SSR molecular marker closely linked with wheat gibberellic disease-resistant main-effect QTL and application of SSR molecular marker

Abstract

The invention discloses an SSR marker closely linked with a wheat gibberellic disease-resistant main-effect QTL and application of the SSR marker. A gibberellic disease-resistant major QTL is positioned at a position close to an endpoint of a long arm of a wheat 1B chromosome, the number is QFhb-1BL, 25%-32% of disease phenotypic variation can be explained, and a disease-resistant site comes from a wheat variety, namely, red miscanthus; the SSR marker Xbua684 closely linked with the QTL QFhb-1BL is developed and obtained, and the marker can be used for rapidly and accurately screening plants with the gibberellic disease resistant QTL QFhb-1BL, so that the marker is applied to molecular marker-assisted selective breeding of wheat, technical support is provided for wheat gibberellic disease character improvement and disease-resistant breeding, and the process of wheat disease-resistant breeding is accelerated.

Description

Technical Field

[0001] The invention relates to a major QTL for wheat resistance to scab and a SSR marker closely linked thereto in the field of crop genetic breeding. Background Art

[0002] Wheat is one of the three major food crops, and Fusarium head blight seriously threatens wheat production safety (Ren et al., 2019; Ma et al., 2020). The genetic research of Fusarium head blight resistance has received widespread attention both at home and abroad. So far, scientists have discovered more than 500 Fusarium head blight resistance QTLs covering five types of resistance through linkage mapping and association analysis, which are distributed throughout all chromosomes of wheat (Buerstmayr et al., 2009; Liu et al., 2009; Ma et al., 2020; Zhang Hongli, 2024) and concentrated in 44 chromosome intervals. Although the number is huge, most QTLs have weak effects, are easily affected by the environment, and many are repeated sites. Although hundreds of QTLs related to Fusarium head blight resistance have been identified, only 9 (Fhb1-Fhb9) have been widely used and named internationally. They are Fhb1 (Cuthbert et al., 2006; Jia et al., 2018; Li, Zhou et al., 2019; Liu et al., 2006; Rawat et al., 2016; Schweiger et al., 2016; Su et al., 2019), Fhb2 (Cuthbert et al., 2007; Jia et al., 2019), respectively. al., 2018), Fhb3 (Qi et al., 2008), Fhb4 (Xue et al., 2010; Jia et al., 2017), Fhb5 (Xue et al., 2011; Jia et al., 2018), Fhb6 (Cainong et al., 2015), Fhb7 (Guo et al., 2015; Wang et al., 2020), Fhb8 (Wang et al. al., 2023) and Fhb9 (Zhang et al., 2024). Due to the complex genetic mechanism of fusarium resistance, the cloning of fusarium resistance genes has been slow, and only Fhb1 and Fhb7 have been successfully cloned.

[0003] Over the past few decades, through the unremitting efforts of researchers around the world, great progress has been made in the genetics of wheat fusarium head blight resistance. However, it is inevitable that most of the disease resistance QTLs are weak and easily affected by the environment. There are very few major effect QTLs that are truly strong and can stably resist diseases, and excellent resistance source materials are very scarce. Except for a few such as Fhb1, they are rarely used in breeding practice and improving variety resistance. Therefore, continuing to find better disease resistance genes and using existing major effect QTLs for stable disease resistance to cultivate excellent disease-resistant varieties, improve the overall resistance of planting resources and serve production practice, is still the most important issue at present. Summary of the invention

[0004] After years of large-scale screening, we found a major QTL (QFhb-1BL) that not only has a high degree of disease resistance (explaining more than 25% of phenotypic variation), but also has outstanding disease resistance stability, which can significantly reduce the disease in different environmental conditions and genetic backgrounds, and found a molecular marker closely linked to it. This QTL has not yet been used in wheat production at home and abroad, and has great potential application value.

[0005] Based on this, the purpose of the present invention is to provide an SSR specific primer set for identifying or assisting wheat fusarium head blight resistance; the SSR specific primer pair consists of primer F1 and primer R1;

[0006] The primer F1 is a single-stranded DNA molecule shown in Sequence 1 of the sequence table;

[0007] The primer R1 is a single-stranded DNA molecule shown in Sequence 2 of the sequence table;

[0008] The present invention also provides an application of the SSR specific primer set, which is at least one of the following (b1)-(b4):

[0009] (b1) identifying or assisting in the identification of wheat fusarium head blight resistance;

[0010] (b2) screening or assisting in screening wheat resistant to Fusarium head blight;

[0011] (b3) preparing a kit for identifying or assisting in identifying wheat fusarium head blight resistance;

[0012] (b4) preparing reagents for screening or assisting in screening wheat resistant to Fusarium fusarium.

[0013] The kit for the SSR specific primer set also falls within the protection scope of the present invention.

[0014] Furthermore, the present invention provides a method for identifying or assisting in identifying wheat scab resistance, comprising the following steps:

[0015] (c1) using the genomic DNA of the wheat to be tested, wheat A and wheat B as templates, respectively, and performing PCR amplification using the SSR specific primer pair to obtain the PCR product of the wheat to be tested, the PCR product of wheat A and the PCR product of wheat B; the wheat to be tested is a hybrid offspring obtained by hybridizing the wheat A and the wheat B, and the fusarium head blight diseased ear rate of the wheat A is significantly lower than the fusarium head blight diseased ear rate of the wheat B;

[0016] (c2) subjecting the PCR product of the wheat to be tested, the wheat A PCR product and the wheat B PCR product to electrophoresis, and determining the resistance / susceptibility of the wheat to be tested to Fusarium fusarium based on the electrophoresis results: if the electrophoresis results show that the PCR product of the wheat to be tested contains a band of the same size as the wheat A PCR product, the wheat to be tested is or is a candidate for being Fusarium fusarium resistant wheat; if the electrophoresis results show that the PCR product of the wheat to be tested does not contain a band of the same size as the wheat A PCR product, the wheat to be tested is or is a candidate for being non-Fusarium fusarium resistant wheat.

[0017] The present invention also provides a method for screening or assisting in screening wheat resistant to fusarium scaber, comprising the following steps:

[0018] (d1) identifying the resistance of the tested wheat to fusarium head blight according to the method of claim 4;

[0019] (d2) screening or assisting in screening wheat resistant to ergot based on the result of step (d1).

[0020] The application of the SSR specific primer set in wheat breeding also falls within the protection scope of the present invention.

[0021] The application of the above method in wheat breeding also falls within the protection scope of the present invention.

[0022] Beneficial effects of the present invention:

[0023] The present invention locates a major effect QTL for resistance to fusarium head blight on the wheat chromosome 1BL, numbered QFhb-1BL, which can explain 25% to 32% of the phenotypic variation of the disease, and the disease resistance locus comes from the wheat variety Dahongmang; and the SSR marker Xbua684 closely linked to it is developed and obtained, and the marker can be used to quickly and accurately screen plants with the fusarium head blight resistance QTL QFhb-1BL, which can be used for molecular marker-assisted selection breeding, and provide technical support for wheat fusarium head blight trait improvement and disease resistance breeding. The SSR marker of the present invention has important applications in molecular marker-assisted breeding and resistance improvement of wheat fusarium head blight. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 LOD curve of the main QTL for resistance to Fusarium ergot disease located using SNP markers.

[0025] Figure 2 LOD curve of the major QTL for resistance to Fusarium ergot using SNP and SSR markers.

[0026] Figure 3 Comparison of spikelet rates of two different genotypes of wheat materials tested for Fusarium head blight. Note: Genotype A has a band in the PCR product that is the same size as the PCR product of Dahongmang, and genotype B has a band in the PCR product that is the same size as the PCR product of Aikang 58. DETAILED DESCRIPTION

[0027] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0028] Wheat variety "Dahongmang": This is a local variety of wheat from the northwest of Sichuan Province. The China Agricultural University Wheat Quantitative Disease Resistance Research Collaboration Group has found that this variety is resistant to Fusarium head blight after years of observation. The public can obtain this variety from Beijing Agricultural College.

[0029] Wheat variety Dwarf 58: It is a high-yield, multi-resistant and widely adaptable variety selected and bred by Henan Institute of Science and Technology. Its pedigree is Zhoumai 11 / / Yumai 49 / Zhengzhou 8960. It was once planted on a large scale in the Huanghuaihai wheat region and was severely infected with ergot.

[0030] The recombinant inbred line of Dahongmang × Aikang 58: Dahongmang is used as the female parent and Aikang 58 is used as the male parent. F1 seeds are obtained by hybridization. F2 seeds are harvested from plants grown from F1 seeds. In the following years, the recombinant inbred line is obtained by continuous self-pollination from generation to generation through the single-seed transmission method.

[0031] Wheat variety Sumai No. 3: It is a spring wheat variety recognized both at home and abroad as highly resistant to ergot. It was bred by the Taihu Agricultural Science Institute of Jiangsu Province. For related information, please refer to the literature "Jiangsu Taihu Agricultural Science Institute, Jiangsu Academy of Agricultural Sciences, Institute of Grain Crops and Institute of Plant Protection. The best source of resistance to wheat ergot - Sumai No. 3. Jiangsu Agricultural Science, 1988 (12): 1-4."

[0032] Gibberellic acid: Fusarium graminearum culture) HN5, a highly pathogenic strain collected from Henan Province. For information about this strain, please refer to the literature "Ren J, Wang Z, Du Z, Che M, Zhang Y, Quan W, Wang Y, Jiang X, Zhang Z. Detection and validation of a novel major QTL for resistance to Fusarium head blight from Triticum aestivum in the terminal region of chromosome 7DL. Theoretical and Applied Genetic, 2019, 132: 241–259", which is available to the public from Beijing Agricultural College.

[0033] Example 1. Location of the major QTL for wheat resistance to Fusarium head blight and acquisition of closely linked SSR markers I. Identification of disease phenotypes

[0034] The Dahongmang × Aikang 58 recombinant inbred line population (F8) and the female parent Dahongmang and the male parent Aikang 58 were planted in Suiping County, Zhumadian City, Henan Province in 2018-2019 and 2022-2023. The row sowing, randomized complete block design, and 3 replications were adopted. The experimental plots consisted of a single row of 1m long with a row spacing of 25cm. Each plot was sown with a recombinant inbred line, about 40 seeds, and every 60 recombinant inbred line plots were sown with a group of parents and disease-resistant controls (Dahongmang, Aikang 58, and Sumai No. 3). In the early stage of flowering, spray inoculation was carried out, and the conidia suspension of Fusarium graminearum (concentration of 5×10 per microliter) was sprayed on the canopy of wheat plants using an electric backpack sprayer in the late afternoon. 4 spores), and repeat the process on the second day. Within 72 hours after inoculation, the wheat canopy was sprayed with moisture for three to ten minutes every hour according to the weather conditions during the day, and covered with plastic film at night. Three weeks after inoculation, the number of diseased spikelets and the total number of spikelets in one ear of the recombinant inbred line, parent and disease-resistant control were investigated, and the percentage of symptomatic spikelet (PSS) was calculated. Ten ears of wheat were investigated in each plot, and the average disease value of the crop plot was calculated.

[0035] In addition, the above materials were planted in a greenhouse in Chifeng City, Inner Mongolia from 2022 to 2023. Spot sowing was adopted, and random complete blocks were repeated three times. The experimental plot consisted of a hole with an interval of 25-40cm (25cm in the vertical direction and 40cm in the horizontal direction). Each hole was sown with 12 seeds of a recombinant inbred line, and a group of parents and disease-resistant controls were sown every 40 holes. In the early stage of flowering, single flower injection inoculation was carried out, and 10μL of conidia suspension (approximately 5×10 per microliter) was added. 4 spores) were injected between the lemma and palea of ​​the floret on one side of the middle of the ear. In order to ensure humidity and promote fungal infection, the ear should be sprayed with water to keep it moist and covered with a transparent fresh-keeping bag for 48 hours after inoculation. The diseased spikelet rate was investigated 3 weeks after inoculation.

[0036] As shown in Table 1, the PSS of Dahongmang is significantly lower than that of Aikang 58, but slightly higher than that of Sumai 3; the PSS variation range of the Dahongmai × Aikang 58 recombinant inbred line is relatively wide, for example, from 7.4% to 94.4% in the 2024 greenhouse trial.

[0037] Table 1 Diseased spikelet rate (PSS, %) of wheat parent varieties Dahongmang and Aikang 58 and their F8 recombinant inbred lines (RIL_F8)

[0038]

[0039] 2. Construction of DNA marker genetic map

[0040] The genotypes of 122 recombinant inbred lines of the hybrid combination of Dahongmang×Aikang 58 were determined and the chromosome genetic map was constructed.

[0041] DNA from Dahongmang, Aikang 58 and 122 recombinant inbred lines was extracted by CTAB method, and the concentration of DNA stock solution was determined by NanoDrop2000 ultra-micro spectrophotometer, and then diluted to a working solution with a concentration of 50 ng / μL. The DNA quality was detected by 0.8% agarose gel. Then, SNP analysis was performed using Illumina iSelect90K SNP array technology. This work was completed by Beijing CapitalBio Technology Co., Ltd. (CapitalBio Technology, http: / / www.capitalbiotech.com), including allele clustering and genotype calling of SNP sites.

[0042] To construct a genetic map using these SNP marker data, the original genotype data was first filtered: (1) heterozygous genotype data were considered missing values; (2) marker sites with missing values ​​greater than 5% were removed; (3) marker sites with allele frequencies less than 0.35 or greater than 0.65 were removed; (4) redundant markers were removed using the BIN function in the QTL IciMapping V4.0 software. The genotype of the parent Dahongmang was recorded as "A", the genotype of the parent Aikang 58 was recorded as "B", and the genotypes of the recombinant inbred lines that were consistent with Dahongmang were recorded as "A" and those that were consistent with Aikang 58 were recorded as "B". JoinMap 4.0 software was used to construct linkage groups, and the genetic distance was measured in centimorgans (cM) calculated by the Kosambi function. The flanking sequences of each SNP in the linkage group were compared with the reference genome sequence of the wheat variety Chinese Spring (chromosomal survey sequence map; IWGSC 2018) to determine the corresponding chromosome to which each linkage group belonged and mark the centromere position.

[0043] 3. QTL Positioning

[0044] Based on the genetic map of chromosome 58 of Dahongmang × Aikang and the genotype data and disease phenotype data of 122 recombinant inbred lines, QTL analysis was performed using the composite interval mapping (CIM) method of Windows QTL Cartographer 2.5 software. A major effect QTL ( Figure 1 ), numbered as QFhb-1BL, the LOD peak value was located in the approximately 10cM interval defined by SNP markers IWB5732 and IWA6511, significantly higher than the test standard 3.5, and the disease resistance locus came from the wheat variety Dahongmang.

[0045] Example 2: Acquisition of SSR marker Xbua684, which is tightly linked to the major QTL for wheat resistance to Fusarium head blight

[0046] Based on Example 1, the two flanking markers of the main effect QTL for resistance to Fusarium head blight were obtained, namely IWB5732 and IWA6511. Their physical positions in the wheat reference genome IWGSC RefSeq v2.1 were determined by Blast comparison, namely, they were located at 679.34Mb and 697.29Mb of chromosome arm 1BL, respectively. JBrowse was used to search for the Predicted_SSR repeat motif in this interval, and 467 pairs of SSR primers were designed using Tools / PrimerSever in http: / / 202.194.139.32 / . Five SSR markers close to QFhb-1BL were screened out, and the genotypes of 122 recombinant inbred lines of Dahongmang×Aikang 58 were determined using these five markers, and a linkage map of the region where QFhb-1BL is located in 1BL was constructed. QTL positioning was performed based on the linkage map and disease value data, as shown in FIG. Figure 2 As shown in the figure, QFhb-1BL is limited to 0.7cM, and the two closest flanking markers are Xbua683 and Xbua687. Marker Xbua684 is located at the peak of LOD value, which is closely linked to QFhb-1BL. The peak values ​​of Xbua684 in three different experiments are 10, 12 and 17, respectively, which can explain 25% to 32% of the phenotypic variation of disease. Xbua683 and Xbua687 limit QFhb.cau-1BL to 0.7cM, which corresponds to about 3.2Mb in the physical sequence of the Chinese Spring reference genome. At the Xbua684 site, the recombinant inbred lines containing the amplification product from Dahongmang have lower disease and are resistant to fusarium scurf; the recombinant inbred lines containing the amplification product from Aikang 58 have higher disease and are susceptible to fusarium scurf.

[0047] SSR marker Xbua684 consists of an upstream primer and a downstream primer;

[0048] Upstream primer F: 5'-AGCTGAGTCCAAGTTGAGCC-3' (SEQ ID NO: 1);

[0049] Downstream primer R: 5'-CCACTAGCATCCCCCGTTTT-3' (SEQ ID NO: 2).

[0050] Example 3: Application of the tightly linked marker Xbua684 in the selection of wheat resistance to Fusarium head blight

[0051] The developed QFhb-1BL tightly linked SSR marker Xbua684 was further tested and verified in a hybrid combination population of Dahongmang×Aikang 58, that is, the wheat to be tested was 100 recombinant inbred lines of Dahongmang×Aikang 58 (different from the 122 wheat materials in Example 1).

[0052] The genomic DNA of wheat to be tested was extracted and used as a template, and PCR amplification reaction was performed using primer Xbua684.

[0053] The PCR reaction system (10 μL) was as follows: 10×PCR buffer 1.00 μL, dNTPs (10mM) 0.25μL,

[0054] Taq enzyme (5U / μL) 0.10μL,

[0055] Primers (1.0 μM) 2.00 μL,

[0056] Template DNA (25ng / μL) 2.00μL,

[0057] ddH2O 4.65μL.

[0058] The PCR reaction program was as follows: 94°C for 5 min, followed by 30-35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and finally 72°C for 10 min and storage at 10°C.

[0059] The PCR amplification products were detected by 6% polyacrylamide denaturing gel electrophoresis, with a buffer of 1×TBE, a constant power of 60 W, a voltage of 3000 V, and an electrophoresis time of 50 min. The band type was then read by silver staining. If the PCR product contained a band of the same size as the big red awn PCR product, the genotype of the wheat to be tested was "A"; if the PCR product contained a band of the same size as the dwarf resistance 58 PCR product, the genotype of the wheat to be tested was "B".

[0060] In addition, the resistance test of the wheat to ergot was carried out in a greenhouse in Chifeng City, Inner Mongolia from 2023 to 2024. The experimental design, inoculation, disease investigation and other methods were the same as those described in Example 1.

[0061] The genotype and phenotypic data of 100 recombinant inbred lines of Dahongmang×Aikang58 were analyzed (see Table 2). It was found that using SSR marker Xbua684, 53 strains containing the female parent Dahongmang band type (A) were screened out in the test population, and the average rate of fusarium head blight was 37.5%; in addition, 47 strains containing the male parent Aikang58 band type (B) were screened out, and the average rate of diseased spikelets was 61.8%. Statistical tests (see Table 3, Figure 3 ) showed that the gene effect of QFhb-1BL reached a significant level (P<0.0001).

[0062] The above results indicate that wheat materials with genotype A at the Xbua684 locus in the wheat genome have higher or potential higher resistance to Fusarium fusarium than wheat materials with genotype B at the Xbua684 locus in the wheat genome. This shows that the SSR marker Xbua684 can be used to effectively screen wheat lines containing QFhb-1BL, improve selection efficiency, and be used for disease resistance breeding of wheat.

[0063] Table 2 Genotype detection results and fusarium resistance of 100 recombinant inbred lines of Dahongmang×Aikang58

[0064]

[0065]

[0066]

[0067] Table 3 Effect of QFhb-1BL on Fusarium head blight resistance of 100 recombinant inbred lines of Dahongmang×Aikang58

[0068]

[0069]

[0070] Note: Genotype A means that the PCR product contains a band of the same size as the PCR product of Dahongmang, and genotype B means that the PCR product contains a band of the same size as the PCR product of Aikang 58.

Claims

1. An SSR specific primer set for identifying or assisting wheat fusarium head blight resistance; the SSR specific primer pair consists of primer F1 and primer R1; The primer F1 is a single-stranded DNA molecule shown in Sequence 1 of the sequence table; The primer R1 is a single-stranded DNA molecule shown in Sequence 2 of the sequence table.

2. The use of the SSR specific primer set according to claim 1 is at least one of the following (b1)-(b4): (b1) identifying or assisting in the identification of wheat fusarium head blight resistance; (b2) screening or assisting in screening wheat resistant to Fusarium head blight; (b3) preparing a kit for identifying or assisting in identifying wheat fusarium head blight resistance; (b4) preparing reagents for screening or assisting in screening wheat resistant to Fusarium fusarium.

3. A kit containing the SSR specific primer set according to claim 1.

4. A method for identifying or assisting in identifying wheat scab resistance, comprising the following steps: (c1) using the genomic DNA of the wheat to be tested, wheat A and wheat B as templates, respectively, and performing PCR amplification using the SSR specific primer pair described in claim 1 to obtain the PCR product of the wheat to be tested, the PCR product of wheat A and the PCR product of wheat B; The wheat to be tested is a hybrid offspring obtained by hybridizing the wheat A and the wheat B, and the rate of fusarium head blight diseased ears of the wheat A is significantly lower than the rate of fusarium head blight diseased ears of the wheat B; (c2) subjecting the PCR product of the wheat to be tested, the wheat A PCR product and the wheat B PCR product to electrophoresis, and determining the resistance / susceptibility of the wheat to be tested to Fusarium fusarium based on the electrophoresis results: if the electrophoresis results show that the PCR product of the wheat to be tested contains a band of the same size as the wheat A PCR product, the wheat to be tested is or is a candidate for Fusarium fusarium resistant wheat; if the electrophoresis results show that the PCR product of the wheat to be tested does not contain a band of the same size as the wheat A PCR product, the wheat to be tested is or is a candidate for non-Fusarium fusarium resistant wheat. The wheat A is the wheat variety Big Red Mango, and the wheat B is the wheat variety Aikang 58.

5. A method for screening or assisting in screening wheat resistant to fusarium head blight, comprising the following steps: (d1) identifying the resistance of the tested wheat to fusarium head blight according to the method of claim 4; (d2) screening or assisting in screening wheat resistant to ergot based on the result of step (d1).

6. Use of the SSR specific primer set described in claim 1 in wheat breeding.

7. Use of the method according to claim 4 or 5 in wheat breeding.

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