Molecular markers tightly linked to QTL Qfcr.sicau.2A for wheat stem rot resistance and their application

Abstract

The present invention provides a molecular marker tightly linked to wheat stem base rot resistance QTL Qfcr.sicau.2A and its application. The molecular marker provided by the present invention is KASP marker KASP97, and marker KASP97 is tightly linked to wheat stem base rot resistance QTL Qfcr.sicau.2A, and the two are co-localized on wheat chromosome 2A. The present invention shows through detection and analysis that the molecular marker KASP97 can accurately track the wheat stem base rot resistance QTL, predict wheat stem base rot resistance, and then facilitate molecular design breeding. The present invention also provides the application of molecular marker KASP97 in wheat breeding. The method provided by the present invention can enhance the accuracy of wheat stem base rot resistance prediction, so as to quickly screen out wheat varieties or strains with QTLs with high resistance to wheat stem base rot for breeding, which can greatly accelerate the breeding process of high-yield wheat varieties.

Description

Technical Field

[0001] The invention relates to the technical field of molecular markers and plant genetic breeding, in particular to a molecular marker tightly linked to wheat stem rot resistance QTL Qfcr.sicau.2A and its application. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the most important staple crops in the world. It provides dietary fiber and a variety of nutrients for humans. About 40% of the world's population relies on wheat as an important source of calories. The safe production of wheat is of great significance to China's food security. Wheat stem rot (Fusarium crown rot, FCR) is a global fungal disease caused by infection with multiple pathogens. When the disease occurs seriously, it greatly reduces the yield and quality of grains. The selection and large-scale application of disease-resistant varieties are the most economical and effective ways to control the disease. The use of resistance sources and stem rot resistance identification technology are the key to ensuring the selection and breeding of disease-resistant varieties.

[0003] At present, QTLs for resistance to crown rot are distributed on 14 of the 21 chromosomes of wheat, but only QTLs on 2D, 3B and 5D can be consistently detected in different genetic backgrounds; Wallwork et al. (2004) used a diploid (DH) population obtained by doubling haploids from the "Kukri / Janz" cross and tested resistance in the adult stage in an outdoor environment. They detected the 4B QTL near the dwarfing gene Rht1. The resistance allele of this locus came from the "Kukri" variety and explained up to 48% of the phenotypic variation (Wallwork, H., M. Butt, et al. Resistance to crown rot in wheat identified through an improved method for screening adult plants. Australas. Plant Pathol. 2004, 33: 1-7.); Bovill et al. (2006) used 95 diploid (DH) populations obtained by doubling haploids from the "W21MMT70 / Mendos" cross in a greenhouse environment for identification at the seedling stage, and detected two QTLs located at 5D and 2B from the "W21MMT70" and "Mendos" resistance genes, respectively, which explained 28% and 20% of the phenotypic variation (Bovill, WD, W. Ma, et al. Identification of novel QTL for resistance to crown rot in the doubled haploid wheat population'W21MMT70'9'Mendos'. Plant Breed. 2006, 125, 538-543.); Ma et al. (2010) analyzed the population obtained by hybridization between "CSCR6" and the Australian variety "Lang". Through greenhouse inoculation at the seedling stage, the resistance allele of one of the QTLs was identified to be derived from "CSCR6". The QTL, called Qcrs.cpi-3B, is located on the long arm of chromosome 3B. According to interval mapping analysis, it can explain up to 48.8% of the phenotypic variation, which is the QTL locus with the largest effect so far. Another QTL with a resistance allele from the "Lang" variety is located on chromosome 4B. This QTL explains up to 22.8% of the phenotypic variation and can be detected in different genetic backgrounds (Ma, J., HBLi, et al. Identification and validation of a major QTLconferring crown rot resistance in hexaploid wheat. Theor. Appl. Genet.2010, 120, 1119-1128.); Zheng et al. (2014) found four QTLs conferring resistance to stem rot in the RIL population "EGAWylie / 'Sumai 3". The one with the greatest impact was located on chromosome arm 5DS, which explained up to 31% of the phenotypic variation with an LOD value of 9.6. Another QTL was located on 2DL, which explained up to 20% of the phenotypic variation with an LOD value of 4.5. When the effect of plant height was considered by covariance analysis, no significant effects of the other two loci (both located on chromosome 4B) were detected (Zheng Z, Kilian A, Yan G, et al. QTL conferring Fusarium crown rotresistance in the elite bread wheat variety EGA Wylie. PLoS One. 2014, 9: e96011.). .

[0004] Tibetan semi-wild wheat (T.aestivum ssp.tibetanum), Yunnan iron-shell wheat (T.aestivumssp.yunnanense), Xinjiang rice wheat (T.petropavlovskyi Udacz et.Migusch) and Sichuan white wheat are unique local wheat germplasm resources in western China (Ward, RW, Yang, ZL, Kim, HS, et al. Comparative analyses of RFLP diversity in landraces of Triticum aestivum and collections of T.tauschii from China and Southwest Asia. Theor. Appl. Genet. 1998, 96:312-318.). These four germplasm resources are evolved from the original hexaploid wheat in different ecological environments through natural and artificial selection in a long-term unique ecological environment in China; they have rich agronomic traits and genetic diversity, and are potential gene resources for enriching wheat genetic variation (Dong, Y., Zheng, D., Qiao, D., Zeng, X., En, Z., Chen, X. 1981. Expedition and investigation of Yunnan wheat (Triticum aestivumssp. yunnanense King). Zuo Wu Xue Bao 3: 145-152.). Therefore, a genome-wide association analysis was conducted on 209 samples of western China's unique local wheat to further locate the wheat stem base rot resistance loci, explore genes with high resistance to stem base rot, and find closely linked molecular markers. At the same time, new gene resources are provided for the creation of wheat high resistance to stem base rot materials and high-yield breeding. Further use of molecular marker-assisted selection will enhance the accuracy of stem base rot resistance prediction, improve breeding efficiency, and accelerate the cultivation of disease-resistant and high-yield wheat.

[0005] Molecular marker-assisted selection does not rely on phenotypic selection, that is, it is not affected by environmental conditions, gene interactions, genotype-environment interactions and other factors, but directly selects genotypes, thus greatly improving breeding efficiency. Competitive allele-specific PCR (Kompetitive Allele Specific PCR) can accurately detect double alleles of SNPs and Indels at specific sites in a wide range of genomic DNA samples. This detection method has the advantages of simple operation, good specificity, high throughput, rapidity, low detection cost, accurate results, and has achieved true closed-tube operation and has received widespread attention. Therefore, screening out molecular markers that are closely linked to the wheat stem base rot resistance QTL and are suitable for the fluorescent quantitative PCR platform KASP technology can not only select wheat stem base rot resistance genes, effectively improve wheat disease resistance, and ensure normal growth and fruiting of wheat, but also improve the selection throughput, speed and accuracy, solve the technical bottleneck of large-scale promotion and application, and have important significance for the large-scale improvement of wheat breeding population quality and yield. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker tightly linked to wheat stem rot resistance QTL Qfcr.sicau.2A and its application.

[0007] To achieve the purpose of the present invention, in a first aspect, the present invention provides a molecular marker KASP97 tightly linked to the wheat stem rot resistance QTL Qfcr.sicau.2A, wherein the molecular marker KASP97 contains a nucleotide sequence in which the polymorphism at the 36th bp of the wheat sequence as shown in SEQ ID NO:1 is A or G.

[0008] When the base at the 36bp position of the molecular marker KASP97 is A, the wheat contains QTL Qfcr.sicau.2A, which corresponds to strong resistance to wheat base stem rot; when the base at the 36bp position of the molecular marker KASP97 is G, the wheat does not contain QTL Qfcr.sicau.2A, which corresponds to weak resistance to wheat base stem rot.

[0009] In the present invention, the wheat stem rot resistance QTL Qfcr.sicau.2A is located in a 3.62Mb segment between 70.43-74.06Mb on wheat chromosome 2A. This interval contains 19 high-confidence genes.

[0010] In a second aspect, the present invention provides a set of specific primers for amplifying the molecular markers developed based on the KASP technology, wherein the primers include primers with sequences as shown in SEQ ID NOs: 2-4.

[0011] Furthermore, different fluorescent probes are connected to the 5' ends of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0012] Preferably, the nucleotide sequence of the fluorescent probe connected to the 5' end of the primer shown in SEQ ID NO:2 is as shown in SEQ ID NO:5, the nucleotide sequence of the fluorescent probe connected to the 5' end of the primer shown in SEQ ID NO:3 is as shown in SEQ ID NO:6, and the 5' ends of the fluorescent probes shown in SEQ ID NO:5 and SEQ ID NO:6 are respectively connected to different fluorescent groups, such as FAM, HEX, etc.

[0013] In a third aspect, the present invention provides a detection reagent or a kit containing the primer set.

[0014] In a fourth aspect, the present invention provides any of the following uses of the molecular marker KASP97, the primer set, or a detection reagent or kit containing the primer set:

[0015] (1) Used to identify the wheat stem rot resistance QTL Qfcr.sicau.2A;

[0016] (2) Used for screening or identifying wheat varieties with high resistance to stem rot;

[0017] (3) Used in wheat molecular marker-assisted breeding;

[0018] (4) Used for wheat germplasm improvement.

[0019] In a fifth aspect, the present invention provides a method for identifying wheat stem rot resistance QTL Qfcr.sicau.2A, using the genomic DNA of the wheat to be tested as a template, using the primer set to perform fluorescent quantitative PCR amplification, and performing genotyping on the wheat to be tested according to the PCR amplification results.

[0020] Preferably, the reaction system of the fluorescent quantitative PCR amplification is: 2×KASP Mastermix 5μL, KASP AssayMix 0.14μL, template DNA 50ng, and DNase / RNase-free deionized water added to a total amount of 10μL; wherein the KASPAssay Mix contains primers shown in SEQ ID NO:2-4, the volume ratio of the three primers is 2:2:5, and the concentration of each primer is 100μM.

[0021] Preferably, the fluorescence quantitative PCR program is: activation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing and extension at 65°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 36 cycles; 37°C for 60 s, collecting fluorescence signals.

[0022] According to the above method, wheat varieties containing the wheat stem rot resistance QTL Qfcr.sicau.2A show the same fluorescent signal as the fluorescent probe connected to the primers shown in SEQ ID NO:2, while wheat varieties not containing the wheat stem rot resistance QTL Qfcr.sicau.2A show a fluorescent signal that is significantly different from the fluorescent probe connected to the primers shown in SEQ ID NO:2.

[0023] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0024] The present invention discloses for the first time the wheat stem rot resistance QTL Qfcr.sicau.2A located in local wheat, which is located on wheat chromosome 2A and significantly improves wheat stem rot resistance. The QTL has a high utilization value in wheat yield (improving wheat stem rot resistance) breeding. The present invention further discloses a molecular marker KASP97 for accurately detecting wheat stem rot resistance QTL Qfcr.sicau.2A based on a fluorescent quantitative PCR platform, which is located at the 73.62Mb position on wheat chromosome 2A and is a co-dominant marker with accurate and efficient detection and convenient and stable amplification.

[0025] The molecular marker KASP97 provided by the present invention is significantly correlated with the wheat stem rot resistance QTL Qfcr.sicau.2A, presents a co-segregation marker feature, has high accuracy in molecular marker-assisted selection, improves the selection and identification efficiency of wheat specific stem rot resistance varieties adapted to different environments, and has a high success rate.

[0026] The method provided by the present invention can enhance the accuracy of wheat stem rot resistance prediction, so as to quickly screen out wheat varieties or lines with QTLs with high resistance to wheat stem rot for breeding, which can greatly accelerate the breeding process of high-yield wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the physical location of the wheat stem rot resistance QTL Qfcr.sicau.2A on chromosome 2A and the Manhattan plot of the association analysis between the molecular marker KASP97 and the stem rot resistance in a preferred embodiment of the present invention.

[0028] Figure 2 The present invention is a preferred embodiment of the present invention, in which fluorescent quantitative PCR primers are used to genotype the leaf DNA of local wheat stem base rot extremely resistant materials (Longzi Zheda, Chaya Zheda, Lincang Tiekemai, Yutian Daomai) and extremely susceptible materials (Jiacha Broken-ear Wheat, Xinjiang Daomai, Dingmang Tiekemai, China Spring) at the three-leaf stage.

[0029] Figure 3The F constructed using Yutian rice and wheat and Chinese spring in the preferred embodiment of the present invention 6 The results of genotyping of the validation group using fluorescent quantitative PCR primers. DETAILED DESCRIPTION

[0030] The present invention aims to provide a molecular marker KASP97 which is tightly linked to wheat stem base rot resistance QTL Qfcr.sicau.2A, and provides application of the molecular marker in wheat breeding.

[0031] The present invention adopts the following technical solution:

[0032] In the first aspect, the present invention provides a molecular marker tightly linked to the wheat stem rot resistance QTL Qfcr.sicau.2A, which is the molecular marker KASP97, and the nucleotide sequence is shown in SEQ ID NO:1 (5'-AGTTGTATCCCATAGTTGTATACCAAAATGGCATCNTGCATGTATACATGTTTGTAT AGCACTGATCTACA-3'; wherein N is A or G), that is, the polymorphism of the 36th base of the sequence is A / G, and the polymorphism is related to wheat stem rot resistance.

[0033] The molecular marker KASP97 of the present invention is closely linked to the wheat stem rot resistance QTL Qfcr.sicau.2A and co-localized in a 3.62Mb segment between 70.43-74.06Mb on wheat chromosome 2A. The molecular marker KASP97 is located in the confidence interval of the wheat stem rot resistance QTL Qfcr.sicau.2A.

[0034] Furthermore, the wheat stem rot resistance QTL Qfcr.sicau.2A provided by the present invention can significantly increase wheat stem rot resistance, with a maximum LOD value of 5.49, explaining 14.48% of the phenotypic variation.

[0035] More specifically, the single base difference site of the molecular marker KASP97 provided by the present invention is A, corresponding to strong resistance to wheat base stem rot; the single base difference site of the molecular marker KASP97 is G, corresponding to weak resistance to wheat base stem rot.

[0036] In a second aspect, the present invention provides a specific primer set for fluorescent quantitative PCR amplification of the molecular marker KASP97.

[0037] A person skilled in the art can design primers for amplifying the molecular marker KASP97 based on the KASP detection platform technology. Preferably, the specific primer pair includes primers with sequences as shown in SEQ ID NO: 2-4. The 5' ends of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 are respectively connected to different fluorescent probes.

[0038] KASP97-1:5'-AGTTGTATACCAAAATGGCATCA-3'(SEQ ID NO:2)

[0039] KASP97-2:5'-AGTTGTATACCAAAATGGCATCG-3'(SEQ ID NO:3)

[0040] KASP97-3: 5'-GGGTCAATCACGGGCATGTA-3' (SEQ ID NO: 4)

[0041] Furthermore, different fluorescent probes were attached to the 5′ ends of primers KASP97-1 and KASP97-2;

[0042] The sequence of the fluorescent probe is as follows:

[0043] F probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO: 5). In the embodiment of the present invention, the probe is combined with a FAM fluorescent group.

[0044] H probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO: 6) In the embodiment of the present invention, the probe is combined with a HEX fluorescent group.

[0045] The present invention also provides any of the following applications of the molecular marker KASP97 or the above-mentioned specific primers:

[0046] (1) Application in identification of QTL Qfcr.sicau.2A for wheat stem rot resistance;

[0047] (2) Application in screening or identifying wheat varieties resistant to wheat stem rot;

[0048] (3) Application in wheat molecular marker-assisted breeding;

[0049] (4) Application in wheat germplasm improvement.

[0050] In a third aspect, the present invention provides a method for identifying wheat stem rot resistance QTL Qfcr.sicau.2A, using the genomic DNA of the wheat to be tested as a template, using the above-mentioned specific primer set to perform fluorescent quantitative PCR amplification, and performing genotyping on the wheat to be tested according to the PCR amplification results.

[0051] Preferably, the reaction system of the fluorescent quantitative PCR amplification is: 2×KASP Mastermix 5μL, KASP AssayMix 0.14μL, template DNA 50ng, and DNase / RNase-free deionized water added to a total amount of 10μL; wherein the nucleotide sequence of the primers contained in KASP Assay Mix is ​​as shown in SEQ ID NO:2-4, the volume ratio of the three primers is 2:2:5, and the concentration of each primer is 100μM.

[0052] In the embodiment of the present invention, the fluorescence quantitative PCR program is as follows: activation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing and extension at 65°C for 60 s, for 10 cycles, with the annealing and extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, for 36 cycles; and collection of fluorescence signals at 37°C for 60 s.

[0053] The judgment criterion of the method for identifying the wheat stem rot resistance QTL Qfcr.sicau.2A provided by the present invention is that wheat varieties containing the wheat stem rot resistance QTL Qfcr.sicau.2A all show the same fluorescent signal as the fluorescent probe labeled with the primers shown in SEQ ID NO:2, while wheat varieties not containing the wheat stem rot resistance QTL Qfcr.sicau.2A all show fluorescent signals that are significantly different from the fluorescent probe labeled with the primers shown in SEQ ID NO:2.

[0054] In the present invention, the wheat stem rot resistance QTL Qfcr.sicau.2A and the molecular marker KASP97 are obtained by the following method:

[0055] (1) 209 local wheat varieties were used as genetic mapping populations.

[0056] (2) The DNA of each strain of the genetic population was extracted using the CTAB method, and the qualified DNA samples of all materials were genotyped using a wheat 55K SNP chip to obtain the genotype data of the population.

[0057] (3) The disease grade of stem rot of 209 local wheat varieties was identified by greenhouse immersion inoculation method at wheat seedling stage, and the disease index (DI) was calculated.

[0058] (4) Combining the molecular marker data and phenotypic data of 209 local wheat varieties, a genome-wide association analysis was performed using TASSEL v5.0 association analysis software under a mixed linear model, and the wheat stem rot resistance QTL Qfcr.sicau.2A was located in a 3.62 Mb segment between 70.43-74.06 Mb on chromosome 2A.

[0059] (5) The SNP sites most closely associated with stem base rot resistance in the target segment were converted into fluorescent quantitative PCR primers for subsequent screening. Nine pairs of fluorescent quantitative PCR primers were designed using the PolyMarker website (Table 1). Fluorescent quantitative PCR primer design standards: amplification primer length 18-25 bp, amplification product length 45-60 bp, annealing temperature 57-62°C, GC content between 40% and 60%. The synthesized primer sequences are:

[0060] Forward primer 1: F probe + amplification primer sequence

[0061] Forward primer 2: H probe + amplification primer sequence

[0062] Reverse primer: Amplification primer sequence

[0063] F probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (can bind to FAM fluorescent group)

[0064] H probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (can be combined with HEX fluorescent group)

[0065] Table 1 Sequences of 9 pairs of KASP primers and lengths of amplified fragments

[0066]

[0067]

[0068] (6) Competitive allele-specific PCR (KASP) analysis

[0069] a) Screening of polymorphic molecular markers: The 9 pairs of primers designed above were selected, and DNA of extremely disease-resistant materials (Longzi Zheda, Chaya Zheda, Lincang Tiekemai, Yutian Daomai) and extremely susceptible materials (Jiacha Broken-ear Wheat, Xinjiang Daomai, Dingmang Tiekemai, China Spring) of the unique local wheat stem base rot was used as a template for PCR amplification, and a pair of molecular marker primers with good effects were obtained, the primers shown in SEQ ID NO: 2-4, which can amplify SEQ ID NO: 1, and amplification was named KASP97-1 / 2 / 3 (the nucleotide sequences are shown in SEQ ID NO: 2-4 respectively). The amplified product is a molecular marker KASP97 with polymorphism, and the nucleotide sequence is shown in SEQ ID NO: 1.

[0070] b) KASP analysis of unique local wheat populations: The PCR primers of the molecular marker KASP97 with polymorphism obtained in the above steps were used to amplify the DNA of the local wheat population, perform genotype identification, and obtain molecular marker data. The type of the extremely resistant materials to stem base rot (Longzi Zheda, Chaya Zheda, Lincang Tiekemai, Yutian Daomai) was recorded as A, the amplified fragment size was 71 bp, and the single base difference site was A. The type of the extremely susceptible materials (Jiacha Broken Ear Wheat, Xinjiang Daomai, Dingmang Tiekemai, and China Spring) was recorded as B, the amplified fragment length was 71 bp, and the single base difference site was G. The genotypes of the strains of the unique local wheat population were consistent with the extremely resistant materials (Longzi Zheda, Chaya Zheda, Lincang Tiekemai, and Yutian Daomai) and were consistent with the extremely susceptible materials (Jiacha Broken Ear Wheat, Xinjiang Daomai, Dingmang Tiekemai, and China Spring) as A, and those were consistent with the extremely susceptible materials (Jiacha Broken Ear Wheat, Xinjiang Daomai, Dingmang Tiekemai, and China Spring) as B.

[0071] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions. Example 1 Acquisition of wheat stem rot resistance QTL Qfcr.sicau.2A and molecular marker KASP97

[0072] The wheat stem rot resistance QTL Qfcr.sicau.2A and molecular marker KASP97 of the present invention are obtained by the following method:

[0073] (1) 209 local wheat varieties were used as genetic mapping populations.

[0074] (2) The DNA of each strain of the genetic population was extracted using the CTAB method, and the qualified DNA samples of all materials were genotyped using a wheat 55K SNP chip to obtain the genotype data of the population.

[0075] (3) The disease grade of stem rot of 209 local wheat varieties was identified by greenhouse immersion inoculation method at wheat seedling stage, and the disease index (DI) was calculated.

[0076] (4) Combining the molecular marker data and phenotypic data of 209 local wheat varieties, a genome-wide association analysis was performed using TASSEL v5.0 association analysis software under a mixed linear model, and the wheat stem rot resistance QTL Qfcr.sicau.2A was located in a 3.62 Mb segment between 70.43-74.06 Mb on chromosome 2A.

[0077] (5) Convert the SNP sites in the target segment into fluorescent quantitative PCR primers for subsequent screening. Nine pairs of fluorescent quantitative PCR primers were designed using the PolyMarker website (Table 1). Fluorescent quantitative PCR primer design standards: amplification primer length 18-25 bp, amplification product length 45-60 bp, annealing temperature 57-62°C, GC content between 40% and 60%. The synthetic primer sequences are:

[0078] Forward primer 1: F probe + amplification primer sequence

[0079] Forward primer 2: H probe + amplification primer sequence

[0080] Reverse primer: Amplification primer sequence

[0081] F probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (can bind to FAM fluorescent group)

[0082] H probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (can be combined with HEX fluorescent group)

[0083] (6) Competitive allele-specific PCR (KASP) analysis

[0084] a) Screening of polymorphic molecular markers: The 9 pairs of primers designed above were selected, and DNA of extremely disease-resistant materials (Longzi Zheda, Chaya Zheda, Lincang Tiekemai, Yutian Daomai) and extremely susceptible materials (Jiacha Broken-ear Wheat, Xinjiang Daomai, Dingmang Tiekemai, China Spring) of the unique local wheat stem base rot was used as a template for PCR amplification, and a pair of molecular marker primers with good effects were obtained, the primers shown in SEQ ID NO: 2-4, which can amplify SEQ ID NO: 1, and amplification was named KASP97-1 / 2 / 3 (the nucleotide sequences are shown in SEQ ID NO: 2-4 respectively). The amplified product is a molecular marker KASP97 with polymorphism, and the nucleotide sequence is shown in SEQ ID NO: 1.

[0085] b) KASP analysis of unique local wheat populations: The PCR primers of the molecular marker KASP97 with polymorphism obtained in the above steps were used to amplify the DNA of the local wheat population, perform genotype identification, and obtain molecular marker data. The type of the extremely resistant materials to stem base rot (Longzi Zheda, Chaya Zheda, Lincang Tiekemai, Yutian Daomai) was recorded as A, the amplified fragment size was 71 bp, and the single base difference site was A. The type of the extremely susceptible materials (Jiacha Broken Ear Wheat, Xinjiang Daomai, Dingmang Tiekemai, and China Spring) was recorded as B, the amplified fragment length was 71 bp, and the single base difference site was G. The genotypes of the strains of the unique local wheat population were consistent with the extremely resistant materials (Longzi Zheda, Chaya Zheda, Lincang Tiekemai, and Yutian Daomai) and were consistent with the extremely susceptible materials (Jiacha Broken Ear Wheat, Xinjiang Daomai, Dingmang Tiekemai, and China Spring) as A, and those were consistent with the extremely susceptible materials (Jiacha Broken Ear Wheat, Xinjiang Daomai, Dingmang Tiekemai, and China Spring) as B.

[0086] c) Using the mixed linear model calculation method of the software TASSEL v5.0 and combining the phenotypic data of stem rot resistance in local wheat populations, it was found that the molecular marker KASP97 was closely linked to the wheat stem rot resistance QTL Qfcr.sicau.2A locus. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that the wheat stem rot resistance QTL Qfcr.sicau.2A is located in the 3.62Mb segment between 70.43-74.06Mb on chromosome 2A, and the molecular marker KASP97 is closely linked to the QTL.

[0087] Example 2 Application of molecular marker KASP97 tightly linked to wheat stem rot resistance QTL Qfcr.sicau.2A

[0088] 1. DNA extraction

[0089] The experimental materials were selected from Longzi Zheda, Chaya Zheda, Lincang Tiekemai, Yutian Daomai, Jiacha Broken-spiked Wheat, Xinjiang Daomai, Dingmang Tiekemai and Zhongguochun. Longzi Zheda, Chaya Zheda, Lincang Tiekemai and Yutian Daomai were resistant varieties of wheat stem base rot, while Jiacha Broken-spiked Wheat, Xinjiang Daomai, Dingmang Tiekemai and Zhongguochun were susceptible varieties of wheat stem base rot. The leaf DNA of wheat samples at the three-leaf stage was extracted using the CTAB method.

[0090] 2. Screening of primers for detecting wheat stem rot resistance QTL Qfcr.sicau.2A

[0091] 2.1 Primer design

[0092] The SNP marker KASP97 was converted to design fluorescent quantitative PCR primers for subsequent screening. Nine pairs of fluorescent quantitative PCR primers were designed using the PolyMarker website (Table 1). Fluorescent quantitative PCR primer design standards: amplification primer length 18-25 bp, amplification product length 45-60 bp, annealing temperature 57-62°C, GC content between 40% and 60%. The synthetic primer sequences are:

[0093] Forward primer 1: F probe + amplification primer sequence

[0094] Forward primer 2: H probe + amplification primer sequence

[0095] Reverse primer: Amplification primer sequence

[0096] F probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (can bind to FAM fluorescent group)

[0097] H probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (can be combined with HEX fluorescent group)

[0098] 2.2 Fluorescence quantitative PCR platform to test the differences between primers and their parents

[0099] (1) Extract leaf DNA from local wheat cultivars extremely resistant to stem rot (Longzi Zheda, Chaya Zheda, Lincang Tiekemai, Yutian Daomai) and extremely susceptible to stem rot (Jiacha Shattered-Ear Wheat, Xinjiang Daomai, Dingmang Tiekemai, and China Spring) at the three-leaf stage.

[0100] (2) Using the genomic DNA of the wheat to be tested as a template, primers were designed based on the KASP detection platform technology to perform fluorescence quantitative PCR amplification;

[0101] Wherein, the primer sequence of step 2 is as follows (SEQ ID NO:2-4):

[0102] KASP97-1: 5'-AGTTGTATACCAAAATGGCATCA-3';

[0103] KASP97-2: 5'-AGTTGTATACCAAAATGGCATCG-3';

[0104] KASP97-3: 5'-GGGTCAATCACGGGCATGTA-3';

[0105] Furthermore, different fluorescent probes were attached to the 5′ ends of primers KASP97-1 and KASP97-2;

[0106] The sequence of the fluorescent probe is as follows (SEQ ID NO: 5-6):

[0107] F probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (can bind to FAM fluorescent group)

[0108] H probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (can be combined with HEX fluorescent group)

[0109] (3) Fluorescence quantitative PCR amplification reaction system: 2×KASP Mastermix 5μL, KASP Assay Mix 0.14μL, template DNA 50ng, DNase / RNase-free deionized water added to a total of 10μL; wherein the KASP Assay Mix contains primers KASP97-1, KASP97-2 and KASP97-3 in a volume ratio of 2:2:5. That is, in the KASP Assay Mix, the primers KASP97-1, KASP97-2 and KASP97-3 with a concentration of 100μM are mixed in a volume ratio of 2:2:5.

[0110] (4) Fluorescence quantitative PCR program: activation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing and extension at 65°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 36 cycles; 37°C for 60 s, collecting fluorescence signals.

[0111] (5) The specific method for analyzing PCR products is as follows: Wheat samples containing the wheat stem rot resistance QTL Qfcr.sicau.2A all showed the same type as the extremely resistant materials to the wheat stem rot (Longzi Zheda, Chaya Zheda, Lincang Tiekemai, Yutian Daomai) and were recorded as type A, while wheat samples not containing the wheat stem rot resistance QTL Qfcr.sicau.2A all showed fluorescent signals that were significantly different from the extremely resistant materials to the wheat stem rot (Longzi Zheda, Chaya Zheda, Lincang Tiekemai, Yutian Daomai), such as the extremely susceptible materials (Jiacha Broken-Ear Wheat, Xinjiang Daomai, Dingmang Tiekemai, and China Spring), which were recorded as type B. The results of genotyping using KASP primers for Longzi Zheda, Chaya Zheda, Lincang Tiekemai, Yutian Daomai, Jiacha Broken-Ear Wheat 39, Xinjiang Daomai 8, Dingmang Tiekemai, and China Spring are shown in the table. Figure 2 .

[0112] 3. Applicability of primer sequences KASP97-1 / 2 / 3 in population detection

[0113] (1) The Yutian rice and wheat were used as the female parent and the Chinese spring was used as the male parent to obtain the F1 hybrid. The F1 was self-pollinated continuously to obtain the F6 as the verification population. The leaf DNA of each strain in the population was extracted at the three-leaf stage.

[0114] (2) Using the DNA obtained in step (1) as a template, using the primers provided by the present invention to perform fluorescent quantitative PCR amplification, and using SsoFast EvaGreen as the fluorescent dye.

[0115] (3) Fluorescence quantitative PCR amplification reaction system: 2×KASP Mastermix 5μL, KASP Assay Mix 0.14μL, template DNA 50ng, DNase / RNase-free deionized water added to a total of 10μL; wherein the KASP Assay Mix contains primers KASP97-1, KASP97-2 and KASP97-3 in a volume ratio of 2:2:5. That is, in the KASP Assay Mix, the primers KASP97-1, KASP97-2 and KASP97-3 with a concentration of 100μM are mixed in a volume ratio of 2:2:5, and then 0.14μL of the mixed solution is aspirated.

[0116] (4) Fluorescence quantitative PCR program: activation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing and extension at 65°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 36 cycles; 37°C for 60 s, collecting fluorescence signals.

[0117] (5) The specific method for analyzing PCR products is as follows: Wheat samples containing the wheat stem rot resistance QTL Qfcr.sicau.2A all showed the same type as the extremely resistant material Yutiandaomai, which was marked as A, while wheat samples not containing the wheat stem rot resistance QTL Qfcr.sicau.2A all showed fluorescent signals that were significantly different from the extremely resistant material Yutiandaomai, which was marked as the extremely susceptible material China Spring, which was marked as B. The results are as follows: Figure 3 As shown. A total of 125 lines were randomly sampled, and 58 lines were able to amplify the same type of fragments as Yutian Daomai, which are plants with the wheat stem base rot resistance QTL Qfcr.sicau.2A. It is predicted that the lines have strong resistance to stem base rot. 61 lines were able to amplify the same B-type fragments as Sanyuehuang, which are plants that do not contain the wheat stem base rot resistance QTL Qfcr.sicau.2A. It is predicted that these plants have poor resistance to stem base rot in the predicted lines. Another 6 lines were heterozygous.

[0118] (6) Identification of 73 F 2The results of the plant's resistance to stem rot are shown in Table 2. The molecular marker KASP97 of the wheat stem rot resistance QTL Qfcr.sicau.2A predicted the results of the wheat stem rot resistance in the verification population. The average stem rot disease index of plants of the same type as Yutian rice and wheat is 42.13, which is significantly lower than the average stem rot disease resistance grade of 51.24 of plants of the same type as Sanyuehuang. The actual results are consistent with the expected results, indicating that the wheat stem rot resistance QTL Qfcr.sicau.2A of the present invention does have the effect of significantly improving wheat stem rot resistance, and the molecular marker KASP97 can be used to track and identify the wheat stem rot resistance QTL Qfcr.sicau.2A.

[0119] Table 2

[0120]

[0121]

[0122]

[0123]

[0124] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Any of the following applications of the molecular marker KASP97, a primer set for amplifying the molecular marker developed based on the KASP technology, or a detection reagent or kit containing the primer set: (1) Used to screen or identify wheat varieties with high resistance to stem rot; (2) Used for molecular marker-assisted breeding of wheat stem rot resistance; (3) Used for improving wheat germplasm resources resistant to stem rot; The molecular marker KASP97 has a nucleotide sequence in which the polymorphism at the 36 bp position of the sequence as shown in SEQ ID NO: 1 is A or G; When the base at the 36th bp of the molecular marker KASP97 is A, it corresponds to strong resistance to wheat base stem rot; when the base at the 36th bp of the molecular marker KASP97 is G, it corresponds to weak resistance to wheat base stem rot.

2. The use according to claim 1, characterized in that: The primer set includes primers whose sequences are shown in SEQ ID NOs: 2-4.

3. The use according to claim 2, characterized in that: The 5' ends of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 are connected to different fluorescent probes respectively.

4. The use according to claim 3, characterized in that: The nucleotide sequence of the fluorescent probe connected to the 5' end of the primer shown in SEQ ID NO:2 is shown in SEQ ID NO:5, the nucleotide sequence of the fluorescent probe connected to the 5' end of the primer shown in SEQ ID NO:3 is shown in SEQ ID NO:6, and the 5' ends of the fluorescent probes shown in SEQ ID NO:5 and SEQ ID NO:6 are respectively connected to different fluorescent groups.

Images