Molecular marker of wheat scab resistance gene fhb8 and application thereof

Abstract

This invention belongs to the field of crop breeding and discloses a wheat Fusarium head blight resistance gene. Fhb8 Molecular markers and their applications. This invention discloses a wheat Fusarium head blight resistance gene. Fhb8 The primer pairs labeled with molecular markers are selected from any one of the following (1)-(3): (1) WGRB1500-F: as shown in SEQ ID NO.1, WGRB1500-R: as shown in SEQ ID NO.2; (2) WGRB1587-F: as shown in SEQ ID NO.3, WGRB1587-R: as shown in SEQ ID NO.4; (3) WGRB1559-F: as shown in SEQ ID NO.5, WGRB1559-R: as shown in SEQ ID NO.6. Detection is performed using primer pairs labeled with WGRB1500, WGRB1587 and WGRB1559. Fhb8 Genes can be determined Fhb8 The presence and state of existence of pathogens can be used to predict wheat resistance to Fusarium head blight, thereby enabling rapid screening for pathogens carrying pathogens. Fhb8 The plants were used for breeding disease-resistant varieties.

Description

Technical Field

[0001] This invention belongs to the field of crop breeding and relates to the molecular marker of wheat Fusarium head blight resistance gene Fhb8 and its application. Background Technology

[0002] Fusarium head blight, caused by the facultative parasitic fungus Fusarium spp., is a highly devastating disease in wheat production (Dean et al. 2012; Figueroa et al. 2018). In recent years, due to abundant inoculum in the field, improved water and fertilizer conditions, and hot and humid weather during the susceptible period (flowering stage), the occurrence of Fusarium head blight in wheat worldwide has become increasingly serious (Ma et al. 2020). Currently, 80% of wheat-producing areas in my country are threatened by Fusarium head blight, with frequent outbreaks in major wheat-producing areas. The average annual affected area exceeds 80 million mu (approximately 5.3 million hectares), causing direct economic losses of billions of yuan annually, making it the most serious disease affecting wheat production in my country (Cheng et al. 2012; Zhang et al. 2018; Ma et al. 2020). The breeding and application of disease-resistant varieties are the most economical and effective methods for controlling Fusarium head blight. The discovery of disease-resistant genes is the prerequisite and foundation for disease-resistant breeding, and the development of molecular markers closely linked to disease-resistant genes is the key to the application of disease-resistant genes.

[0003] Current research indicates that there are no wheat varieties immune to Fusarium head blight, and most Fusarium head blight resistant germplasm has poor agronomic traits. In addition, Fusarium head blight resistance is diverse, including five types: resistance to Fusarium head blight infection (resistance to infection), resistance to Fusarium head blight spread (resistance to spread), low toxin accumulation, low diseased grain rate, and varietal resistance (Mesterházy 1995), with complex mechanisms. Moreover, it is controlled by quantitative trait loci (QTLs) with minor effects, and the phenotype is greatly influenced by the environment (Bai and Shaner 2004).

[0004] To date, over 500 QTLs for resistance to Fusarium head blight have been identified. Among them, Qfdk.nau-7D, located in the Xwmc405-Xwmc702 region on chromosome 7D, is a major QTL for low grain disease rate, explaining 16% of the phenotypic variation (Li et al. 2008). This QTL segment has also been associated with the spread of Fusarium head blight resistance in varieties such as sea salt (Li et al. 2011), Kenyon (McCartney et al. 2016), and Catbird (Cativelli et al. 2013). We used marker-assisted backcrossing to transfer it into the highly susceptible Fusarium head blight varieties PH691 and Yangmai 15. Multi-year, multi-location resistance assessments showed that it reduced the diseased spikelet percentage, number of diseased spikelets, and diseased grain percentage by 58.4%, 64.8%, and 57.8%, respectively, significantly improving wheat's resistance to infection and spread, and reducing the diseased grain percentage. This indicates that Qfdk.nau-7D is a major-effect QTL with comprehensive Fusarium head blight resistance; and this QTL has been tentatively named Fhb8. However, its initial mapping interval is relatively large, and it lacks closely linked molecular markers, which could easily lead to linkage redundancy in breeding selection, affecting the efficiency of the resistance gene application. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a molecular marker that is more closely linked to the wheat scab resistance gene Fhb8.

[0006] Another object of the present invention is to provide the application of the molecular marker of the wheat Fusarium head blight resistance gene Fhb8.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention first protects a primer pair for a molecular marker of the wheat scab resistance gene Fhb8, selected from any one of the following (1)-(3):

[0009] (1) WGRB1500-F: as shown in SEQ ID NO.1; WGRB1500-R: as shown in SEQ ID NO.2;

[0010] (2) WGRB1587-F: as shown in SEQ ID NO.3; WGRB1587-R: as shown in SEQ ID NO.4;

[0011] (3) WGRB1559-F: as shown in SEQ ID NO.5, WGRB1559-R: as shown in SEQ ID NO.6.

[0012] Secondly, this invention protects a molecular marker for the wheat scab resistance gene Fhb8, selected from any one of WGRB1500, WGRB1587, and WGRB1559; wherein,

[0013] The genomic DNA of wheat varieties was amplified using marker primers WGRB1500-F (SEQ ID NO.1) and WGRB1500-R (SEQ ID NO.2). The amplified fragment obtained was 289 bp, which is the molecular marker WGRB1500 linked to the wheat resistance gene Fhb8. This marker is a codominant marker and is tightly linked to Fhb8. The genetic distance between this marker and the Fhb8 gene was measured to be 0.6 cM using Mapmaker Macintosh V 3.0.

[0014] Alternatively, wheat cultivar DNA was amplified using marker primers WGRB1587-F (SEQ ID NO.3) and WGRB1587-R (SEQ ID NO.4). The amplified fragment obtained was 141 bp, which is the molecular marker WGRB1587 linked to the wheat Fusarium head blight resistance gene Fhb8. This marker showed polymorphism in the Fusarium head blight resistant cultivar Wangshuibai and the Fusarium head blight susceptible cultivar PH691 after digestion with HinfI. This marker is a codominant molecular marker and is tightly linked to Fhb8. The genetic distance between this marker and the Fhb8 gene was measured to be 0 cM using Mapmaker Macintosh V 3.0.

[0015] Alternatively, wheat varietal DNA was amplified using marker primers WGRB1559-F (SEQ ID NO.5) and WGRB1559-R (SEQ ID NO.6). The amplified fragment obtained was 114 bp, which is the molecular marker WGRB1559 linked to the wheat resistance gene Fhb8. This marker is a codominant molecular marker and is tightly linked to Fhb8. The genetic distance between this marker and the Fhb8 gene was measured to be 0.5 cM using Mapmaker Macintosh V 3.0.

[0016] Thirdly, the present invention also protects reagents or kits containing the primer pairs described above, and / or the molecular markers described above.

[0017] Fourthly, the present invention also protects the use of the primer pairs described above, and / or the molecular markers described above, and / or the reagents or kits described above in the identification of the Fusarium head blight resistance gene Fhb8 in wheat germplasm resources.

[0018] Fifthly, the present invention also protects the use of the primer pairs described above, and / or the molecular markers described above, and / or the reagents or kits described above, in the preparation of products for the identification of the Fusarium head blight resistance gene Fhb8 in wheat germplasm resources.

[0019] Sixthly, this invention also protects a molecular marker method for detecting the wheat resistance gene Fhb8. The method involves PCR amplification of the wheat genomic DNA using the primer pairs described above, followed by detection of the amplification products. If a 289 bp amplification fragment can be amplified using primers WGRB1500-F and WGRB1500-R (molecular marker WGRB1500), or a 141 bp amplification fragment can be amplified using primers WGRB1587-F and WGRB1587-R (molecular marker WGRB1587), or a 114 bp amplification fragment can be amplified using primers WGRB1559-F and WGRB1559-R (molecular marker WGRB1559), then the wheat being tested is found to contain the Fhb8 resistance gene.

[0020] Seventhly, the present invention also protects the use of the primer pairs described above, and / or the molecular markers described above, and / or the reagents or kits described above, in screening wheat resistant to Fusarium head blight.

[0021] In a specific implementation plan, the genomic DNA of the wheat to be tested is amplified by PCR using the primer pairs described above, and the amplification products are detected. If a 289bp amplification fragment can be amplified using primers WGRB1500-F and WGRB1500-R for the molecular marker WGRB1500, or a 141bp amplification fragment can be amplified using primers WGRB1587-F and WGRB1587-R for the molecular marker WGRB1587, or a 114bp amplification fragment can be amplified using primers WGRB1559-F and WGRB1559-R for the molecular marker WGRB1559, then the wheat to be tested is identified as a Fusarium head blight resistant wheat containing the Fhb8 resistance gene.

[0022] Eighthly, the present invention also protects the use of the primer pair described above in cloning the Fusarium head blight resistance gene Fhb8.

[0023] Ninthly, the present invention also protects the use of the primer pairs described above, and / or the molecular markers described above, and / or the reagents or kits described above, in the breeding of wheat resistant to Fusarium head blight.

[0024] The molecular marker for the wheat Fusarium head blight resistance gene Fhb8 was obtained through the following method:

[0025] (I) Wangshui Bai and its reincarnation parent PH691 BC4F 2：3 Population creation and screening of Fhb8 segment recombinants:

[0026] (1) Wangshui white (♀) was crossed with wheat variety PH691 (♂) to obtain hybrid F1. F1 was backcrossed with PH691 for three generations, and then self-crossed to produce BC3F2 population;

[0027] (2) Use the boundary marker of Fhb8 to screen for heterozygous individuals that recombined in this segment in the BC4F2 population, and use the same marker to screen for homozygous individuals that recombined in this segment in the BC3F3 generation.

[0028] (II) Identification of disease-resistant phenotypes in recombinant organisms

[0029] (3) About ten days before flowering, infected wheat grains were sown in the field for inoculation with the recombinant strain, and the inoculation was repeated one week later. The diseased spikelet rate was investigated 15 days after inoculation to evaluate its resistance; the spikes were harvested at maturity, and the diseased grain rate was investigated after manual threshing to evaluate its resistance.

[0030] (III) Molecular Marker Analysis

[0031] (4) DNA was extracted from the resistant parent Wangshuibai, the susceptible parent PH691, and individual plants in the F2 population using the SDS method; molecular markers were developed using sequences located on chromosome 7D of the Chinese spring reference genome to perform polymorphism and specificity analysis on Wangshuibai and PH691.

[0032] (5) Select polymorphic molecular markers that can be amplified between parents and amplify them in the F2 generation population to obtain the genotype data of each individual in the population. Use these markers to detect the genotype of each individual in the F3 family of the heterozygous recombinant offspring.

[0033] The PCR reaction system consisted of 12.5 μl of 10× buffer, 0.75 μl of 25 mM MgCl2, 1 μl of 2.5 mM dNTPs, 0.2 μM each of the left and right primers, 0.1 μl of Taq enzyme (5 u / μl), 10 ng of template DNA, and water to a final volume of 12.5 μl.

[0034] The PCR amplification program was as follows: pre-denaturation at 94℃ for 3 min, followed by denaturation at 94℃ for 30 sec, annealing at 60℃ for 1 min, extension at 72℃ for 1 min, for 35 cycles, and a final extension at 72℃ for 8 min. PCR amplification was performed on a PE9600 amplification instrument. The amplification products were separated by electrophoresis on an 8% non-denaturing polyacrylamide gel and then photographed on a UV transilluminator to record the results.

[0035] (iv) Molecular marker acquisition

[0036] (6) Based on the linkage exchange law, combined with the genotype data of each individual plant in the F2 generation population and the field disease resistance phenotype of the F3 lineage of the heterozygous recombinant, the genetic linkage map of Wangshui White Fhb8 was constructed using the software Mapmaker Macintosh V3.0, and the molecular markers WGRB1500, WGRB1587 and WGRB1559 most closely linked to Fhb8 were obtained.

[0037] Beneficial effects

[0038] This invention is the first in the world to obtain molecular markers WGRB1500, WGRB1587, and WGRB1559 that are closely linked to Fhb8. This can accelerate the application of the disease resistance gene Fhb8 in wheat disease resistance breeding and can also be used for cloning the Fhb8 gene.

[0039] 1. Molecular markers WGRB1500, WGRB1587, and WGRB1559, which are closely linked to Fhb8, were obtained. Among the known molecular markers, WGRB1587 is the most closely linked to Fhb8, with a genetic distance of only 0 cM. ​​This marker can help transfer the gene to promoted varieties and aggregate with other disease resistance genes.

[0040] 2. Convenient identification. All three molecular markers are co-dominant, offering advantages such as convenient detection, stable amplification, and simplicity. Detecting the Fhb8 gene using the WGRB1587 marker can determine the presence and status of Fhb8, predict wheat resistance to Fusarium head blight, and rapidly screen for Fhb8-carrying plants for breeding resistant varieties. Furthermore, using molecular markers for laboratory testing avoids the influence of environmental factors on the variety.

[0041] 3. Improve the efficiency of disease-resistant variety selection and identification, and save costs. Traditional Fusarium head blight resistance breeding is time-consuming, labor-intensive, difficult, and costly. By detecting molecular markers closely linked to the Fusarium head blight resistance gene Fhb8, the work of phenotypic identification can be greatly reduced, and individual plants carrying the disease-resistant gene Fhb8 can be identified at the seedling stage, thereby eliminating non-target plants. This not only saves breeding costs but also greatly improves the efficiency of disease-resistant variety selection.

[0042] 4. Reduce linkage burden. Since wheat resistance to Fusarium head blight is often correlated with some undesirable agronomic traits, early marker-assisted selection of Fusarium head blight resistant lines carrying Fhb8 generally exhibited long ears and tall stems. This may be due to the relatively large initial QTL mapping interval, leading to a large selection interval and some linkage burden. Utilizing molecular markers tightly linked to the Fusarium head blight resistance gene Fhb8 can reduce linkage burden during selection and improve selection efficiency.

[0043] 5. It can be used for cloning the Fusarium head blight resistance gene Fhb8. Map-based cloning of the Fhb8 gene requires obtaining a molecular marker tightly linked to Fhb8. WGRB1587 is the most tightly linked to Fhb8 among all known molecular markers. Attached Figure Description

[0044] Figure 1 Disease incidence rate of spikelets in 11 homozygous recombinants ( Figure 1 a) and disease incidence ( Figure 1 Distribution map of b).

[0045] Figure 2 Genetic linkage map of WGRB1500, WGRB1587, and WGRB1559 with wheat scab resistance gene Fhb8. The right side shows the markers on the genetic linkage map, and the left side shows the genetic distance between the markers.

[0046] Figure 3 WGRB1500 amplification band pattern. M stands for PUC19 / MspI, and the left side shows the molecular weight marker band size (bp). 1 represents Wangshui White, 2 represents PH691, 3, 5, 6, 9, and 10 represent disease-resistant genotypes, and 4, 7, 8, 11, and 12 represent disease-susceptible genotypes. The arrows indicate specific amplified bands.

[0047] Figure 4 WGRB1587 amplification band pattern. M is D2000, and the left side shows the molecular weight marker band size (bp). 1 is Wangshui White, 2 is PH691, 3, 5, 6, 9, and 10 are disease-resistant genotypes, and 4, 7, 8, 11, and 12 are disease-susceptible genotypes. The arrows indicate specific amplified bands.

[0048] Figure 5 WGRB1559 amplification band pattern. M stands for PUC19 / MspI, and the left side shows the molecular weight marker band size (bp). 1 represents Wangshui White, 2 represents PH691, 3, 5, 6, 9, and 10 represent disease-resistant genotypes, and 4, 7, 8, 11, and 12 represent disease-susceptible genotypes. The arrows indicate specific amplified bands. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to the embodiments. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0050] Example 1: Obtaining the molecular marker for the wheat Fusarium head blight resistance gene Fhb8

[0051] (I) Wangshui Bai and its reincarnation parent PH691 BC4F 2：3 Population creation and screening of Fhb8 segment recombinants:

[0052] (1) Wangshuibai (♀) was crossed with wheat variety PH691 (♂) to obtain hybrid F1. F1 was backcrossed with PH691 for three generations, and self-crossed to produce BC3F2 population containing 97 individual plants.

[0053] (2) Eleven heterozygous plants that recombined in this segment were screened using the boundary markers WMC405 and WMC702 of Fhb8. Eleven homozygous plants that recombined in this segment were screened using the same markers in their BC4F3 generation.

[0054] (II) Identification of disease-resistant phenotypes in recombinant organisms

[0055] About ten days before flowering, infected wheat grains were sown in the field to inoculate these homozygous recombinants, and the inoculation was repeated one week later. The incidence of diseased spikelets was assessed 15 days after inoculation, and the incidence of diseased grains was assessed after harvest. Identification results showed that these recombinants exhibited significant segregation of resistance. Figure 1 The strains carrying the Fhb8 gene showed significantly increased resistance compared to the susceptible parents (Table 1).

[0056] (III) Screening of polymorphic molecular markers and analysis of recombinant genotypes

[0057] (1) The developed markers were screened for polymorphism in Wangshui White and PH691, and three molecular markers with polymorphism between the parents were finally obtained, namely WGRB1500, WGRB1587 and WGRB1559.

[0058] (2) The genotypes of all homozygous recombinants were analyzed using the molecular markers WGRB1500, WGRB1587 and WGRB1559, which are polymorphic among the parents (Table 1).

[0059] The PCR reaction system consisted of 12.5 μl of 10× buffer, 0.75 μl of 25 mM MgCl2, 1 μl of 2.5 mM dNTPs, 0.2 μM each of the left and right primers, 0.1 μl of Taq enzyme (5 u / μl), 10 ng of template DNA, and water to a final volume of 12.5 μl.

[0060] The PCR amplification program was as follows: pre-denaturation at 94℃ for 3 min, followed by denaturation at 94℃ for 30 sec, annealing at 60℃ for 1 min, extension at 72℃ for 1 min, for 35 cycles, and a final extension at 72℃ for 8 min. PCR amplification was performed on a PE9600 amplification instrument. The amplification products were separated by electrophoresis on an 8% non-denaturing polyacrylamide gel and then photographed on a UV transilluminator to record the results.

[0061] (iv) Obtaining molecular markers

[0062] Based on linkage and recombination, and combining the genotype data of individual plants in the F2 generation with the field disease resistance phenotypes of the F3 families of heterozygous recombinants (Table 1), a genetic linkage map of Wangshui White Fhb8 was constructed using Mapmaker Macintosh V3.0 software. Molecular markers WGRB1500, WGRB1587, and WGRB1559, which are closely linked to Fhb8, were obtained, with distances of 0.6 cM, 0 cM, and 0.5 cM from the Fhb8 gene, respectively. The primer amplification band patterns for WGRB1500, WGRB1587, and WGRB1559 are shown in [Table 1]. Figure 3 , Figure 4 and Figure 5 .

[0063] Table 1. Wangshui Bai – PH691 BC4F 2:3 Genotypes and phenotypes of homozygous recombinants in a population

[0064]

[0065] W represents the Wangshuibai genotype, P represents the PH691 genotype, the susceptible control is PH691, and the resistant control is the Fhb8 near-isogenic line (NIL). **, *** indicate significant phenotypic differences from the resistant control at the P=0.01 and 0.001 levels.

[0066] Example 2: Application of Molecular Markers in Fusarium Head Blight Resistance Wheat Breeding

[0067] Foreground selection of Fhb8 was performed on a BC2F2 population containing 97 individuals obtained by crossing *Bryum oxysporum* (♀) with PH691 (♂) using molecular markers WGRB1500, WGRB1587, and WGRB1559. Twenty-three individuals homozygous for the *Bryum oxysporum* genotype in the Fhb8 region were selected for background recovery rate testing. A total of 107 pairs of SSR markers distributed across 21 chromosomes were selected to assess the background recovery rate of these individuals. Finally, two individuals with background recovery rates of 93.5% and 92.5%, respectively, were selected for self-pollination to obtain near-isogenic lines 7DL1 and 7DL2. The resistance of these near-isogenic lines to Fusarium head blight was evaluated using two inoculation methods under two different environmental conditions over two years at three locations (see Example 1). The results showed that the constructed near-isogenic lines had significantly improved disease resistance. Compared with the susceptible control PH691, the near-isogenic lines showed an average reduction of 51% in type I resistance index PIS (infected ear rate), 45% in type II resistance index LDR (infected axis length), and 59% in type IV resistance index FDK (infected grain rate) (Table 2). This indicates that the three molecular markers WGRB1500, WGRB1587, and WGRB1559 can be effectively used to select for the Fusarium head blight resistance gene Fhb8, and have significant application value in breeding.

[0068] Table 2. Evaluation of the resistance to Fusarium head blight of the constructed PH691 background Fhb8 near-isogenic line.

[0069]

[0070] Numerical values ​​represent the mean ± SD across the three environments; ** and *** indicate significant differences at the P = 0.001 and 0.0001 levels compared to PH691 when performing Dunnett's test.

[0071] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. Wheat Fusarium head blight resistance gene Fhb8 The application of molecularly labeled primer pairs or reagents or kits containing said primer pairs in screening wheat resistant to Fusarium head blight, wherein said primer pairs are selected from any one of the following (1)-(3): (1) WGRB1500-F: as shown in SEQ ID NO.1; WGRB1500-R: as shown in SEQ ID NO.2; (2) WGRB1587-F: as shown in SEQ ID NO.3; WGRB1587-R: as shown in SEQ ID NO.4; (3) WGRB1559-F: as shown in SEQ ID NO.5; WGRB1559-R: as shown in SEQ ID NO.6; The genomic DNA of the wheat to be tested was amplified by PCR using the primer pairs described above, and the amplification products were detected. If a 289 bp amplification fragment could be amplified using primers WGRB1500-F and WGRB1500-R (molecular marker WGRB1500), or a 141 bp amplification fragment could be amplified using primers WGRB1587-F and WGRB1587-R (molecular marker WGRB1587), or a 114 bp amplification fragment could be amplified using primers WGRB1559-F and WGRB1559-R (molecular marker WGRB1559), then the wheat to be tested was identified as having a gene for resistance to Fusarium head blight. Fhb8 wheat resistant to Fusarium head blight; The Fhb8 is a major QTL for resistance to Fusarium head blight, located in the Xwmc405-Xwmc702 region of wheat chromosome 7D, namely Qfdk.nau-7D.

2. Wheat Fusarium head blight resistance gene Fhb8 The application of molecularly labeled primer pairs or reagents or kits containing said primer pairs in the breeding of wheat resistant to Fusarium head blight, wherein said primer pairs are selected from any one of the following (1)-(3): (1) WGRB1500-F: as shown in SEQ ID NO.1; WGRB1500-R: as shown in SEQ ID NO.2; (2) WGRB1587-F: as shown in SEQ ID NO.3; WGRB1587-R: as shown in SEQ ID NO.4; (3) WGRB1559-F: as shown in SEQ ID NO.5; WGRB1559-R: as shown in SEQ ID NO.6; The genomic DNA of the wheat to be tested was amplified by PCR using the primer pairs described above, and the amplification products were detected. If a 289 bp amplification fragment could be amplified using primers WGRB1500-F and WGRB1500-R (molecular marker WGRB1500), or a 141 bp amplification fragment could be amplified using primers WGRB1587-F and WGRB1587-R (molecular marker WGRB1587), or a 114 bp amplification fragment could be amplified using primers WGRB1559-F and WGRB1559-R (molecular marker WGRB1559), then the wheat to be tested was identified as having a gene for resistance to Fusarium head blight. Fhb8 wheat resistant to Fusarium head blight; The Fhb8 is a major QTL for resistance to Fusarium head blight, located in the Xwmc405-Xwmc702 region of wheat chromosome 7D, namely Qfdk.nau-7D.

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