 x 10 plug trays in a greenhouse with 25℃ / 16h during the day and 20℃ / 8h at night. When the wheat seedlings reached the one-leaf-one-heart stage (about 7 days), the spore suspension with adjusted concentration was added to 0.5 v / v volume of Tween 20 and injected into the interior of the wheat seedling stems with a 1 ml syringe. After the top liquid came out, the injection was stopped. Later, drought treatment was combined. The disease-resistant control was 04zhong36 (disclosed in the literature "Jin J, Duan S, Qi Y, et al. Identification of a novel genomic region associated with resistance to Fusarium crown rot in wheat. Theor Appl Genet. 2020;133:2063-2073"), and the disease-susceptible control was Xinmai26 (disclosed in the literature "Jin J, Duan S, Qi Y, et al. Identification of a novel genomic region associated with resistance to Fusarium crown rot in wheat. Theor Appl Genet. 2020;133:2063-2073"). When the disease-susceptible control reached grade 6 (22 days after transplanting), the disease grade of the wheat stem base was investigated according to Table 1, and the disease index was calculated.

[0034] Table 1 Wheat stem base rot resistance grading standards and evaluation standards

[0035]

[0036] The disease grade of 223 hexaploid wheat (153 cultivars, 36 advanced lines, 25 foreign materials, and 9 farmer varieties) was investigated as shown in Table 2, and the results are shown in Table 3. Figure 1 and Table 3.

[0037] Table 2 223 wheat materials

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] Note: CRS1, the phenotypic disease index of the first experiment; CRS2, the phenotypic disease index of the second experiment; CRS3, the phenotypic disease index of the third experiment; BLUP, the phenotypic disease index of the BLUP value of the three experiments.

[0044] Table 3 Statistical analysis of disease index of 223 hexaploid wheat

[0045]

[0046] After extracting DNA from 223 wheat materials using the CTAB method, genotyping was performed using a wheat 90k SNP array. The phenotype data and filtered 90K SNP data were jointly analyzed using the GAPIT language package in R4.0.2 program, and it was found that 54 SNP sites were significantly associated with FCR resistance. These 54 SNPs were divided into 10 QTLs, and Qfcr.cau.5D stable expression was identified (Table 4).

[0047] Table 4 QTLs against FCR identified by genome-wide association analysis

[0048]

[0049] 2. Transcriptome analysis of resistant and susceptible materials

[0050] The 36 of the resistant control 04 and the susceptible control Xinmai 26 were inoculated by injection method and seed soaking method respectively when the wheat seedlings grew to the one-leaf-one-heart stage (about 7 days). Stem base tissues of inoculated and non-inoculated were taken for transcriptome sequencing 72 hours after inoculation. The differentially expressed genes up-regulated after inoculation were selected for Wayne diagram enrichment analysis, and a total of 186 candidate genes were identified. Figure 2 ).

[0051] 3. Mining of TaLRK-D1 locus and development of KASP marker

[0052] Based on the candidate interval of Qfcr.cau.5D located by GWAS and the transcriptome sequencing results, an anti-disease candidate gene TraesCS5D02G525400.1 was analyzed, which encodes Leucine-rich repeat receptor-like protein kinase, so it is named TaLRK-D1. Sequencing of TaLRK-D1 gene in the resistant control 04 36 and the susceptible material Xinmai 26 was performed, and the sequence differences were analyzed. The results showed that SNP sites were found at the "86", "93", "193" bases of the gene, which had two haplotypes, AAG haplotype and GCA haplotype. The genotype of the resistant material was "AAG", and the genotype of the susceptible material was "GCA". Figure 3). Sequence extension analysis of 223 materials of GWAS found only two haplotypes of AAG and GCA. According to the differences of these SNPs, KASP marker KASP5DTaLRK was developed to distinguish the genotypes of resistant and susceptible parents (Table 5).

[0053] Table 5 Primer sequence of KASP5DTaLRK marker

[0054]

[0055] Note: The detection site is the 193rd base of TaLRK-D1 gene.

[0056] Method for using KASP marker:

[0057] Mix: Beijing Jiacheng Biotechnology Co., Ltd. HiGeno 2 × Probe Mix A (version number: E01 / 2020) was used to prepare the KASP system, and 10 μL of mineral oil was used to seal the top.

[0058] The KASP amplification system is shown in Table 7, and the KASP amplification program is shown in Table 8.

[0059] The primer concentration was diluted to 100 μM / μL, and the KASP primer system (Primer Mix) was prepared according to Table 6:

[0060] Table 6 KASP primer system

[0061]

[0062] Table 7 KASP amplification system

[0063]

[0064] Table 8 KASP amplification program

[0065]

[0066] After amplification, FAM and HEX fluorescence signals in Bio-Rad instrument were used for scanning and fluorescence signal reading.

[0067] Example 2

[0068] Verification of the function of KASP marker:

[0069] In order to verify whether the gene is associated with stem base rot resistance, another 223 wheat materials were detected by KASP5DTaLRK marker, and the method was the same as that in Example 1. Due to the limitation of the amount of samples detected by KASP, 223 samples were divided into three batches for experiment. As shown in Table 9, the results of the three batches of experiments were consistent, and the KASP5DTaLRK marker was verified to be associated with stem base rot resistance. Figure 4As shown, 163 single plants presented the diseased control Xinmai 26 genotype (HEX fluorescence), i.e. the diseased genotype (AA); 60 single plants presented the resistant control 04zhong36 genotype (FAM fluorescence), i.e. the resistant genotype (GG). According to the genotype grouping, it was found that the disease index of the resistant genotype group was significantly lower than that of the diseased genotype group. TaLRK-D1 could reduce the FCR severity by 5.26% Figure 5 ).

[0070] The above-described embodiments are only used to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A KASP marker primer set linked to a wheat stem base rot resistance gene, characterized in that, The upstream primer F1 comprises a nucleotide sequence as shown in SEQ ID NO. 1, the upstream primer F2 comprises a nucleotide sequence as shown in SEQ ID NO. 2, and the universal downstream primer R comprises a nucleotide sequence as shown in SEQ ID NO.

3.

2. The KASP marker primer set according to claim 1, wherein, The upstream primer F1 and the upstream primer F2 are labeled with different fluorescent groups.

3. The KASP marker primer set according to claim 2, wherein, The upstream primer F1 is labeled with a FAM fluorescent group, and the upstream primer F2 is labeled with a HEX fluorescent group.

4. Use of a KASP marker primer set as claimed in any one of claims 1 to 3 in the identification of resistance to wheat foot rot, characterised in that, The wheat variety with a genotype of GG has higher resistance to stem base rot than the wheat variety with a genotype of AA.

5. Use of the KASP marker primer set according to any one of claims 1-3 in the preparation of a product for identifying the resistance of wheat to stem base rot.

6. Use according to claim 5, characterized in that, The product is a kit.

7. A product for identifying resistance to wheat foot rot, characterized in that, The product comprises the KASP marker primer set according to any one of claims 1-3.

8. The product of claim 7, wherein, The product is a kit.

9. Use of a product according to claim 7 or 8 in a product for identifying resistance to wheat foot rot, characterized in that, The wheat variety with a genotype of GG has higher resistance to stem base rot than the wheat variety with a genotype of AA.

10. A method of identifying resistance to wheat foot rot, characterized in that, The product comprises the following steps: The KASP marker primer set according to any one of claims 1-3 is used to perform KASP amplification on the genomic DNA of the wheat to be tested, the wheat to be tested is genotyped according to the amplification results, and the resistance of the wheat to stem base rot is determined according to the genotyping results: the wheat variety with a genotype of GG has higher resistance to stem base rot than the wheat variety with a genotype of AA.

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