Screening of KASP primer sets for wheat resistant to scab infection and its application

Abstract

The present invention discloses a KASP primer set for screening wheat resistant to fusarium head blight infection and its application. The present invention uses wheat Wheat55K high-throughput gene chip to obtain genotype data, detects fusarium head blight infection resistance sites QFhi-yaas-Y4-6B and QFhi-yaas-Y5-3A on wheat chromosomes 6B and 3A, respectively, which are derived from Yangmai No. 4 and Yangmai No. 5, and develops two KASP marker primer sets KASP.Y4.6B and KASP.Y5.3A based on their tightly linked marker information. Experiments have proved that the KASP molecular markers KASP.Y4.6B and KASP.Y5.3A described in the present invention can be used for molecular marker-assisted selection breeding of wheat resistant to fusarium head blight infection. The development and utilization of KASP.Y4.6B and KASP.Y5.3A provide a powerful tool for wheat fusarium head blight infection resistance breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of wheat molecular breeding methods and relates to a KA SP primer set for screening wheat resistant to fusarium head blight infection and an application thereof. Background Art

[0002] Fusarium head blight is a global disease that seriously endangers wheat yield and food safety. It is mainly caused by Fusarium graminearum and other fungi. The manifestations of wheat resistance to fusarium head blight can be divided into five categories: the first category is resistance to infection, the second category is resistance to expansion; the third category is grain resistance to infection, the fourth category is disease tolerance, and the fifth category is resistance to toxin accumulation. Among them, the first and second categories are the types of wheat that scholars at home and abroad have been committed to studying for a long time. Fusarium head blight resistance is the first barrier for the host to resist fusarium head blight, and it is even more important for breeding for fusarium head blight resistance. In addition to the resistance of the variety itself, the agronomic traits of wheat also have a certain impact on fusarium head blight. The plant height is high, the ear is far away from the soil surface where the pathogen is hidden, and the ability to be infected is weak. There are many reports on the relationship between wheat dwarfing genes Rht-B1, Rht-D1 and Rht8 and resistance to fusarium head blight. Studies have shown that the dwarfing alleles at these three loci have a significant effect on increasing the severity of fusarium head blight. The relationship between other dwarfing genes and wheat fusarium head blight resistance has not been fully studied. In the study of the relationship between growth period and wheat fusarium head blight, scholars have found that genes or loci related to wheat growth period have a significant effect on fusarium head blight resistance under a certain genetic background (Zhu et al., 2021). At present, there are few reports on wheat infection resistance loci (Quantitative traits loci, QTL), only from Fhb4 on chromosome 4B of Wangshuibai (Xue et al., 2010) and Fhb5 on chromosome 5A (Xue et al., 2011). Wangshuibai is a local wheat variety with high plant height, easy lodging, and a long growth period, which makes it difficult to use as fusarium head blight resistant germplasm. Therefore, it is very important to mine excellent fusarium head blight resistance sites from varieties with good agronomic traits for comprehensive breeding of fusarium head blight resistance and high yield. Yangmai No. 4 and Yangmai No. 5 were once the dominant varieties in the middle and lower reaches of the Yangtze River. They have high yield potential, excellent agronomic traits, and good resistance to fusarium head blight. Yanzhan No. 1 is a wheat variety in the southern part of the Huanghuai region. It has weak spring characteristics, short stems and large spikes, high yield, excellent agronomic traits, and is susceptible to fusarium head blight. Therefore, the two RIL (Recombinant inbred lines) populations of Yangmai No. 4 / Yanzhan No. 1 and Yangmai No. 5 / Yanzhan No. 1 can be used to mine the fusarium head blight resistance sites of Yangmai No. 4 and Yangmai No. 5, providing genetic resources for wheat breeding for fusarium head blight resistance.

[0003] KASP technology is based on the specific matching of primer terminal bases and universal fluorescent probes to perform SNP typing detection. The typing can be completed by a fluorescent quantitative PCR instrument or an ordinary PCR instrument combined with an enzyme marker. KASP technology is similar to TaqMan (fluorescent probe method for detecting oligonucleotides). Both are based on the reading and judgment of terminal fluorescent signals. Each well reaction uses two-color fluorescence to detect two genotypes of each SNP site, and different SNPs correspond to different fluorescent signals. However, it does not require the synthesis of specific fluorescent primers for each SNP site. All site detections can eventually be amplified using universal fluorescent primers, which reduces the reagent cost of KASP technology and makes it more practical. After many agronomic traits or disease resistance and stress resistance-related genes / sites in modern wheat are mined or precisely located, researchers will develop KASP markers based on the closely linked marker flanking sequences on both sides of the site to facilitate breeders (Su et al., 2018; Zhang et al., 2020; Hu Wenjing et al., 2022). KASP technology has the advantages of high throughput and convenient detection (Rasheed et al., 2019). Genotyping with SNP chips and SNPs linked to target traits obtained through QTL mapping or GWAS analysis can be converted into KASP markers for easy application in breeding. (Rasheed et al., 2019) 70 KASP markers for wheat functional genes have been developed and widely used. Summary of the invention

[0004] In order to overcome the defects in the prior art, a linked molecular marker is provided for molecular marker-assisted selection of wheat breeding for ergot resistance genotypes. The present invention uses Wheat 55KSNP wheat high-throughput gene chip to obtain genotype data, and detects QTL regions QFhi-yaas-Y4-6B and QFhi-yaas-Y5-3A significantly associated with ergot resistance from wheat varieties Yangmai No. 4 and Yangmai No. 5. Further, by screening the effectiveness and reliability of nearby SNP sequences, two KASP marker primer sets are developed near the two QTL peaks to efficiently screen wheat germplasm for ergot resistance genotypes.

[0005] In a first aspect, the present invention provides a KASP primer set for screening wheat resistant to ergot infection, the KASP primer set being a combination of a set of primers X1 for detecting whether the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID No.4 on chromosome 6B in the wheat genome is TT, GG, or T and G, and a set of primers X2 for detecting whether the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID No.8 on chromosome 3A in the wheat genome is TT, CC, or T and C.

[0006] Further, the set of primers X1 contains two upstream primers and one downstream primer;

[0007] The upstream primer is designed according to the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 4 on chromosome 6B in the wheat genome and its upstream sequence, and the 3' terminal deoxyribonucleotide of one of the upstream primers is T, and the 3' terminal deoxyribonucleotide of the other upstream primer is G;

[0008] The downstream primer is designed according to the downstream sequence of the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 4 on chromosome 6B in the wheat genome.

[0009] Further, the set of primers X2 contains two upstream primers and one downstream primer;

[0010] The upstream primer is designed according to the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 8 on chromosome 3A in the wheat genome and its upstream sequence, and the 3' terminal deoxyribonucleotide of one of the upstream primers is T, and the 3' terminal deoxyribonucleotide of the other upstream primer is C;

[0011] The downstream primer is designed according to the downstream sequence of the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 8 on chromosome 3A in the wheat genome.

[0012] Furthermore, the primer set X1 consists of the upstream primer sequences SEQ ID No.1 and SEQ ID No.2 and the downstream primer sequence SEQ ID No.3; the primer set X2 consists of the upstream primer sequences SEQ ID No.5 and SEQ ID No.6 and the downstream primer sequence SEQ ID No.7.

[0013] The second aspect of the present invention provides the use of the above-mentioned KASP primer set in any of the following:

[0014] (A) Identifying or assisting in identifying wheat's resistance to infection by fusarium head blight;

[0015] (B) Comparing the resistance of tested wheat to infection by fusarium head blight;

[0016] (C) breeding or selecting wheat plants, strains, lines or varieties that are relatively resistant to infection by fusarium head blight;

[0017] (D) breeding or selecting wheat plants, strains, lines or varieties that are relatively less resistant to infection by fusarium head blight;

[0018] (E) preparing a product for identifying or assisting in identifying the resistance to infection by fusarium head blight of wheat;

[0019] (F) preparing a product for breeding or screening wheat plants, strains, lines or varieties with relatively strong resistance to infection by fusarium head blight;

[0020] (G) preparing a product for breeding or screening wheat plants, strains, lines or varieties that are relatively weak in resistance to infection by ergot.

[0021] The third aspect of the present invention provides any of the following methods:

[0022] Method A: A method for comparing the resistance of wheat scab to infection, comprising the following steps (A1) or (A2):

[0023] (A1) detecting whether the genotype of the 36th position of the molecular marker represented by SEQ ID No.4 on chromosome 6B in the wheat genome is TT, GG or TG, and simultaneously detecting whether the genotype of the 36th position of the molecular marker represented by SEQ ID No.8 on chromosome 3A in the wheat genome is TT, CC or TC;

[0024] (A2) determining the strength of the fusarium head blight resistance of the wheat to be tested as follows: if the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No. 4 on chromosome 6B in the genome is GG and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No. 8 on chromosome 3A in the genome is a homozygous CC, the wheat to be tested has a strong fusarium head blight resistance;

[0025] Method B: A method for breeding or screening wheat plants or strains or lines or varieties with relatively strong resistance to infection by fusarium head blight, comprising the following steps:

[0026] (B1) detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the wheat genome is TT, GG or TG, and simultaneously detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the wheat genome is TT, CC or TC;

[0027] (B2) selecting a wheat plant to be tested in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the genome is G and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the genome is C as a parent for breeding, and selecting a wheat plant in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the genome is G and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the genome is C in each breeding generation, and finally obtaining a wheat plant or strain or variety with relatively strong resistance to infection by ergot;

[0028] Method C: A method for breeding or screening wheat plants or strains or lines or varieties with relatively weak resistance to ergot infection, comprising the following steps:

[0029] (C1) detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the wheat genome is TT, GG or TG, and simultaneously detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the wheat genome is TT, CC or TC;

[0030] (C2) selecting a wheat plant to be tested in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the genome is T and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the genome is T as a parent for breeding, and selecting a wheat plant in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the genome is T and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the genome is T in each breeding generation, and finally obtaining a wheat plant or strain or variety with relatively weak resistance to infection by ergot.

[0031] Furthermore, said (A1)(B1)(C1)

[0032] The specific operation is:

[0033] The KASP primer set described in the first aspect of the present invention is used to perform PCR amplification on the wheat genomic DNA to be tested, the amplified product is subjected to fluorescence signal scanning, the scanning data is analyzed, and then the type of the 36th deoxyribonucleotide shown in SEQ ID No.4 on chromosome 6B in the wheat gene to be tested and the type of the 36th deoxyribonucleotide shown in SEQ ID No.8 on chromosome 3A in the wheat gene to be tested are determined as follows;

[0034] If the fluorescent signal data of the amplified product of the wheat to be tested is displayed as blue, the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the genome of the wheat to be tested is a homozygous G, or the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the genome of the wheat to be tested is a homozygous C;

[0035] If the fluorescent signal data of the amplified product of the wheat to be tested is displayed as red, the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the genome of the wheat to be tested is a homozygous T, or the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the genome of the wheat to be tested is a homozygous T.

[0036] Compared with the prior art, the present invention uses Wheat 55KSNP wheat high-throughput gene chip to obtain genotype data, detects QTL regions QFhi-yaas-Y4-6B and QFhi-yaas-Y5-3A significantly associated with fusarium head blight resistance from wheat varieties Yangmai No. 4 and Yangmai No. 5, and further develops two KASP marker primer sets near the two QTL peaks by screening the effectiveness and reliability of nearby SNP sequences, so as to efficiently screen wheat germplasm with fusarium head blight resistance genotypes. The present invention provides a good tool for the effective use of two fusarium head blight resistance sites QFhi-yaas-Y4-6B and QFhi-yaas-Y5-3A in fusarium head blight resistance breeding. The two KASP markers can quickly screen wheat fusarium head blight resistance characteristics, provide convenience for screening wheat materials carrying excellent alleles for resistance to infection, and improve the efficiency of wheat fusarium head blight resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific implementation manner of the present invention or the technical solution in the prior art, the drawings required for describing the specific implementation manner are briefly introduced below.

[0038] Figure 1 This is a schematic diagram of the QTL location for wheat fusarium head blight resistance in Example 1, wherein: Figure 1 a is a partial genetic linkage map of chromosome 6B and a schematic diagram of the location of QFhi-yaas-Y4-6B; Figure 1 b is a partial genetic linkage map of chromosome 3A and a schematic diagram of the location of QFhi-yaas-Y5-3A.

[0039] Figure 2 This is a schematic diagram of the results of applying the KASP.Y4.6B marker and the KASP.Y5.3A marker in Example 2 to the genotyping of a natural wheat population. Figure 2 a is the result of genotype amplification detection of KASP.Y4.6B marker in natural wheat population; Figure 2 b shows the genotype amplification detection result of KASP.Y5.3A marker in natural wheat population. DETAILED DESCRIPTION

[0040] The embodiments of the technical solution of the present invention will be described in detail below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only used as examples and cannot be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be the common meanings understood by technicians in the field to which the present invention belongs. As a professional agricultural research institution, the applicant has long-term preservation of relevant germplasm materials, and the relevant wheat varieties are all publicly available in the market or existing germplasm banks.

[0041] Example 1 Screening for stable loci significantly associated with wheat scab resistance

[0042] In this example, 151 recombinant inbred lines (F 10 ) and 143 recombinant inbred lines derived from “Yangmai 5×Yanzhan 1” (F 10 ) was used as the material, and the recombinant inbred line and its parents were planted in the fusarium induced identification greenhouse of Wanfu Experimental Base in Lixiahe District, Jiangsu Province for three consecutive growing seasons in 2020, 2021 and 2022. A randomized block design was adopted, with 2 rows of 25 seeds per row, 2m long, 0.20m row spacing, two replicates, conventional greenhouse management, and no prevention and control of pests and diseases. The investigation of fusarium scabbard of field parents, families, and varieties (lines) was resistant to infection. The strain used was a mixed strain of F. graminearum (F4, F15, F34 and F0609). Diseased wheat kernels infected with Fusarium fusogenum were sown in the experimental field at the booting stage (18 days before heading). For each wheat material, 40 ears with the same growth process were randomly selected for marking. Ten days after the wheat bloomed, the number of diseased spikelets (at least one spikelet showed disease symptoms) and the total number of spikelets on each infected ear were investigated. At least 30 ears were investigated for each family or variety. The number of diseased spikelets / total number of spikelets*100%=average diseased spikelet rate (PDS). PDS was used as an indicator to measure the severity of Fusarium fusogenum (Lin et al., 2006; Xue et al., 2011).

[0043] Genomic DNA was extracted from fresh leaf tissues by the CTAB method (Doyle et al., 1987), and DNA integrity and quantity were detected by gel electrophoresis. First, PCR markers related to four major ergot resistance genes / QTLs in common wheat, including WGRB619 (Fhb1), WMC398 and WGRB969 (Fhb2), GWM149 and GWM513 (Fhb4), WMC752 and MAG9482 (Fhb5), were used to screen for polymorphisms between parents (Li et al., 2019b; Zhang et al., 2021). The results showed that the parental materials of this study, Yangmai 4, Yangmai 5 and Yanzhan 1, did not carry these four disease resistance genes / QTLs.

[0044] The wheat 55K single nucleotide polymorphism (SNP) array containing 53,063 SNPs provided by Zhongyujin Marker (Beijing) Biotechnology Co., Ltd. was used to genotype the parents and recombinant inbred line populations. Then, a total of 15 KASP and SSR markers related to other reported functional genes in wheat were selected to detect polymorphism (Rasheed et al., 2016; Zhang et al., 2019; Xu et al., 2020; Zhu et al., 2021). All polymorphic SNP flanking sequences were aligned with the International Wheat Genome Sequencing Consortium (IWGSC) Ensembl Plants database (http: / / plants.ensembl.org) Chinese Spring reference genome v2.1 (Ref v2.1) to obtain their physical positions, and an expected value (E) of 1E-10 was used as the significance threshold.

[0045] First, the SNP chip results were subjected to preliminary quality control. In IciMapping v4.1, the "BIN" function was used to simultaneously delete SNPs with a missing rate greater than 20%, a minimum allele frequency less than 5%, and partial separation (χ2 ≥ 33.3%), and to delete redundant SNPs. All polymorphic markers were used for population genotyping, and the marker flanking sequences were placed in the International Wheat Genome Sequencing Consortium database v2.1 (International Wheat Genome Sequencing Consortium, IWGSC) for physical position comparison (Ma et al., 2021) (WheatOmics 1.0, http: / / 202.194.139.32 / blast / blast.html). The "MAP" function was used to filter the markers for grouping (Li et al., 2007; 2008; Meng et al., 2015). The Kosambi mapping function was used in JoinMap 4.0 software to calculate the genetic distance of each population (Kosambi et al., 1944; Van et al., 2006). The complete interval mapping (ICIM) algorithm was used to detect QTLs for resistance to Fusarium head blight using the “BIP” function of IciMapping v4.1, and the LOD threshold was set to 3.0 (Li et al., 2021). A genetic map covering the QTL region was drawn using MapChart v2.32 (Voorrips, 2002). QTLs located within overlapping confidence intervals were considered to be identical. The physical positions of QTL flanking markers were compared with previously reported genes / QTLs (http: / / 202.194.139.32 / blast / blast.html and http: / / wheatomics.sdau.edu.cn / genes / ).

[0046] A stable infection resistance locus QFhi-yaas-Y4-6B was detected in the Yangmai 4 / Yanzhan 1 population in 3 years and BLUE values. Yangmai 4 provided a PDS-reducing effect. This locus contributed 10.67–12.99% to the phenotype, with an additive effect of 6.18–8.13 ( Figure 1 , Table 1), and after comparison with previous studies, this is a new wheat ergot resistance site.

[0047] Table 1 QTL mapping results for resistance to infection in Yangmai 4 / Yanzhan 1

[0048]

[0049]

[0050] Notes: a2020, 2021 and 2022 represent the Yangzhou Fusarium fusarium infection resistance identification nurseries in 2020, 2021 and 2022 respectively, and BLUE represents the optimal linear unbiased value. b Represents a physical location based on the Chinese Spring 2.1 version. c represents the common logarithm of the maximum likelihood function. d represents the phenotypic contribution rate. e represents additive effects (negative values ​​represent alleles derived from Yangmai 4 that increase resistance to fusarium scurf., and positive values ​​represent alleles derived from Yanzhan 1 that increase resistance to fusarium scurf.).

[0051] In the Yangmai 5 / Yanzhan 1 population, a stable resistance locus QFhi-yaas-Y5-3A was detected in the 2-year and BLUE values. The resistance effect originated from Yangmai 5, which could explain 7.49–10.56% of the phenotypic variation, with an additive effect of 3.92–6.44 ( Figure 1 , Table 2), and after comparison with previous studies, this is a new wheat ergot resistance site.

[0052] Table 2 QTLs for infection resistance in Yangmai 5 / Yanzhan 1

[0053]

[0054] Notes: a 2020, 2021 and 2022 represent the Yangzhou Fusarium fusarium infection resistance identification nurseries in 2020, 2021 and 2022 respectively, and BLUE represents the optimal linear unbiased value. b Represents a physical location based on the Chinese Spring 2.1 version. c Represents the common logarithm of the maximum likelihood function. d represents the phenotypic contribution rate. e represents additive effects (negative values ​​represent alleles derived from Yangmai 4 that increase resistance to fusarium scurf., and positive values ​​represent alleles derived from Yanzhan 1 that increase resistance to fusarium scurf.).

[0055] Example 2: Clarifying the relationship between fusarium head blight resistance QTL and agronomic traits

[0056] The effects of the three infection resistance QTLs on various agronomic traits were analyzed using QFhi-yaas-Y4-6B and QFhi-yaas-Y5-3A flanking markers and the BLUE values ​​of plant height, heading date, panicle length, spikelet number, spikelet density, number of grains per spike, 1000-grain weight, grain length and grain width. The results showed that QFhi-yaas-Y4-6B and QFhi-yaas-Y5-3A had no significant effects on various agronomic traits and were two ergot resistance loci with high utilization value (Table 3).

[0057] Table 3 Effect analysis of QFhi-yaas-Y4-6B and QFhi-yaas-Y5-3A on agronomic traits (BLUE value)

[0058]

[0059] Note: BLUE stands for best linear unbiased estimator. ns means the phenotype is not significantly different from that of Yanzhan 1.

[0060] Example 3 Development of KASP markers linked to QFhi-yaas-Y4-6B and QFhi-yaas-Y5-3A

[0061] The above research results show that QFhi-yaas-Y4-6B and QFhi-yaas-Y5-3A have no significant correlation with agronomic traits. We used the wheat reference genome information to convert the flanking SNP markers AX109922067 and AX110621643 of the peak interval of these two loci into KASP markers KASP.Y4.6B and KASP.Y5.3A. The primer sequences of KASP.Y4.6B and KASP.Y5.3A are shown in Table 4.

[0062] Table 4 Primer sequences of KASP.Y4.6B and KASP.Y5.3A

[0063]

[0064]

[0065] Note: F1 and F2 are forward primers and R is reverse primer. Competitive primers are underlined.

[0066] Preparation of KASP primers and PCR reaction

[0067] The flanking SNPs of the peak interval of the Fusarium fusarium resistance site with breeding value were converted into KASP markers using wheat reference genome information, and the primers were designed using the online software Primer 3.0. According to the KASP design principle, two specific primers (F1 / F2) and one universal primer (R) were designed for each marker. A specific sequence that binds to FAM fluorescence was added to the tail of F1, and a specific sequence that binds to HEX fluorescence was added to the tail of F2. The primers were synthesized by Beijing Jiacheng Biotechnology Co., Ltd. The total KASP reaction system was 6μL, including 2×KASP Master Mix 3.5μL, KASP primer mixed working solution 0.1μL, and a concentration of 20ngμL -1The template DNA was 2.4 μL. The first step of the KASP reaction procedure was 94°C, 15 min; the second step was 94°C, 25 s, 61–55°C, 1 min, with a decrease of 0.6°C in each cycle, for a total of 10 cycles; the third step was 94°C, 20 s, 55°C, 45 s, for a total of 29 cycles. The KASP typing results were analyzed using a KASP fluorescence analyzer (LGC, PHERAstar plus).

[0068] The two sets of RIL populations and their parents were amplified with KASP.Y4.6B and KASP.Y5.3A as described above. The fluorescence signal data of the amplified product of KASP.Y4.6B were analyzed by Kluster Caller software and clustered at a position close to the X-axis in the fluorescence signal coordinate system of the typing result (blue), which was the same as Yangmai No. 4, proving that the genotype of these wheats at the 36th base (SNP site) of the molecular marker KASP.Y4.6B flanking nucleotide sequence (such as SEQ ID NO.4) was G; while the fluorescence signal data of the amplified product were analyzed by Kluster Caller software and clustered at a position close to the Y-axis in the coordinate system (red), which was different from the typing of Yangmai No. 4, proving that the genotype of these wheats at the SNP site was T. The material was well typed, and the KASP primer set was successfully designed.

[0069] The fluorescence signal data of the amplified products of KASP.Y5.3A were analyzed by Kluster Caller software and clustered near the X-axis position (blue) in the fluorescence signal coordinate system of the typing results, which was the same as Yangmai No. 5, proving that the genotype of these wheats at the 36th base (SNP site) of the molecular marker KASP.Y5.3A flanking nucleotide sequence (such as SEQ ID NO.4) was C; while the fluorescence signal data of the amplified products were analyzed by Kluster Caller software and clustered near the Y-axis position (red) in the coordinate system, which was different from the typing of Yangmai No. 5, proving that the genotype of these wheats at the SNP site was T. The typing of the material was good, and the KASP primer set was successfully designed.

[0070] Table 5 Results of t-tests of RIL families carrying different genotypes

[0071]

[0072] Notes: a Different lowercase letters after the numbers indicate extremely significant differences (P<0.01); b The ** after the t value indicates a very significant difference (P<0.01).

[0073] As shown in Table 5, using the two-sample t test of Excel 2019, it can be seen that the PSS of the family with KASP.Y4.6B genotype GG is significantly lower than that of the family with TT (reduced by 26.80%) (P<0.01); the PSS of the family with KASP.Y5.3A genotype CC is significantly lower than that of the family with TT (reduced by 24.39%) (P<0.01) (the statistical method is a conventional method in this field, and the details can also be found in the document "Gai Junyi, "Experimental Statistical Methods", China Agriculture Press, September 2000").

[0074] The above two sets of KASP primer sets and genotype detection systems were used separately or simultaneously in the selection of materials with strong or weak resistance to wheat ergot infection. The effects of QFhi-yaas-Y4-6B and QFhi-yaas-Y5-3A were significant, indicating that the KASP marker was successfully developed and can be further used for breeding material detection.

[0075] Example 4 KASP primer set breeding application

[0076] Field test: In this example, 211 wheat materials from the middle and lower reaches of the Yangtze River were used as research objects. In 2020, 2021 and 2022, 211 wheat materials were planted in the fusarium induced identification greenhouse of Wanfu Experimental Base in Lixiahe District, Jiangsu Province for three consecutive growing seasons. A randomized block design was adopted, with 2 rows planted in each line, 25 grains per row, 2m long, 0.20m row spacing, two replicates, conventional greenhouse management, and no prevention and control of pests and diseases. The field fusarium investigation was resistant to infection. The strain used was a mixed strain of F.graminearum (F4, F15, F34 and F0609). Diseased wheat kernels infected with Fusarium fusogenum were sown in the experimental field at the booting stage (18 days before heading). For each wheat material, 40 ears with the same growth process were randomly selected for marking. Ten days after the wheat bloomed, the number of diseased spikelets (at least one spikelet showed disease symptoms) and the total number of spikelets in the infected ears were investigated. At least 30 ears were investigated for each variety (line). The number of diseased spikelets / total number of spikelets*100%=average diseased spikelet rate (Percentage of diseased spikelets, abbreviated as PDS). PDS was used as an indicator to measure the severity of Fusarium fusogenum (Lin et al., 2006; Xue et al., 2011).

[0077] The KASP primer set obtained in Example 1 was used to genotype 211 wheat varieties (lines). The average values ​​of the fusarium head blight identification results and the genotype detection results of the field trials in 2020, 2021 and 2022 are shown in Tables 5 and Figure 2As shown. The fluorescence signal data of the amplified product of KASP.Y4.6B was analyzed by Kluster Caller software and clustered at a position close to the X-axis in the fluorescence signal coordinate system of the typing result (blue), which is the same as Yangmai No. 4, which proves that the genotype of these wheats at the 36th base (SNP site) of the molecular marker KASP.Y4.6B flanking nucleotide sequence (such as SEQ ID NO.4) is G. The fluorescence signal data of the amplified product was analyzed by Kluster Caller software and clustered at a position close to the Y-axis in the coordinate system (red), which is different from the typing of Yangmai No. 4, which proves that the genotype of these wheats at the SNP site is T; the fluorescence signal data of the amplified product of KASP.Y5.3A was analyzed by Kluster Caller software and clustered at a position close to the X-axis in the fluorescence signal coordinate system of the typing result (blue), which is the same as Yangmai No. 5, which proves that the genotype of these wheats at the molecular marker KASP.Y5.3A flanking nucleotide sequence (such as SEQ ID The genotype of the 36th base (SNP site) of NO.4) is C; and the fluorescence signal data of the amplified products are clustered near the Y-axis in the coordinate system (red) after analysis by KlusterCaller software, which is different from the typing of Yangmai No. 5, proving that the genotype of these wheats at this SNP site is T.

[0078] Table 6 Genotypes and average phenotypic values ​​of 211 wheat varieties (lines)

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Table 7 t test results of tested varieties (lines) carrying different genotypes

[0086]

[0087] Note: *** after the number indicates that there is a significant difference relative to "TT+TT" at P<0.001, and ** indicates that there is a significant difference relative to "TT+TT" at P<0.01.

[0088] Table 6 shows the typing and phenotyping results of 211 wheat materials. Table 7 shows the results of the two-sample t test using Excel 2019. Among the 211 wheat varieties (lines), when QFhi-yaas-Y4-6B was the Yangmai 4 genotype (GG) and QFhi-yaas-Y5-3A was the Yangmai 5 genotype (CC), the PSS was the lowest, which was 29.87% lower (P<0.001) than when QFhi-yaas-Y4-6B and QFhi-yaas-Y5-3A were both the Yanzhan 1 genotype (TT+TT); when QFhi-yaas-Y4-6B was the Yangmai 4 genotype (GG) and QFhi-yaas-Y5-3A was the Yangmai 5 genotype (CC), the PSS was the lowest. When QFhi-yaas-Y4-6B was the Yangmai 4 genotype (GG) and QFhi-yaas-Y5-3A was the Yanzhan 1 genotype, the PSS of the variety (line) was reduced by 17.11% (P<0.01) relative to "TT+TT"; when QFhi-yaas-Y4-6B was the Yanzhan 1 genotype (TT) and QFhi-yaas-Y5-3A was the Yangmai 5 genotype (CC), the PSS of the variety (line) was reduced by 18.91% (P<0.001) relative to "TT+TT". This indicates that the Yangmai 4 genotype (GG) of QFhi-yaas-Y4-6B and the Yangmai 5 genotype (CC) of QFhi-yaas-Y5-3A have a significant additive effect and are most effective in enhancing resistance to fusarium head blight. The above two sets of KA SP primer sets and genotype detection systems can be used separately or simultaneously in the screening of wheat fusarium resistant germplasm materials and molecular marker-assisted selection breeding.

[0089] It can be concluded from the above experimental results that by performing PCR amplification on wheat genomic DNA using the two sets of KASP primer sets of the present invention, it can be directly determined by KASP typing whether the wheat carries the ergot infection-resistant genotype of Yangmai No. 4 and the ergot infection-resistant genotype of Yangmai No. 5, the detection method is simple to operate, the detection result is very intuitive, and the detection effect is obvious and effective. The use of these two sets of KASP primer sets can greatly improve the efficiency of molecular marker-assisted selection breeding of wheat resistant to ergot infection.

[0090] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. In all examples shown and described here, unless otherwise specified, any specific value should be interpreted as merely exemplary, rather than as restriction, and therefore, other examples of exemplary embodiments may have different values.

Claims

1. A KASP primer set for screening wheat resistant to fusarium head blight infection, It is characterized in that The KASP primer set is a combination of a set of primers X1 for detecting whether the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID No.4 on chromosome 6B in the wheat genome is TT, GG, or T and G, and a set of primers X2 for detecting whether the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID No.8 on chromosome 3A in the wheat genome is TT, CC, or T and C, wherein the set of primers X1 consists of upstream primer sequences SEQ ID No.1 and SEQ ID No.2 and a downstream primer sequence SEQ ID No.3; and the set of primers X2 consists of upstream primer sequences SEQ ID No.5 and SEQ ID No.6 and a downstream primer sequence SEQ ID No.

7.

2. The KASP primer set according to claim 1, It is characterized in that The set of primers X1 contains two upstream primers and one downstream primer; The upstream primer is designed according to the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 4 on chromosome 6B in the wheat genome and its upstream sequence, and the 3' terminal deoxyribonucleotide of one of the upstream primers is T, and the 3' terminal deoxyribonucleotide of the other upstream primer is G; The downstream primer is designed according to the downstream sequence of the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 4 on chromosome 6B in the wheat genome.

3. The KASP primer set according to claim 1, It is characterized in that The set of primers X2 contains two upstream primers and one downstream primer; The upstream primer is designed according to the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 8 on chromosome 3A in the wheat genome and its upstream sequence, and the 3' terminal deoxyribonucleotide of one of the upstream primers is T, and the 3' terminal deoxyribonucleotide of the other upstream primer is C; The downstream primer is based on the sequence shown in SEQ ID No.8 on chromosome 3A in the wheat genome. The downstream sequence of the 36th deoxyribonucleotide in the column is designed.

4. Use of the KASP primer set according to any one of claims 1 to 3 in any of the following: (A) Identifying or assisting in identifying wheat's resistance to infection by fusarium head blight; (B) Comparing the resistance of tested wheat to infection by fusarium head blight; (C) breeding or selecting wheat plants, strains, lines or varieties that are relatively resistant to infection by fusarium head blight; (E) preparing a product for identifying or assisting in identifying the resistance to infection by fusarium head blight of wheat; (F) preparing a product for breeding or screening wheat plants, strains, lines or varieties with relatively strong resistance to infection by ergot.

5. Any of the following methods: Method A: A method for comparing the resistance of wheat scab to infection, comprising the following steps: (A1) detecting whether the genotype of the 36th position of the molecular marker represented by SEQ ID No.4 on chromosome 6B in the wheat genome is TT, GG or TG, and simultaneously detecting whether the genotype of the 36th position of the molecular marker represented by SEQ ID No.8 on chromosome 3A in the wheat genome is TT, CC or TC; (A2) the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the genome is GG and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the genome is CC, and the wheat to be tested has a strong ability to resist infection by fusarium head blight; Method B: A method for breeding wheat plants or strains or lines or varieties with relatively strong resistance to infection by fusarium head blight, comprising the following steps: (B1) detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the wheat genome is TT, GG or TG, and simultaneously detecting whether the genotype of the 36th position of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the wheat genome is TT, CC or TC; (B2) selecting a wheat plant to be tested in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the genome is G and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the genome is C as a parent for breeding, and selecting wheat plants in which the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the genome is G and the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the genome is C in each breeding generation, and finally obtaining wheat plants, strains, lines or varieties with relatively strong resistance to infection by ergot.

6. The method according to claim 5, It is characterized in that The specific operations of (A1) and (B1) are: Using the KASP primer set described in any one of claims 1 to 3 to perform PCR amplification on the wheat genomic DNA to be tested, scanning the amplified product for fluorescence signals, analyzing the scanning data, and then determining the type of the 36th deoxyribonucleotide shown in SEQ ID No. 4 on chromosome 6B in the wheat gene to be tested and the type of the 36th deoxyribonucleotide shown in SEQ ID No. 8 on chromosome 3A in the wheat gene to be tested as follows; If the fluorescent signal data of the amplified product of the wheat to be tested is displayed as blue, the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the genome of the wheat to be tested is a homozygous G, or the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the genome of the wheat to be tested is a homozygous C; If the fluorescent signal data of the amplified product of the wheat to be tested is displayed as red, the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.4 on chromosome 6B in the genome of the wheat to be tested is a homozygous T, or the 36th deoxyribonucleotide of the molecular marker shown by SEQ ID No.8 on chromosome 3A in the genome of the wheat to be tested is a homozygous T.

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