AQP molecular markers, primers, and applications of the major QTL for wheat stripe rust resistance at the seedling stage

Abstract

This application discloses an AQP molecular marker for a major QTL for wheat stripe rust seedling resistance, its primers and its application, aiming to solve the current technical problem of lack of a stable QTL for CYR34 resistance. AX‑111630305~AX‑109458466 Within the 0.34 cM range QYr.hau‑S‑7BL , in this QYr.hau‑S‑7BL A SNP marker was found at 718.77 Mb on the physical map within the physical interval tightly linked to the major QTL controlling stripe rust resistance at the seedling stage. AX‑ 108877192 , with a genetic distance of 0.76 cM to the major QTL. The AQP molecular markers located in this application have a large and stable genetic effect (with a mean genetic effect of 32.4). The AQP molecular markers are closely linked to the target QTL, meeting the needs of molecular breeding.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to an AQP molecular marker for a major QTL of wheat stripe rust resistance at the seedling stage, a primer thereof, and an application thereof. Background Art

[0002] Wheat is one of my country's most important staple crops. With the rigid growth of food demand and the increasing difficulty of increasing grain production, my country's grain supply and demand will be in a tight balance for a long time. Among the many biotic stresses, stripe rust is a major fungal disease worldwide, posing a serious threat to the safe production of wheat.

[0003] Wheat stripe rust is caused by Puccinia striatum wheat-specific type ( Puccinia striiformis f. sp. tritici , Pst Stripe rust is a major airborne foliar disease of wheat, often causing 5-25% yield losses and, in years with a high incidence of stripe rust, even 100% total crop failure. The wheat stripe rust pathogen, Puccinia streakinsis, mutates frequently, making it highly likely that wheat varieties will lose their resistance to the disease, leading to outbreaks. Wheat resistance to stripe rust is generally categorized into two types: non-race-specific and race-specific. Race-specific resistance, because it maintains resistance throughout the entire growth period, is also known as seedling resistance or all-stage resistance (ASR). ASR is typically controlled by a single or oligogene and manifests as a quality trait, conferring high resistance to a specific race throughout the growth period. Therefore, wheat breeders often utilize these genes for wheat cultivar improvement. However, these genes suffer from a significant drawback: their resistance is not durable. After widespread field use, resistance may be lost within 3-5 years. When several ASR genes are combined with adult-stage disease resistance genes, they often provide sufficient and long-lasting resistance.

[0004] With the emergence of CYR34, it has quickly become the dominant species in my country. It has the characteristics of high frequency, strong toxicity, wide pathogenicity, high temperature resistance, and disease resistance genes. Yr26 、 Yr10All are toxic, with a toxicity spectrum similar to that of Stripe 32 and Stripe 33, but with a wider toxicity spectrum. This has caused most wheat varieties in my country's main wheat-producing areas to lose their disease resistance, leaving fewer resistant varieties, posing a potential threat to wheat production. There is an urgent need to find resistance sources carrying new genes for resistance to CYR34 races for the improvement of new varieties. Therefore, increasing efforts to screen stripe rust-resistant germplasm, broadening the genetic basis of resistance, creating excellent new wheat germplasm with resistance to CYR34, discovering QTL loci with stable resistance to CYR34 and developing molecular markers closely linked to them for molecular marker-assisted selection breeding of stripe rust resistance, and then cultivating new wheat varieties that aggregate multiple stripe rust-resistant genes suitable for promotion and cultivation in the Huanghuai wheat region have become major scientific research tasks that urgently need to be addressed in the Huanghuai wheat region.

[0005] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0006] In view of at least one of the above technical problems, this application develops and provides an AQP molecular marker and its amplification primer based on the major QTL of wheat stripe rust seedling resistance based on the discovery of markers closely linked to the major QTL of wheat stripe rust resistance, and provides a technical approach for its utilization, aiming to solve the current technical problem of the lack of QTL with stable resistance to CYR34.

[0007] According to one aspect of the present disclosure, an AQP molecular marker for a major QTL for wheat stripe rust resistance at the seedling stage is provided. The major QTL is located on the long arm of wheat chromosome 7. QYr.hau-S-7BL , which contains two linked SNP molecular markers AX-111630305 and AX-109458466 , QYr.hau-S-7BL Positioned in AX-111630305~AX- 109458466 The 0.34 cM interval between the two loci corresponds to the physical map of the Chinese Spring RefSeq_v1.0 reference genome of 717.89 to 720.32 Mb, explaining 27.1 to 38.8% of the phenotypic variation, with an additive effect on the stripe rust response type of -1.0 to -1.3.

[0008] According to another aspect of the present disclosure, an AQP molecular marker for a major QTL for wheat stripe rust resistance at the seedling stage is provided, wherein the major QTL is located on the long arm of wheat chromosome 7. AX-111630305~AX-109458466 Within the 0.34cM range QYr.hau-S-7BL , in this QYr.hau-S-7BL A SNP marker was found at 718.77 Mb on the physical map within the physical interval tightly linked to the major QTL controlling stripe rust resistance at the seedling stage. AX-108877192, and its genetic distance to the major effect QTL was 0.76 cM.

[0009] According to another aspect of the present disclosure, an AQP molecular marker primer is provided, and the primer sequence is as follows:

[0010] .

[0011] According to another aspect of the present disclosure, a detection kit containing the above-mentioned AQP molecular marker primers is provided.

[0012] According to another aspect of the present disclosure, the AQP molecular marker, the AQP molecular marker primer or the detection kit is respectively applied to wheat breeding, identification of wheat stripe rust resistance, and genotyping of major effect QTL of wheat stripe rust resistance at the seedling stage.

[0013] In some embodiments of the present disclosure, the main method steps for genotyping the major effect QTL for wheat stripe rust resistance at the seedling stage include:

[0014] (1) Extraction of wheat genomic DNA;

[0015] (2) AX-108877192 The extracted genomic DNA was amplified by PCR using AQP-labeled primers, and the end-point fluorescence signal of the amplification product was collected for corresponding analysis;

[0016] (3) Genotyping: Cluster the samples and further determine the genotype based on the sample clusters.

[0017] In some embodiments of the present disclosure, the step of PCR amplification includes:

[0018] PCR reaction system: including 4 uL of template DNA at a concentration of 100 ng / uL, 4 uL of HiGeno 2x Probe Mix A and AQP Primer Mix (take 420 uL of HiGeno 2x Probe Mix A and 12.5 uL of AQP Primer Mix, gently vortex and mix, then centrifuge), a total of 8 uL;

[0019] PCR reaction procedure: pre-denaturation at 95°C for 10 min; first amplification reaction: denaturation at 95°C for 20 s, gradient annealing and extension at 61°C for 40 s, 10 cycles, with the annealing temperature decreasing by 0.6°C each cycle; second amplification reaction: denaturation at 95°C for 20 s, annealing and extension at 55°C for 40 s, 28–34 cycles; storage at 25°C;

[0020] The AQP primer mixture includes: a FAM forward primer, a HEX forward primer, a reverse primer, and purified water; the concentration of the FAM forward primer is 12 uM, the concentration of the HEX forward primer is 12 uM, and the concentration of the reverse primer is 30 uM;

[0021] Analysis of detection results: A real-time fluorescence quantitative PCR instrument (Thermo Fisher, QuantStudio™ 5) was used to collect the endpoint fluorescence signal of the amplification product at 35°C, and the data were visualized using QuantStudio™ Design & Analysis Software v1.4.3.

[0022] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0023] The average genetic effect of the QTL located by the AQP molecular marker obtained in this application reached 32.4, which is large and stable. The genetic distance between the AQP molecular marker and the QTL is close (the genetic distance is 0.76 cM), and the linkage disequilibrium coefficient D' value with the markers on both sides of the QTL is 0.98, which has a close linkage disequilibrium relationship and can meet the needs of molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the main effect QTL for stripe rust resistance in YN1813 seedling stage in one embodiment of the present application ( QYr.hau-S -7BL ) positioning map.

[0025] Figure 2 In one embodiment of this application AQP-AX-108877192 Genotyping results of 344 germplasm resources from different sources; in the left figure, red dots indicate that the genotype of the sample is homozygous for the HEX-marked allele; blue dots indicate that the genotype of the sample is homozygous for the FAM-marked allele; green dots indicate that the genotype of the sample is heterozygous; black squares are negative controls (NTC); X indicates that the material has not been typed; in the right figure, the markers AQP-AX108877192 Genotyping of 344 germplasm resources from different sources (genotype identification was homozygous), box plot of the significant difference between resistant and susceptible groups, TT genotype is the resistant type. DETAILED DESCRIPTION

[0026] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific implementation methods.

[0027] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; the reagents and raw materials involved are all conventional commercially available products unless otherwise specified; the detection and test methods involved are all conventional methods unless otherwise specified.

[0028] Example 1

[0029] (1) QTL mapping population:

[0030] The new high-resistance line YN1813 to stripe rust was hybridized with the stripe rust-susceptible local variety China Spring to produce F1, and the F2 generation was self-fertilized and then the recombinant inbred line F containing 308 lines was constructed by single seed fertilization. 7:8 Genetic population.

[0031] (2) Identification of resistance to stripe rust at the seedling stage:

[0032] Seedling resistance characterization of recombinant inbred line populations, parents, and 344 wheat germplasm accessions from different sources was conducted at the Xuchang campus of Henan Agricultural University in the spring of 2021. The single subspecies of the stripe rust fungus, CYR34, was used. The test materials were planted in 50-well trays, with 10 seeds per well. The susceptible control, Mingxian 169, was planted in two narrow rows on each tray. When the wheat reached the two-leaf stage, the leaves were inoculated using a dusting method followed by a spore spray. First, a mixture of Tween 20 diluted approximately 1:1000 was sprayed onto the leaves for dewaxing. The leaves were then inoculated with a spore spray containing a mixture of uredia spores (1:2000). Talc powder (approximately 1:30) containing uredia spores was placed in a wide-mouthed plastic bottle. Two to three layers of gauze were placed around the bottle mouth to secure it. The bottle was then inverted and gently tapped to release the talc onto the leaves for inoculation. After inoculation, the seedlings were incubated in a dark room at 9-11°C and relative humidity greater than 80% for 36 hours. They were then transferred to a greenhouse and incubated at approximately 17°C with 14 hours of light (high-light cultivation). After approximately 15 days, the susceptible controls developed complete disease, at which point the response type was investigated. The response type was determined using a 0-9 scale. According to the 9-scale, 0-6 indicates resistance, and 7-9 indicates susceptible. Based on actual disease activity, the response type was recorded twice, with an interval of 2-3 days between each. Resistance testing at the seedling stage was replicated twice, and the results of the two replicates (IT1 and IT2), the maximum value of the two replicates (ITmax), and the mean of the two replicates (ITmean) were used as the four data points for QTL mapping.

[0033] (3) DNA extraction and QTL mapping:

[0034] DNA was extracted from parents, RILs, and germplasm using the CTAB method. Genotyping of the "YN1813 / Chinese Spring" RIL population was performed using a wheat 55K SNP array. A genetic linkage map was constructed using the Kosambimapping function in the QTL mapping software IciMapping v4.2. QTL mapping was performed using the Inclusive Composite Interval Mapping (ICIM) method using the Biparental Populations (BIP) function.

[0035] The major QTL controlling stripe rust resistance in YN1813 was located on the long arm of chromosome 7 (7BL) of wheat using the "YN1813 / Chinese Spring" RIL population. QYr.hau-S-7BL ( Figure 1 The QTL was stable in different environments, explaining 27.1-38.8% of the phenotypic variation and had an additive effect on the stripe rust response type of -1.0 to -1.3. QYr.hau-S- 7BL Mapped to SNP markers AX-111630305 ~ AX-109458466 The 0.34 cM interval between them corresponds to the physical map of the 2.43 Mb (717.89-720.32 Mb) Chinese Spring RefSeq_v1.0 reference genome.

[0036] (4) AQP label development and verification:

[0037] According to the positioning interval on both sides ( AX-111630305 、 AX-109458466 ) and physical maps ( AX-108820558 、 AX-108877192 、 AX-109955495 ) Polymorphism analysis of SNP markers between parents and haplotype analysis in RIL population to infer resistance gene loci and markers AX-111630305 , AX-108820558 and AX-108877192, and the SNP marker located in the physical interval closely linked to the major effect QTL controlling stripe rust seedling resistance on 7BL AX- 108877192 (located at 718.77 Mb on the physical map, with a genetic distance of 0.76 cM to the major QTL) was developed into a simple, high-throughput AQP marker and validated in RIL populations and germplasm resources (because AX-111630305 and AX-108820558 The specific process and primer sequence information are as follows:

[0038] Using the tightly linked SNP markers within the physical interval of the newly mapped QTL locus in this study and referring to the wheat Chinese Spring RefSeq_v1.0 (http: / / wheatomics.sdau.edu.cn / ) genome sequence, AQP primers were designed using DNAMAN software and the PolyMarker online tool (http: / / www.polymarker.info / ) (Table 1). After primer design, a different adapter sequence was added to the 5' end of each forward primer. These adapter sequences are recognized by fluorescent probes labeled with FAM (5'-GAAGGTGACCAAGTTCATGCT-3', marked in blue) and HEX (5'-GAAGGTCGGAGTCAACGGATT-3', marked in red), respectively, to generate fluorescent signals during the PCR amplification reaction.

[0039] Table 1 AQP labeling primer sequences

[0040] .

[0041] Dilute each of the three primers to 100 μM using the amount of ddH2O indicated on the primer label, then configure primers according to Table 2. AQP reactions were performed in a 96-well fluorescent quantitative PCR plate. The total reaction volume was 8 μL, including 4 μL of 100 ng / μL template DNA and 4 μL of a mixture of HiGeno 2x Probe Mix A and AQP Primer Mix (420 μL of HiGeno 2x Probe Mix A and 12.5 μL of AQP Primer Mix, gently vortexed, and centrifuged). PCR reactions were performed using a standard PCR instrument (Thermo Fisher, MiniAmp™ Thermal Cycler) with the following protocol: pre-denaturation at 95°C for 10 min; first amplification: 10 cycles of denaturation at 95°C for 20 s, followed by a gradient annealing and extension at 61°C for 40 s, with the annealing temperature decreasing by 0.6°C each cycle; second amplification: 33 cycles of denaturation at 95°C for 20 s, followed by a gradient annealing and extension at 55°C for 40 s; and storage at 25°C. After the PCR reaction, endpoint fluorescence signals of the amplified products were collected using a real-time quantitative PCR instrument (Thermo Fisher, QuantStudio™ 5) at 35°C, and data visualization was performed using QuantStudio™ Design & Analysis Software v1.4.3. Two negative controls (no template control (NTC)) and two positive controls (dd HO and parental DNA, respectively) were added to each PCR plate.

[0042] Table 2 AQP TM Genotyping system primer configuration method

[0043] .

[0044] After genotyping based on the “YN1813 / Chinese Spring” RIL population and 344 germplasm resources from different sources, it was found that AQP-AX-108877192 344 germplasm resources from different sources can be clearly divided into two groups, and the difference in resistance and susceptibility is significant ( Figure 2 ).

[0045] The average genetic effect of the QTL located by the AQP molecular marker obtained in this application reached 32.4, which is large and stable. The genetic distance between the AQP molecular marker and the QTL is close, with a genetic distance of 0.76 cM, and the linkage disequilibrium coefficient D' value with the markers on both sides of the QTL is 0.98, which has a close linkage disequilibrium relationship and can meet the needs of molecular breeding.

[0046] Although some preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0047] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications of the present invention fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Use of an AQP molecular marker for a major QTL for wheat stripe rust seedling resistance, the following marker primers, or a detection kit containing the marker primers in wheat stripe rust seedling resistance breeding or wheat stripe rust resistance identification, characterized in that: The major QTL is located on the long arm of wheat chromosome 7, within the physical map of the Chinese Spring RefSeq_v1.0 reference genome at 717.89 to 720.32 Mb, as shown in the website http: / / wheatomics.sdau.edu.cn / . A SNP marker is present at 718.77 Mb on the physical map. AX-108877192 , its genetic distance to the major effect QTL is 0.76 cM; and the SNP marker AX-108877192 Positioning was performed using the following labeled primers: 。 2. The use according to claim 1, characterized in that The steps include: (1) Extraction of wheat genomic DNA; (2) AX-108877192 The extracted genomic DNA was amplified by PCR using AQP-labeled primers, and the end-point fluorescence signal of the amplification product was collected for corresponding analysis; (3) Genotyping: Cluster the samples and further determine the genotype based on the sample clusters.

3. The use according to claim 2, characterized in that The steps of the PCR amplification include: PCR reaction system: including 4 uL of template DNA at a concentration of 100 ng / uL, 4 uL of HiGeno 2x Probe Mix A and AQP Primer Mix, a total of 8 uL; PCR reaction procedure: pre-denaturation at 95°C for 10 min; first amplification reaction: denaturation at 95°C for 20 s, gradient annealing and extension at 61°C for 40 s, 10 cycles, with the annealing temperature decreasing by 0.6°C each cycle; second amplification reaction: denaturation at 95°C for 20 s, annealing and extension at 55°C for 40 s, 28–34 cycles; storage at 25°C; The AQP primer mixture includes: a FAM forward primer, a HEX forward primer, a reverse primer, and purified water; the concentration of the FAM forward primer is 12 uM, the concentration of the HEX forward primer is 12 uM, and the concentration of the reverse primer is 30 uM; Analysis of test results: The endpoint fluorescence signal of the amplification product was collected using a real-time fluorescence quantitative PCR instrument at 35°C, and the data was visualized using QuantStudio™ Design & Analysis Software v1.4.3.

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