Molecular marker for detecting wheat powdery mildew resistance QTL and application thereof

Abstract

The invention belongs to the technical field of wheat disease resistance breeding, and discloses a molecular marker for detecting wheat powdery mildew resistance QTL and application of the molecular marker. On the basis of a three-year group powdery mildew adult-plant stage resistance identification result, the powdery mildew resistance QTL is detected through a complete interval mapping method (ICIM) of IciMapping v4.2, two major QTLs, namely Qpm.histwrc-1AS and Qpm.histwrc-2BS-1, are detected in total, and the resistance contribution rates are 19.4%-34.5% and 19.7%-21.3%. Related molecular markers are developed through Bainong dwarf 58 sequencing data and can be applied to wheat disease-resistant molecular breeding, and a foundation is laid for exploring an Edinburgh-B disease-resistant gene and a resistance genetic mechanism.

Description

Technical Field

[0001] The invention belongs to the technical field of wheat disease resistance breeding, and relates to a molecular marker for detecting wheat powdery mildew resistance QTL and an application thereof. Background Art

[0002] Wheat powdery mildew is caused by the biotype fungus Blumeria graminis f.sp.tritici (Bgt) infecting plants, which causes the host immune system to over-defend and produce an allergic reaction, causing necrosis of cells in the infected area, resulting in curling and withering of leaves. When the disease is extremely serious, wheat tends to die. When wheat leaves are infected by powdery mildew, respiration increases, stomatal transpiration intensity increases, photosynthesis efficiency decreases, and normal growth and development of wheat is seriously hindered, causing wheat leaves to fall prematurely, wheat ears and ear rate to decrease, and thousand-grain weight to decrease, which has a significant impact on per-acre yield, generally reducing yield by 10% to 30%, and reducing yield by more than 50% in recurrent areas.

[0003] In the 1930s, Australian scientist Waterhouse first reported that a pair of dominant powdery mildew resistance genes existed in the Tew wheat variety; in the 1950s, Sears, ER used missing materials to locate Pm1 in 7AL, and then researchers in many countries began to study the genetic characteristics of powdery mildew resistance genes. Especially in the early 21st century, due to the continuous progress of molecular biology, many scientists at home and abroad have devoted themselves to studying the molecular markers of disease resistance genes to find new sources of wheat resistance. There are 69 officially named powdery mildew resistance genes (Pm1-Pm69). The powdery mildew resistance genes named in common wheat are: Pm1a, Pm1c (Pm18), Pm1d, Pm1e (Pm22), Pm2a, Pm2b, Pm3a, Pm3b, Pm3c, Pm3d, Pm3e, Pm3f, Pm3g, Pm3h, Pm3s, Pm3j, Pm4a, Pm4b, Pm5a, Pm5b, Pm5c, Pm5 d. Pm5e, Pm9, Pm10, Pm11, Pm14, Pm15, Pm16, Pm23, Pm24, Pm25, Pm26, Pm28, Pm30, Pm 31. Pm33, Pm36, Pm38, Pm39, Pm41, Pm42, Pm44, Pm45, Pm46, Pm47, Pm50, Pm54, Pm64. As well as several genes with temporary names, such as MlZec1, MlIW72, PmG16, Ml3D232, MlIW170, PmAs846, PmG3M, MlIW172, MlIW30 and MlIW39; wild relatives of wheat also have rich genetic resources for resistance to powdery mildew, such as Secale cereale (Pm7, Pm8, Pm17 and Pm20), Einkorn wheat (Pm1b, Pm4d and Pm25), Aegilops speltatum (Pm12, Pm32 and Pm53), Triticum timophiu (Pm2, Pm6, Pm27 and Pm37), Triticum persianum (Pm4b and Pm33), Elymus intermedia (Pm40 and Pm43) and Aegilops tauschii (Pm34). According to statistics, the varieties that have lost their disease resistance in the field and have lost their usable value are: Pm1, Pm3a, Pm3b, Pm3c, Pm3f, Pm4a, Pm5, Pm6, Pm7, Pm8, and Pm17; the varieties that have low immunity to diseases in the field and are not suitable for use as single parents are: Pm2, Pm19, Pm4+8, Pm4, Pm5+6, and Pm13.

[0004] QTL positioning is to locate one or more QTLs and gene markers on the same chromosome by studying gene markers or finding the association between gene markers and the traits of the target individual. In other words, the markers are associated with the QTL. Due to the rapid development of biological detection technology, the study of molecular markers and the emergence of a large number of wheat genetic linkage maps, QTL mapping through linkage analysis has been widely used to locate target characteristics. Hui Jian et al. used the additive-epistatic genetic model controlled by the major effect gene of powdery mildew resistance in adult plants suitable for RIL populations to locate two stable disease resistance QTLs QPm.naafs-4D and QPm.naafs-7D from Ningchun 27 and Ningchun 4, respectively. Among them, the LOD of QPm.naafs-4D under different environments was 5.9-15.4, the additive effect was -1.1--1.2, and the phenotypic contribution explanation rate was 18.6%-34.5%. The genetic distance between this site and the flanking markers was 4cM. Shi et al. used BSR-Seq analysis to identify the powdery mildew resistance gene Pm52 controlling the entire growth period located on chromosome 2BL and a new QTL locus qAPRpm.caas.7A for powdery mildew resistance in the adult stage located on chromosome 7AL; the resistance locus for the entire growth period located on 2BL was located in the 1.3 cM genetic interval between the molecular markers Xicscl726 and XicsK128, with a LOD value of up to 28.1-34.7, which can explain 45-52% of the phenotypic variation; qAPRpm.caas_7A was located in the 4.6 cM genetic interval between the molecular markers XicsK7A8 and XicsK7A26.

[0005] In summary, the development of tightly linked molecular markers is of great significance for the accurate and rapid screening and analysis of potential disease-resistant genes in breeding populations, as well as for the selection of new germplasm (new lines) resistant to powdery mildew. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker for detecting wheat powdery mildew resistance QTL and its application. Based on the results of three-year population powdery mildew resistance identification in the adult stage, the present invention detects powdery mildew resistance QTL by using the complete interval mapping method (ICIM) of IciMapping v4.2, and detects two main effect QTLs: Qpm.histwrc-1AS and Qpm.histwrc-2BS-1, with resistance contribution rates of 19.4%-34.5% and 19.7%-21.3%. Then, the relevant molecular markers are developed through the sequencing data of Bainong Aikang 58, which can be applied to molecular breeding of wheat disease resistance, and also lay the foundation for discovering Edinburgh-B disease resistance genes and resistance genetic mechanisms.

[0007] In one aspect, the present invention provides a primer pair for identifying or assisting in identifying wheat powdery mildew resistance, wherein the amplified DNA fragment of the primer pair is closely linked to QTL sites Qpm.histwrc-1AS and / or Qpm.histwrc-2BS-1 related to wheat powdery mildew resistance.

[0008] Furthermore, the site Qpm.histwrc-1AS is between markers 60K_6293890-60K_9361171, with a genetic distance of 1.3 cM and a physical distance of 3.07 Mb, and the site Qpm.histwrc-2BS-1 is between markers 60K_23762001-60K_26341302, with a genetic distance of 5.9 cM and a physical distance of 2.58 Mb.

[0009] Furthermore, the primer pair is:

[0010] 1) Primer pair 1 consisting of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2; and / or

[0011] 2) Primer pair 2 consisting of the sequences shown in SEQ ID NO.3 and SEQ ID NO.4; and / or

[0012] 3) Primer pair 3 consisting of the sequences shown in SEQ ID NO.5 and SEQ ID NO.6; and / or

[0013] 4) Primer pair 4 consisting of the sequences shown by SEQ ID NO.7 and SEQ ID NO.8.

[0014] Furthermore, the DNA fragment of the amplified product of the primer pair 1 and / or the primer pair 2 is closely linked to the QTL site Qpm.histwrc-1AS related to the powdery mildew resistance trait of wheat.

[0015] Furthermore, the DNA fragment of the amplified product of the primer pair 3 and / or the primer pair 4 is closely linked to the QTL site Qpm.histwrc-2BS-1 related to the wheat powdery mildew resistance trait.

[0016] On the other hand, the present invention also provides a kit for identifying or assisting in identifying the powdery mildew resistance trait of wheat, comprising the primer pairs 1-4 and at least one of the following: dNTP, DNA polymerase and PCR amplification buffer.

[0017] On the other hand, the present invention also provides a method for identifying or assisting in identifying the powdery mildew resistance trait of wheat hybrid offspring, comprising the following steps:

[0018] (a1) using the genomic DNA of wheat A, wheat B and each individual in a wheat hybrid offspring population obtained by hybridizing wheat A and wheat B as templates, respectively, and performing PCR amplification using the primer pairs described in any one of claims 1 to 5 to obtain a PCR product of wheat A, a PCR product of wheat B and a PCR product of each individual in the wheat hybrid offspring population;

[0019] The wheat A is a powdery mildew resistant variety, and the wheat B is a powdery mildew non-resistant variety;

[0020] (a2) performing electrophoresis on the PCR product of wheat A, the PCR product of wheat B, and the PCR product of each individual in the wheat hybrid offspring group, and determining the powdery mildew resistance trait of the wheat hybrid offspring according to the electrophoresis results as follows: the wheat hybrid offspring having the same band pattern as the electrophoresis band of the PCR product of wheat A has the powdery mildew resistance trait, and the wheat hybrid offspring having the same band pattern as the electrophoresis band of the PCR product of wheat B does not have the powdery mildew resistance trait.

[0021] On the other hand, the present invention also provides a method for identifying or assisting in identifying whether the wheat powdery mildew resistance QTL site Qpm.histwrc-1AS and / or Qpm.histwrc-2BS-1 exists in the genome of the wheat to be tested, comprising the following steps:

[0022] (b1) using the genomic DNA of the wheat to be tested as a template, and using the primer pair 1-2 to perform PCR amplification to obtain a PCR product;

[0023] (b2) subjecting the PCR product to electrophoresis, and determining whether the wheat powdery mildew resistance QTL site Qpm.histwrc-1AS exists in the genome of the wheat to be tested according to the electrophoresis result as follows: if the band pattern of the electrophoresis band of the PCR product is the same as the reference band pattern A, or contains both the reference band pattern A and the reference band pattern B, then the wheat powdery mildew resistance QTL site Qpm.histwrc-1AS exists or is a candidate for existence in the genome of the wheat to be tested; if the band pattern of the electrophoresis band of the PCR product is the same as the reference band pattern B, then the wheat powdery mildew resistance QTL site Qpm.histwrc-1AS does not exist or is a candidate for existence in the genome of the wheat to be tested; and / or,

[0024] (c1) using the genomic DNA of the wheat to be tested as a template, and using the primer pairs 3-4 to perform PCR amplification to obtain a PCR product;

[0025] (c2) performing electrophoresis on the PCR product, and determining whether the wheat powdery mildew resistance QTL site Qpm.histwrc-2BS-1 exists in the genome of the wheat to be tested according to the electrophoresis result as follows: if the band pattern of the electrophoresis band of the PCR product is the same as the reference band pattern A, or contains both the reference band pattern A and the reference band pattern B, then the wheat powdery mildew resistance QTL site Qpm.histwrc-2BS-1 exists or is a candidate for existence in the genome of the wheat to be tested; if the band pattern of the electrophoresis band of the PCR product is the same as the reference band pattern B, then the wheat powdery mildew resistance QTL site Qpm.histwrc-2BS-1 does not exist or is a candidate for existence in the genome of the wheat to be tested.

[0026] Furthermore, the site Qpm.histwrc-1AS is between markers 60K_6293890-60K_9361171, with a genetic distance of 1.3 cM and a physical distance of 3.07 Mb.

[0027] Furthermore, the site Qpm.histwrc-2BS-1 is between markers 60K_23762001-60K_26341302, with a genetic distance of 5.9 cM and a physical distance of 2.58 Mb.

[0028] Furthermore, the reference band type A is a band type obtained by using the genome DNA of the wheat variety Edinburgh-B as a template, using the primer pair for PCR amplification, and performing electrophoresis on the obtained PCR product.

[0029] Furthermore, the reference band type B is a band type obtained by using the genomic DNA of the wheat variety Bainong's Avian Kang 58 as a template, using the primer pair for PCR amplification, and performing electrophoresis on the obtained PCR product.

[0030] On the other hand, the present invention also provides a method for breeding a wheat variety with resistance to powdery mildew, which comprises selecting wheat that meets the following conditions as a parent for breeding: using genomic DNA as a template, using the primer pair to perform PCR amplification, performing electrophoresis on the obtained PCR product, and the band pattern of the electrophoresis band is the same as the reference band pattern A;

[0031] The reference band type A is a band type obtained by using the genome DNA of the wheat variety Edinburgh-B as a template, using the primer pair for PCR amplification, and performing electrophoresis on the obtained PCR product.

[0032] On the other hand, the present invention also provides a molecular marker related to the powdery mildew resistance trait of wheat, characterized in that it is a DNA fragment obtained by PCR amplification using the primer pair with wheat genomic DNA as a template.

[0033] On the other hand, the present invention also provides the use of the primer pair or the kit or the molecular marker in any of the following:

[0034] (1) Identify or assist in identifying wheat powdery mildew resistance traits;

[0035] (2) Breeding of wheat varieties resistant to powdery mildew.

[0036] Compared with the prior art, the present invention has the following technical effects.

[0037] 1) The present invention uses the QTL positioning method, combined with the constructed genetic linkage map and population to locate the resistance-susceptibility phenotypes of powdery mildew and leaf rust, and the highly reliable new powdery mildew resistance genes Qpm.histwrc-1AS and Qpm.histwrc-2BS-1 discovered can explain 19.4%-34.5% and 19.7-21.3% of the phenotypic variation rates, respectively, laying a foundation for the subsequent continued research on the gene.

[0038] 2) The present invention uses SSR molecular markers to encrypt the genetic linkage map and develops four molecular markers that are tightly linked to Qpm.histwrc-1AS and Qpm.histwrc-2BS-1, contributing to the use of these molecular markers for auxiliary selection in breeding practice, accelerating the breeding process and improving selection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] 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.

[0040] Figure 1 The phenotypic evaluation diagram of the response of parents and RILs to powdery mildew, where A is the response of Edinburgh-B, Bainong's Aikang 58 and F1 plants to powdery mildew, and B is the degree of powdery mildew infection in the field recorded by Edinburgh-B / Bainong's Aikang 58 RILs in 3 years.

[0041] Figure 2 The diagram shows the corresponding genetic location region of the powdery mildew QTL on chromosome arms 1As and 2Bs (B) under the three-year environment.

[0042] Figure 3 The effects of different QTL combinations on reducing the severity of powdery mildew.

[0043] Figure 4 The results are the validation results of the marker in the Edinburgh-B / Bainong dwarf 58BCF6 population and in Bainong 5822. DETAILED DESCRIPTION

[0044] 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 the protection scope of the present invention cannot be limited by this. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs.

[0045] Example 1 Phenotypic evaluation of the response of parents and RILs to powdery mildew

[0046] 203 F 6:7 The recombinant inbred line population (RIL population) was derived from the hybridization of Edinburgh-B, a powdery mildew resistant material, and Bainong's Aikang 58, a powdery mildew susceptible material. Edinburgh-B was used as the powdery mildew resistant control, and Bainong's Aikang 58 was used as the powdery mildew susceptible control. The recombinant inbred line population was planted in the Langgongmiao experimental base. A completely randomized design experiment was adopted, with one row area, 60 seeds per row, 1m row length, 2 replicates, and row spacing of 25cm. The field was managed according to the local normal cultivation method. The seedlings were planted in the greenhouse in the climate room of the wheat central experimental base of Henan Institute of Science and Technology. 10 seeds were planted per hole\bag (bucket), and 5 seedlings were finally determined.

[0047] Powdery mildew resistance identification: Resistant and susceptible parents were planted in the middle of each tray as resistant and susceptible controls, respectively. The disease was naturally caused by the invasion of Bgt strains in the greenhouse. Recording began when the primary lesions appeared on the seedlings of the first leaf of the susceptible parent. Recording was done 4 times with a time interval of 5 days. When the third record was made, the susceptible parent and the susceptible control had reached 80% or more. The infection types (ITs) (refer to Liu 1999, etc.) were divided into two groups according to the 0-4 standard (Table 1), resistant (R, IT 0-2) and susceptible (S, IT 3-4). Disease evaluation was performed at least twice, and the highest IT of each line was used for phenotype and QTL.

[0048] Table 1 Powdery mildew identification specification reference

[0049]

[0050] Processing of phenotypic data: The mean IT of individual disease resistance levels in the RIL population in each environment was subjected to variance analysis to determine the effects of genotype (G), environment (E), and G×E interaction. Pearson correlation coefficient (r) analysis and variance analysis were performed using the "AOV" function in QTL IciMapping software 4.2 with default parameters. In addition, the average phenotypic values ​​of all three environments were used to evaluate the genetic effects and find the best confidence interval for each QTL.

[0051] Using the above method, this experiment investigated the response of Bainong 4199, Bainong 5819, Bainong 5822, Zhoumai 22 and other varieties to powdery mildew at the seedling and adult stages, and investigated the response of Edinburgh-B / Bainong Aikang 58 F1 and F2 to powdery mildew. After comparative analysis of several varieties, it was clearly found that Bainong 5822 and Edinburgh-B with Edinburgh-B background had the same resistance to powdery mildew, while the rest were not resistant. The results are shown in Table 2.

[0052] Table 2 Responses of six varieties to powdery mildew at seedling and adult stages

[0053]

[0054] The genetic analysis of the F1 generation showed that the resistance of the F1 generation was consistent with that of the parent, Bainong'aikang 58; the genetic analysis of the F2 generation showed that the powdery mildew resistance was controlled by a recessive gene and did not conform to the segregation ratio of a pair of genes (R / S=150 / 141), so the disease resistance was expressed by multiple genes. As shown in Table 3.

[0055] Table 3 Investigation on the response of Edinburgh-B, Bainong's Akang 58 and their F1 and F2 to powdery mildew

[0056]

[0057] The Edinburgh-B / Bainong's Aikang 58 recombinant inbred line population (RIL population) was identified for powdery mildew resistance in adult plants under three environments at the Langgongmiao Experimental Base of Henan Institute of Science and Technology in Xinxiang County in 2021-2022 and in the greenhouse on campus of the Wheat Center of Henan Institute of Science and Technology in 2023. Edinburgh-B is resistant to powdery mildew infection, Bainong's Aikang 58 is not resistant to powdery mildew infection, and F1 is not resistant to powdery mildew infection (such as Figure 1 Figure A in the middle); the IT data of RILs presents a continuous distribution (e.g. Figure 1 B), indicating that the resistance of Edinburgh-B is quantitatively inherited. The Pearson correlation coefficient of powdery mildew in the three environments was 0.651-0.666 (P<0.001) (Table 4), and the variance analysis P value of IT was very significant (P<0.001) (Table 5). These results indicate that the resistance QTL is effective in these three environments.

[0058] Table 4 Correlation coefficients (r) of powdery mildew infection types of Edinburgh-B×Bainong'aikang 58 recombinant inbred lines under different field environments

[0059]

[0060] Table 5 The Langgongmiao Edinburgh-B×Bainongdwarf RIL population and

[0061]

[0062] Analysis of variance (ANOVA) of powdery mildew infection type data of HC population in 2023

[0063] Example 2 Linkage Mapping of Powdery Mildew Resistance Genes Based on Genetic Linkage Maps of RILs

[0064] Fresh and tender wheat leaves were taken to extract genomic DNA. The purity and concentration of DNA were detected by NanoDrop 8000, and the integrity of samples was detected by agarose gel electrophoresis.

[0065] The wheat exon capture sequencing technology detection platform (Chengdu Tiancheng Future Technology Co., Ltd.) wheat 60K wheat liquid phase chip (GBTS) was used to detect the genotypes of parents and populations. Referring to Zhu Dongmei et al., the original genotype data analyzed by 60K liquid phase chip were screened using IciMapping v4.2 software (http: / / www.isbreeding.net) developed by the Chinese Academy of Agricultural Sciences, and the Binning function was used to remove redundancy. At the same time, polymorphic markers with a missing rate of 80% were set to be screened, and the markers were grouped using the MAP function. The Kosambi function was used to sort the markers in each group and calculate the genetic distance; the genetic linkage map was drawn using MapChart 2.32 (https: / / www.wur.nl / en / show / Mapchart.htm); the disease resistance QTL was detected using the complete interval mapping method (ICIM) of IciMapping v4.2 in combination with the disease resistance phenotypic data, and the LOD threshold was set to 2.5; QTLs with peak values ​​detected on the same chromosome less than 10 cM apart in genetic position were regarded as the same locus, and the naming method of QTL was referred to Hu Wenjing et al. The flanking marker sequences of the located QTL and the flanking marker sequences of similar loci reported before were compared with the Chinese spring reference genome sequence version 2.1 in the Ensembl Plants database (http: / / plants.ensembl.org / ) to determine the novelty of the QTL loci.

[0066] Combining the RILs genetic linkage map and the susceptible phenotype of the population, QTL Icimapping 4.2 software was used to locate QTLs. In the result analysis, a total of 11 powdery mildew resistance QTLs were detected, located on 1A, 1D, 2B, 2D, 4B, and 7B, and the Add values ​​were all negative, so the 11 QTLs were all from Edinburgh-B. As shown in Table 6, Qpm.histwrc-1AS was detected simultaneously in three different years and means, with LOD values ​​of 10.99-28.35 and resistance contribution rates (PVE) of 19.4%-34.5%. The LOD values ​​and PVE were the highest, and this site was the main effect site; Qpm.histwrc-2BS-1 was detected simultaneously in two different years and means, with LOD values ​​of 7.20-12.85 and resistance contribution rates of 19.7%-21.3%. The LOD values ​​and PVE were relatively high, and this site was also the main effect site; the above two QTLs are stable main effect sites for powdery mildew resistance detected in this study.

[0067] Table 6 QTL location table for powdery mildew resistance in Edinburgh-B / Bainong's dwarf 58 RILs population

[0068]

[0069] like Figure 2 As shown, the main effect locus Qpm.histwrc-1AS is between markers 60K_6293890-60K_9361171, with a genetic distance of 1.3 cM and a physical distance of 3.07 Mb. Figure 3 As shown, Qpm.histwrc-2BS-1 is between markers 60K_23762001-60K_26341302, with a genetic distance of 5.9 cM and a physical distance of 2.58 Mb.

[0070] Example 3 Interactions between major disease resistance QTLs

[0071] In order to explore the combined effects of different main effect disease resistance QTLs, RILs were divided into three genotype groups based on the field experimental data (Table 7).

[0072] Table 7 Effects of different QTL combinations on RIL population

[0073]

[0074] The RILs containing Qpm.histwrc-1AS and Qpm.histwrc-2BS-1 were more resistant than all other QTLs and showed similar resistance to Edinburgh-B ( Figure 3). The combination of the two resistance loci, Qpm.histwrc-1AS and Qpm.histwrc-2BS-1, has a significant effect on reducing the severity of powdery mildew infection. The average IT value without the presence of two stable QTLs is 3, the average IT value with only one disease resistance QTL (1AS or 2BS) is 1.2, the average IT value at the 1AS locus is 1, and the average IT value at the 2BS locus is 1.5; when both disease resistance QTL loci, Qpm.histwrc-1AS and Qpm.histwrc-2BS-1, are present, the average IT value is 0.6, which has a significant effect on reducing IT. Wheat lines containing both 1AS-2BS are on average more resistant than lines without them, indicating that the effects of two separate QTLs may be affected by recombination between markers, genetic background, and environment.

[0075] Example 4 Tightly linked SSR markers of disease resistance QTL loci

[0076] The sequence of the stable disease resistance QTL interval was compared with the sequence of Bainong Aikang 58 to find the corresponding interval position of the sequence of the stable disease resistance QTL interval on Bainong Aikang 58, and the 4Mb and 8Mb DNA sequences of 5085462-9848861bp on chromosome 1A and 15834495-23393460bp on chromosome 2B of Bainong Aikang 58 were downloaded, and the SSRHunter software was used to search for SSR sites containing different nucleotide types (the number of repetitions of the nucleotide sequence is greater than or equal to 5 times). The distribution of SSRs of each repeated nucleotide on different chromosomes is shown in Table 8.

[0077] Table 8 SSR distribution of repeated nucleotides on different chromosomes

[0078]

[0079]

[0080] Primer 5.0 software was used to design SSR primers based on the 150bp DNA sequences on both sides of the searched SSR locus. The conditions for designing primers are: ① The primer length range is 18-22bp; ② The annealing temperature range is 50-65℃, and the difference between the upstream and downstream primer annealing temperatures is no more than 5℃; ③ The PCR amplification fragment length range is 100-400bp, and the appearance of secondary structure and hairpin structure should be avoided as much as possible. The designed primer sequence was synthesized by General Bio (Anhui) Co., Ltd., and the primer was named HISTWCR plus serial number. After screening, the polymorphic molecular markers developed were determined as shown in Table 9.

[0081] Table 9 Developed polymorphic molecular markers

[0082]

[0083] Example 5 Validation of markers in the BCF6 population

[0084] Tightly linked markers were used to verify the effects of Qpm.histwrc-1AS and Qpm.histwrc-2BS-1 loci on powdery mildew resistance in 65 Edinburgh-B / Bainong's Avian Kang 58BCF6 population test materials, and the presence of resistance genes was verified in Bainong 5822 (Bainong's Avian Kang 58 / Edinburgh-B / / Zhoumai 22).

[0085] In the BCF6 population, four markers HISTWRC238, HISTWRC235, HISTWRC195, and HISTWRC317 that are closely linked to the two loci Qpm.histwrc-1AS and Qpm.histwrc-2BS-1 were verified. Among them, the application of the two co-segregation markers HISTWRC238 and HISTWRC235 on the 1AS chromosome will more finely distinguish the homozygous resistant plants, homozygous susceptible plants, and heterozygous plants in the individual plants for the Qpm.histwrc-1AS gene. The two co-segregation markers HISTWRC195 and HISTWRC317 on the 2BS chromosome will finely distinguish the difference plants in the individual plants for the Qpm.histwrc-2BS-1 gene.

[0086] As shown in Table 10, in the BCF6 population, when both sites are present, the average IT is 0.5, when one site is present, the average IT is 1.9, and when both sites are absent, the average IT is 3.17. The effect of reducing IT is consistent with that in the RIL population.

[0087] Table 10 Verification of the mutual influence of different QTL combinations in BCF6 population

[0088]

[0089] Secondly, in the new variety Bainong 5822 (Bainong Aikang 58 / Edinburgh-B / / Zhoumai 22) with Edinburgh-B as the background, HISTWRC238, HISTWRC235, HISTWRC195, and HISTWRC317 were specifically amplified ( Figure 4 ).

[0090] In summary, based on the verification results, the effects of the powdery mildew resistance gene Qpm.histwrc-1AS on chromosome 1AS and the powdery mildew resistance gene Qpm.histwrc-2BS-1 on chromosome 2BS are very stable, and can play a stabilizing role when introduced into other backgrounds. Moreover, in the high-generation breeding lines, their excellent allele variations have undergone strong positive selection, which fully demonstrates their application value. In breeding practice, the use of closely linked molecular markers for auxiliary selection will inevitably accelerate the breeding process and improve the selection efficiency. The four co-segregated markers designed and developed, namely HISTWRC238, HISTWRC235, HISTWRC195, and HISTWRC317, can also be well verified in the BCF6 population and can be well used in the breeding of powdery mildew-resistant wheat.

[0091] 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 primer pair for identifying or assisting in identifying the powdery mildew resistance trait of wheat, characterized in that: The DNA fragment of the amplified product of the primer pair is closely linked to the QTL sites Qpm.histwrc-1AS and / or Qpm.histwrc-2BS-1 related to the powdery mildew resistance trait of wheat.

2. The primer pair according to claim 1, characterized in that The site Qpm.histwrc-1AS is between markers 60K_6293890-60K_9361171, with a genetic distance of 1.3 cM and a physical distance of 3.07 Mb. The site Qpm.histwrc-2BS-1 is between markers 60K_23762001-60K_26341302, with a genetic distance of 5.9 cM and a physical distance of 2.58 Mb.

3. The primer pair according to claim 1 or 2, characterized in that: The primer pair is, 1) Primer pair 1 consisting of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2; and / or 2) Primer pair 2 consisting of the sequences shown in SEQ ID NO.3 and SEQ ID NO.4; and / or 3) Primer pair 3 consisting of the sequences shown in SEQ ID NO.5 and SEQ ID NO.6; and / or 4) Primer pair 4 consisting of the sequences shown by SEQ ID NO.7 and SEQ ID NO.

8.

4. The primer pair according to claim 3, characterized in that: The DNA fragments of the amplified products of the primer pair 1 and / or the primer pair 2 are closely linked to the QTL site Qpm.histwrc-1AS related to the wheat powdery mildew resistance trait.

5. The primer pair according to claim 3, characterized in that: The DNA fragments of the amplified products of the primer pair 3 and / or the primer pair 4 are closely linked to the QTL site Qpm.histwrc-2BS-1 related to the powdery mildew resistance trait of wheat.

6. A kit for identifying or assisting in identifying wheat powdery mildew resistance traits, characterized in that: Contains the primer pair according to any one of claims 1 to 5 and at least one of the following: dNTP, DNA polymerase and PCR amplification buffer.

7. A method for identifying or assisting in identifying the powdery mildew resistance trait of wheat hybrid offspring, comprising the following steps: (a1) using genomic DNA of wheat A, wheat B, and each individual in a wheat hybrid offspring population obtained by hybridizing wheat A and wheat B as templates, and using the primer pairs described in any one of claims 1 to 5 to perform PCR amplification respectively, to obtain a PCR product of wheat A, a PCR product of wheat B, and a PCR product of each individual in the wheat hybrid offspring population; wheat A is a powdery mildew resistant variety, and wheat B is a non-powdery mildew resistant variety; (a2) performing electrophoresis on the PCR product of wheat A, the PCR product of wheat B, and the PCR product of each individual in the wheat hybrid offspring group, and determining the powdery mildew resistance trait of the wheat hybrid offspring according to the electrophoresis results as follows: the wheat hybrid offspring having the same band pattern as the electrophoresis band of the PCR product of wheat A has the powdery mildew resistance trait, and the wheat hybrid offspring having the same band pattern as the electrophoresis band of the PCR product of wheat B does not have the powdery mildew resistance trait.

8. A method for identifying or assisting in identifying whether there is a wheat powdery mildew resistance QTL site Qpm.histwrc-1AS and / or Qpm.histwrc-2BS-1 in the genome of the wheat to be tested, comprising the following steps: (b1) using the genomic DNA of the wheat to be tested as a template, and using the primer pairs 1-2 described in claims 1-5 to perform PCR amplification to obtain a PCR product; (b2) subjecting the PCR product to electrophoresis, and determining whether the wheat powdery mildew resistance QTL site Qpm.histwrc-1AS exists in the genome of the wheat to be tested according to the electrophoresis result as follows: if the band pattern of the electrophoresis band of the PCR product is the same as the reference band pattern A, or contains both the reference band pattern A and the reference band pattern B, then the wheat powdery mildew resistance QTL site Qpm.histwrc-1AS exists or is a candidate for existence in the genome of the wheat to be tested; if the band pattern of the electrophoresis band of the PCR product is the same as the reference band pattern B, then the wheat powdery mildew resistance QTL site Qpm.histwrc-1AS does not exist or is a candidate for existence in the genome of the wheat to be tested; and / or, (c1) using the genomic DNA of the wheat to be tested as a template and using the primer pairs 3-4 described in claims 1-5 to perform PCR amplification to obtain a PCR product; (c2) performing electrophoresis on the PCR product, and determining whether the wheat powdery mildew resistance QTL site Qpm.histwrc-2BS-1 exists in the genome of the wheat to be tested according to the electrophoresis result as follows: if the band pattern of the electrophoresis band of the PCR product is the same as the reference band pattern A, or contains both the reference band pattern A and the reference band pattern B, then the wheat powdery mildew resistance QTL site Qpm.histwrc-2BS-1 exists or is a candidate for existence in the genome of the wheat to be tested; if the band pattern of the electrophoresis band of the PCR product is the same as the reference band pattern B, then the wheat powdery mildew resistance QTL site Qpm.histwrc-2BS-1 does not exist or is a candidate for existence in the genome of the wheat to be tested; The site Qpm.histwrc-1AS is between markers 60K_6293890-60K_9361171, with a genetic distance of 1.3 cM and a physical distance of 3.07 Mb, and the site Qpm.histwrc-2BS-1 is between markers 60K_23762001-60K_26341302, with a genetic distance of 5.9 cM and a physical distance of 2.58 Mb; The reference band type A is a band type obtained by electrophoresing the PCR product obtained by using the genomic DNA of the wheat variety Edinburgh-B as a template and using the primer pair described in any one of claims 1 to 5 for PCR amplification; The reference band type B is a band type obtained by using the genomic DNA of the wheat variety Bainong's Aikang 58 as a template, performing PCR amplification using the primer pair described in any one of claims 1 to 5, and performing electrophoresis on the obtained PCR product.

9. A method for breeding a wheat variety with resistance to powdery mildew, comprising selecting wheat that meets the following conditions as a parent for breeding: using genomic DNA as a template, using the primer pair described in any one of claims 1 to 5 to perform PCR amplification, performing electrophoresis on the resulting PCR product, and the band pattern of the electrophoresis band is the same as reference band pattern A; The reference band type A is a band type obtained by using the genome DNA of the wheat variety Edinburgh-B as a template, performing PCR amplification using the primer pair described in any one of claims 1 to 5, and performing electrophoresis on the obtained PCR product.

10. A molecular marker associated with wheat powdery mildew resistance, characterized in that: The invention relates to a DNA fragment obtained by PCR amplification using wheat genomic DNA as a template and using any primer pair described in claims 1 to 5.

11. Use of the primer pair according to any one of claims 1 to 5, the kit according to claim 6, or the molecular marker according to claim 10 in any of the following: (1) Identify or assist in identifying wheat powdery mildew resistance traits; (2) Breeding of wheat varieties resistant to powdery mildew.

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