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

Abstract

This application discloses an AQP molecular marker for a major QTL for resistance to wheat stripe rust in the adult stage, its primers, and its application, aiming to solve the technical problem that most QTLs for resistance to stripe rust are difficult to meet the needs of molecular breeding. The major QTL screened in this application is located on the long arm of chromosome 7 of wheat. QYr.hau‐A‐ 7BL , which contains two linked SNP molecular markers SURE- 110908486 and AX‐89658728 , QYr.hau‐A‐7BL Positioned in AX‐110908486 ~ AX‐89658728 The AQP markers located in this application are located within a 0.17 cM interval, corresponding to the physical map of the Chinese Spring RefSeq_v1.0 reference genome at 720.5 to 721.2 Mb. The AQP markers discovered in this application have a large and stable genetic effect, and 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 molecular biology technology, and in particular to an AQP molecular marker for a major QTL of wheat stripe rust resistance in the adult stage, a primer thereof, and an application thereof. Background Art

[0002] Wheat is an important food crop in my country, and its production is closely related to national food security. Puccinia striiformis f. sp. tritici , Pst Stripe rust is a major airborne foliar disease of wheat, often causing 5-25% yield losses and, in recurrent years, even 100% total yield loss. Three main types of wheat stripe rust resistance exist: race-specific all-stage resistance (ASR), non-race-specific adult plant resistance (APR), and broad-spectrum resistance (SBR) mediated by mutations in susceptible genes. Adult plant resistance, which typically manifests only during the adult stage, is controlled by multiple genes and is a quantitative trait regulated by gene-environment interactions. This type of resistance is generally not selective, but rather slows the rate of mutation of the stripe rust pathogen, resulting in durable resistance. Therefore, cultivating adult plant resistance to stripe rust has become a key focus of wheat breeding.

[0003] Quantitative traits are typically studied using linkage analysis in permanent populations and genome-wide association analysis in natural populations. Because RILs are relatively homozygous and have a stable population genetic structure, they can be replicated over multiple years and at multiple locations. This provides highly reliable phenotypic data and significantly improves the accuracy of localization, making them ideal populations for exploring quantitative traits.

[0004] Currently, medium-throughput PCR-based SNP typing technologies are represented by TaqMan fluorescent probes and KASP (competitive allele-specific PCR). Because the TaqMan method requires the design of a specific fluorescent probe for each variant, it is relatively expensive and not suitable for large-scale analysis. KASP, on the other hand, simply adds a master mix consisting of two universal probes with different fluorescent groups and two complementary probes with quenching groups to a conventional PCR reaction system. However, the PCR reaction system is relatively expensive, and the cost per sample increases if the number of samples tested is small. To address this, a domestic company has developed the AQP™ genotyping system, also known as the allele-specific quantitative PCR-based genotyping assay (AQP). Its principle and detection method are similar to KASP labeling, but the domestically produced master mix is relatively affordable, making it suitable for large-scale testing and has been applied in some studies.

[0005] To date, 84 stripe rust resistance genes have been officially named, but most of them confer resistance throughout the entire growth period, making them vulnerable to new stripe rust races. Although over 200 stripe rust resistance QTLs have been mapped at various locations in the wheat genome, some of these QTLs confer partial resistance and are easily affected by the environment, others have lost resistance, and some markers are genetically distant from the QTLs, making it difficult to identify simple, high-throughput markers. Consequently, most of the mapped QTLs are still insufficient for molecular breeding. Therefore, identifying more QTLs with stable resistance and developing molecular markers closely linked to them is crucial for marker-assisted selection of stripe rust-resistant varieties and for developing new varieties with durable resistance.

[0006] 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

[0007] 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 resistance in the adult stage, 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.

[0008] According to one aspect of the present disclosure, an AQP molecular marker for a major QTL for resistance to wheat stripe rust in the adult stage is provided. The major QTL is located on the long arm of chromosome 7 of wheat and is named QYr.hau-A-7BL ; QYr.hau- A-7BL Two linked SNP markers are AX-110908486 and AX-89658728 , QYr.hau-A-7BL The positioning range is AX-110908486 ~ AX-89658728 The 0.17 cM interval between the two loci corresponds to the 0.7 Mb physical map of the Chinese Spring RefSeq_v1.0 reference genome (720.5-721.2 Mb), explaining 0.9-26.1% of the phenotypic variation. The additive effects on stripe rust response type and severity were -0.4 to -0.7 and -9.6 to -15.4, respectively.

[0009] According to another aspect of the present disclosure, there is provided an AQP molecular marker primer, comprising at least one of the following primer groups:

[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 QTLs for wheat stripe rust resistance at the adult stage.

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

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

[0015] (2) AX-110908486 AQP-labeled primers and / or AX-89658728 The extracted genomic DNA was amplified by PCR using AQP-labeled primers, and the fluorescence signal of the amplified product was collected in real time for corresponding analysis;

[0016] (3) Genotyping: Plot the HEX and FAM fluorescence signal values on the x-axis and y-axis respectively. According to the fluorescence signal values, cluster the samples and further determine the genotype based on the sample clusters, as follows: Figure 2 、 Figure 3As shown in the middle left figure, the x-axis is the resistance gene site TT / GG bound to the HEX linker sequence, and the y-axis is the susceptible gene site CC / AA bound to the FAM linker sequence.

[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 test 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 data visualization was achieved 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 study is 14.3, which is large and stable. The genetic distance between the AQP molecular marker and the QTL is close (the genetic distances to the markers on both sides are 0.11 and 0.06 cM, respectively, which are much smaller than the 0.5 cM frequently located), which can meet the needs of molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 In one embodiment of this application AQP-AX-89658728 Genotyping results and comparison of resistance and sensitivity types of 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- AX-89658728 Boxplot showing the significance of differences between resistant and susceptible groups among genotyped 405 germplasm accessions (genotyped as homozygous) from different sources.

[0026] Figure 3 In one embodiment of this application AQP-AX-110908486 Genotyping results of some germplasm resources from different sources and comparison of resistance and sensitivity types; 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, markers and AQP-AX-110908486 Boxplot showing the significance of differences between resistant and susceptible groups among genotyped 405 germplasm accessions (genotyped as homozygous) from different sources. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] Example 1

[0030] (1) QTL mapping population:

[0031] 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.

[0032] (2) Identification of resistance to stripe rust in adult plants:

[0033] Field testing of RIL populations was conducted at the Pidu Experimental Station of the Sichuan Academy of Agricultural Sciences and the Qingshui Experimental Station of the Gansu Academy of Agricultural Sciences in 2019-2020 and 2020-2021, respectively. A randomized block design was used, with two replications in Qingshui and three replications in Chengdu. Seeds were sown in rows spaced 25 cm apart, with 30 seeds planted per row, each 1 m long. For every 40 families, a control parent and a row of a highly susceptible cultivar were added. Triggering rows were planted around the perimeter of the plots and between plots.

[0034] 405 accessions of diverse germplasm were field-tested and planted at the experimental field of the Xuchang campus of Henan Agricultural University in 2020-2021 and 2021-2022. A randomized block design with two replications was used. Rows were planted in 40 / 20 cm wide / narrow rows, each 1.2 m long, with 30 seeds planted per row. The inducible variety, Xuemingxian 169, was planted every 10 rows around the perimeter of the plot and between plots. Artificial inoculation with stripe rust was performed at the jointing stage of wheat. The response and severity of the induced stripe rust were then investigated after the disease had fully developed.

[0035] (3) DNA extraction and QTL mapping:

[0036] 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) and Multi-Environment Trials (MET) functions.

[0037] 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-A-7BL ( Figure 1 This QTL is stable in different environments and can explain 0.9 to 26.1% of the phenotypic variation. Its additive effect on stripe rust response type is -0.4 to -0.7, and its additive effect on severity is -9.6 to -15.4. Based on the average data of stripe rust resistance QTL mapping, polymorphism analysis of SNP markers in the mapping interval between parents, and haplotype analysis, QYr.hau-A-7BL Finally located at the SNP marker AX-110908486 ~ AX- 89658728The 0.17 cM interval between them corresponds to the 0.7 Mb (720.5-721.2 Mb) physical map of the Chinese Spring RefSeq_v1.0 reference genome.

[0038] (4) AQP label development and verification:

[0039] The SNP markers AX-110908486 and AX-89658728, which were located closely linked to the major QTL controlling adult-stage stripe rust resistance in 7BL, were developed into simple, high-throughput AQP markers and validated in RIL populations and germplasm resources. The specific process and primer sequence information are as follows:

[0040] Using the tightly linked SNP markers on both sides of the newly located QTL locus genetic map in this study and referring to the Chinese Spring wheat 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).

[0041] After the primers are designed, a different adapter sequence needs to be added to the 5' end of each of the two forward primers. The two adapter sequences can be 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.

[0042] Table 1 AQP labeling primer sequences

[0043] .

[0044] 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 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).

[0045] The PCR reaction was performed using a conventional PCR instrument (Thermo Fisher, MiniAmp™ thermal cycler) with the following procedure:

[0046] The DNA was pre-denatured at 95°C for 10 minutes. The first amplification step consisted of 10 cycles of denaturation at 95°C for 20 seconds, followed by a gradient annealing and extension at 61°C for 40 seconds, with the annealing temperature decreasing by 0.6°C each cycle. The second amplification step consisted of 33 cycles of denaturation at 95°C for 20 seconds, followed by an annealing and extension at 55°C for 40 seconds. The DNA was then stored at 25°C. After completion of the PCR reaction, the endpoint fluorescence signal of the amplified product was collected using a real-time fluorescence quantitative PCR instrument (ThermoFisher, 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.

[0047] Table 2 AQP TM Genotyping system primer configuration method

[0048] .

[0049] After genotyping the “YN1813 / Chinese Spring” RIL population and 405 germplasm resources, it was found that AQP-AX- 110908486 and AQP-AX-89658728 The identification materials can be clearly divided into two groups, and the difference in resistance and sensitivity is significant ( Figure 2 、 Figure 3 ).

[0050] There is a single base difference at the 3' end of the two forward primers, and the 5' end carries a different adapter sequence. In the subsequent PCR amplification, the probe recognizes the adapter sequence and amplifies as a primer to form a PCR product with a fluorescent signal. If the genotype of the SNP site is homozygous, only one possible fluorescent signal will be generated, and if the SNP site is heterozygous, the result will be a mixed fluorescent signal. The HEX and FAM fluorescence signal values are plotted on the x-axis and y-axis respectively. According to the fluorescence signal value, the samples are clustered and the genotype is further determined based on the sample cluster, as shown in the following example. Figure 2 、 Figure 3 As shown in the middle left figure, the x-axis is the resistance gene site TT / GG bound to the HEX linker sequence, and the y-axis is the susceptible gene site CC / AA bound to the FAM linker sequence.

[0051] The average genetic effect of the QTL located by the AQP molecular marker was 14.3, which was large and stable. The genetic distance between the AQP molecular marker and the QTL was close (the genetic distances to the markers on both sides were 0.11 and 0.06 cM, respectively, which are much smaller than the 0.5 cM frequently located), which can meet the needs of molecular breeding.

[0052] Although some preferred embodiments of the present application 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 application.

[0053] Obviously, those skilled in the art may make various changes and modifications to this application without departing from the spirit and scope of the invention of this application. Thus, if such changes and modifications of this application fall within the scope of the claims of this application and their equivalents, this application is intended to include such changes and modifications.

Claims

1. Use of an AQP molecular marker for a major QTL for wheat stripe rust resistance in the adult stage, its identification primers, or a detection kit containing the identification primers in wheat stripe rust resistance breeding or wheat stripe rust resistance identification, characterized in that: The major QTL is located on the long arm of chromosome 7 of wheat. QYr.hau-A-7BL , which contains two linked SNP molecular markers AX-110908486 and AX-89658728 , QYr.hau-A-7BL Positioned in AX-110908486 ~ AX- 89658728 The SNP molecular marker is within the 0.17 cM interval between the two groups, corresponding to the physical map of the Chinese Spring RefSeq_v1.0 reference genome 720.5 to 721.2 Mb as shown in the website http: / / wheatomics.sdau.edu.cn / ; AX- 110908486 、 AX-89658728 The markers were located by the following primer sets: ； The identification primers include at least one of the primer sets.

2. The use according to claim 1, characterized in that The steps include: (1) Extraction of wheat genomic DNA; (2) AX-110908486 AQP-labeled primers and / or AX-89658728 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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