A functional molecular marker for detecting wheat powdery mildew resistance gene WTK4 and application thereof

Abstract

The application belongs to the field of plant biotechnology and wheat molecular breeding, and discloses a functional molecular marker for detecting a wheat powdery mildew resistance gene WTK4 and application thereof. The wheat powdery mildew resistance gene WTK4 is located on the short arm of the 7D chromosome of Aegilops tauschii, and the WTK4 homologous gene is located on the long arm of the 4A chromosome in the Chinese spring reference genome RefSeq v2.0 version. The functional molecular marker difference site is located inside the WTK4 gene, and the polymorphism is an insertion / deletion (Indel) of 105 bp. The molecular marker provided in the application can accurately detect whether the powdery mildew resistance gene WTK4 exists in the material, and the material containing the WTK4 gene can be distinguished into the Aegilops tauschii disease-resistant genotype and the Chinese spring disease-sensitive genotype according to the size of the amplification band. The application promotes the utilization of the wheat powdery mildew resistance gene WTK4, and can be applied to molecular marker-assisted selection breeding, and accelerates the breeding process of the wheat disease-resistant variety.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant biotechnology and wheat molecular breeding technology, and particularly relates to a functional molecular marker for detecting wheat powdery mildew resistance gene WTK4 and application thereof. BACKGROUND

[0002] Wheat powdery mildew is a fungal disease of wheat caused by Blumeria graminis f. sp. tritici (Bgt), and is also one of the important diseases frequently occurring in wheat production in China. It can cause a reduction of 30%-50% in wheat yield and seriously harm the wheat production in China. Due to the long-term planting of wheat varieties with single resistance in China, directional selection of wheat powdery mildew has occurred, resulting in gradual loss of resistance in most resistant varieties in China. Screening and breeding of disease-resistant wheat varieties is the most basic and effective method for controlling wheat powdery mildew.

[0003] With the progress of sequencing technology and the development of molecular biology technology, although more than 100 (Pm1-Pm69) powdery mildew resistance genes or QTLs have been located in wheat and its close relatives, most of the resistance gene resources have not been fully applied and popularized. It is difficult to apply excellent disease resistance genes in wheat relatives to wheat breeding through traditional breeding methods. Compared with conventional breeding methods, molecular marker-assisted selection technology has many advantages such as clear purpose, shortening of breeding process and improvement of selection efficiency in the process of screening and breeding of wheat disease-resistant varieties. However, most of the molecular markers used for disease resistance sites are linkage molecular markers, and there is a certain distance between the marker and the disease resistance site. Recombination may occur in wheat varieties with different genetic backgrounds, and only linkage molecular markers are used for tracking, which may lead to the loss of the target disease resistance site. Compared with traditional linkage molecular markers, gene-specific molecular markers are co-segregated with the target gene within the gene, and there is no recombination phenomenon. The detection of molecular markers is not affected by the type of material and genetic background, and the detection result is highly reliable, which greatly improves the utilization of disease resistance genes. WTK4 is a wheat powdery mildew resistance gene from Aegilops sharonensis, which has broad-spectrum resistance and still maintains resistance in synthetic hexaploid wheat. It is an excellent powdery mildew resistance gene resource for wheat. At present, WTK4 has not been applied in wheat varieties, and the molecular marker for tracking WTK4 gene has not been reported.

[0004] In view of the above analysis, the technical problems existing in the prior art that need to be solved urgently are:

[0005] How to efficiently and accurately detect and track WTK4 is of great significance to wheat powdery mildew breeding, and it is particularly urgent to develop a WTK4 gene-specific functional molecular marker. SUMMARY

[0006] In view of the problems in the prior art, the application provides a functional molecular marker for detecting a wheat powdery mildew resistance gene WTK4 and application thereof.

[0007] The application is achieved as follows: a functional molecular marker for detecting a wheat powdery mildew resistance gene WTK4, characterized in that the Indel molecular marker is WTK4-Indel, the marker is developed according to the 105bp insertion / deletion site existing at the position of 866bp of the third intron region based on the difference between the WTK4 gene in Aegilops tauschii and the WTK4 homologous gene sequence in Zhonghuichun, and the primer nucleotide sequence of the Indel molecular marker comprises an upstream primer WTK4-Indel-F and a downstream primer WTK4-Indel-R.

[0008] Further, the functional molecular marker for detecting the wheat powdery mildew resistance gene WTK4 has the following upstream and downstream primer nucleotide sequences:

[0009] WTK4-Indel-F: 5'-GTGGGTTTTTGGAGCAGTGG-3'

[0010] WTK4-Indel-R: 5'-AATAACGCACAACCATAGGCA-3'

[0011] Another object of the application is to provide application of the functional molecular marker for detecting the wheat powdery mildew resistance gene WTK4 in identifying wheat powdery mildew resistance, the marker can specifically track the WTK4 gene, and can distinguish the WTK4 genotype into the Zhonghuichun susceptible genotype and the Aegilops tauschii resistant genotype.

[0012] Further, the DNA of the wheat to be detected is detected by using the above molecular marker, if the PCR product appears a specific band of 375bp, it is indicated that the sample to be detected contains the WTK4 gene of the Aegilops tauschii genotype and has the powdery mildew resistance, if the PCR product appears a specific band of 480bp, it is indicated that the wheat to be detected is the WTK4 gene of the Zhonghuichun genotype and does not have the powdery mildew resistance.

[0013] Further, the detection method comprises the following steps:

[0014] S1: extracting the DNA of the sample to be detected by using the CTAB method;

[0015] S2: performing PCR amplification by using the detection primer pair;

[0016] S3: performing electrophoresis analysis on the PCR product.

[0017] S4: In the above identification method, the PCR reaction system and amplification conditions of step S2 are as follows:

[0018] Reaction system: each 10 uL includes: 50 ng / uL genomic DNA 1.0 uL, 5 uL PCR Master Mix,

[0019] Amplification conditions: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 15 seconds, 32 cycles; 72℃ extension for 5 min; 4℃ storage.

[0020] Further, the PCR product is separated by electrophoresis with 1% agarose gel, and the band is read by a gel imaging instrument.

[0021] First, the efficient use of wheat powdery mildew resistance gene resources is the key to breeding disease-resistant varieties. The molecular marker developed in the present application is based on the sequence of the cloned gene WTK4, which belongs to gene-specific molecular marker. Compared with traditional linkage molecular marker, gene-specific molecular marker co-segregates within the gene and does not exist recombination phenomenon, molecular marker detection is not affected by material type and genetic background, the detection result has high reliability, which greatly improves the utilization of gene WTK4.

[0022] Using WTK4 functional marker for amplification in 385 wheat natural populations, only 7 wheat varieties contain WTK4, and the amplification band type is consistent with that of Zhonghua Chun, and the powdery mildew resistance identification result shows that these 7 common wheat are highly susceptible to powdery mildew. The amplification result of the marker in 94 Aegilops lines shows that 12 Aegilops lines have band amplification, and the amplification band is consistent with that of CIae8, and the powdery mildew resistance identification result shows that these 12 Aegilops lines are highly resistant to wheat powdery mildew. It is shown that the frequency of wheat powdery mildew resistance gene WTK4 in wheat germplasm resources is low, which cannot be utilized in wheat breeding, and it is an excellent disease-resistant gene resource of wheat. The marker can specifically track the WTK4 gene, which belongs to a specific functional molecular marker, can accurately and quickly identify whether the material contains the WTK4 gene, and can judge the powdery mildew resistance of the material through the amplification type, and accelerate the efficient use of gene WTK4.

[0023] Second, the technical scheme of the present application fills the technical blank in the industry at home and abroad:

[0024] WTK4 is a wheat powdery mildew resistance gene from Aegilops tauschii, which has a broad-spectrum resistance and is an excellent resistance gene resource for wheat. At present, it has not been used in wheat disease resistance breeding. The WTK4 gene-specific molecular marker developed by the present application can accurately identify the WTK4 gene and the powdery mildew resistance of the material, greatly improving the utilization efficiency of WTK4 in future wheat disease resistance breeding, and filling the domestic and foreign industry gap of lacking WTK4 molecular marker identification.

[0025] Thirdly, the technical scheme of the present application significantly solves several key problems in disease resistance breeding of the prior art by developing a wheat powdery mildew resistance gene detection method based on specific molecular marker WTK4-Indel. Traditional disease resistance identification methods rely on long-period phenotypic observation and environmental condition control, which is time-consuming and laborious, and has significant uncertainty and low efficiency. The present application directly identifies whether the wheat carries the disease resistance gene WTK4 at the molecular level through molecular detection of genomic DNA, thereby improving the detection speed, accuracy and consistency.

[0026] Technically, the present application solves the problem of determination difficulty caused by the polymorphism of powdery mildew resistance traits in the existing breeding detection. Through the specific WTK4-Indel molecular marker, the present application can accurately distinguish between wheat plants carrying disease resistance genotypes (Aegilops tauschii genotypes) and disease susceptible genotypes (Chinese spring genotypes), avoiding the complex phenotype measurement process relied on by traditional detection. In addition, the marker detection method of the present application has no dependence on environmental conditions and growth period for breeding materials, and therefore can be widely used in early disease resistance breeding screening, solving the problems of early judgment difficulty and low breeding efficiency in the breeding process.

[0027] In terms of industrial application, the present application greatly shortens the period of disease resistance breeding, so that breeding institutions can screen a large number of materials in a short time, quickly lock the disease resistance genotype plants, and significantly improve the efficiency and success rate of disease resistance breeding. At the same time, the marker method of the present application can provide disease resistance genotype data at the molecular level, which is convenient for breeding records and the preservation and utilization of disease resistance gene resources, and reduces the consumption of manpower and resources. In the rapid popularization of disease-resistant wheat, the technical scheme supports the screening of large-scale germplasm resources, and provides stable and reliable technical support for the industrialized production of wheat resistance to powdery mildew. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the WTK4 gene sequence Indel site diagram in Aegilops tauschii and Chinese spring provided by the embodiment of the present application: Figure 1 A is a schematic diagram of the gene structure of WTK4, Figure 1 B is a comparison of three Indel sites in CIae8 and Chinese spring, Figure 1C is the amino acid sequence alignment of CIae8 and WTK4 in Chinese Spring, wherein DD-7D-WTK4 and CS-4A-WTK4 represent the homologous protein sequences of CIae8 and WTK4 in Chinese Spring, respectively;

[0029] Figure 2 is part of the detection results of the developed functional molecular marker WTK4-Indel in Aegilops tauschii / wheat natural populations provided by the embodiments of the present application: Figure 2 A is the detection result of the molecular marker in Aegilops tauschii natural populations, Figure 2 B is the detection result of the molecular marker in wheat natural populations, wherein CS, N7AT7B, N7BT7A, N7DT7B and N4AT4B represent Chinese Spring, Chinese Spring null 7A, 7B, 7D and 4A chromosome null tetraploid materials, Fielder, AY61 and PI574467 represent common wheat and Aegilops tauschii without WTK4 gene, respectively;

[0030] Figure 3 is the sequencing result and powdery mildew resistance identification result of Aegilops tauschii and wheat containing WTK4 gene detected by the molecular marker provided by the embodiments of the present application: Figure 3 A is the sequence alignment result of WTK4 gene in 13 Aegilops tauschii and 8 common wheat, Figure 3 B is the seedling stage powdery mildew resistance identification result of 13 Aegilops tauschii and 8 common wheat containing WTK4 gene, wherein the number below WTK4 represents the position in the coding sequence, and Aet and Ta represent Aegilops tauschii and common wheat, respectively. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0032] The WTK4-Indel functional molecular marker of the present application is based on the specific difference of the wheat and Aegilops tauschii powdery mildew resistance gene WTK4, especially the 105 bp Indel site in the third intron region of the gene. Through sequence analysis of the WTK4 gene, specific primers WTK4-Indel-F and WTK4-Indel-R are designed, which can specifically recognize and amplify the WTK4 gene, and then detect whether the wheat plant contains the powdery mildew resistance gene WTK4. The design of the molecular marker effectively makes up for the limitations of phenotype identification in traditional breeding, and provides a rapid and accurate disease resistance gene detection means.

[0033] The working principle of WTK4-Indel marker detection is based on PCR amplification. By using WTK4-Indel-F and WTK4-Indel-R to amplify plant genomic DNA, PCR products of different sizes are generated. In electrophoresis detection, if the WTK4 gene sequence carries the disease-resistant type (genotype of Aegilops tauschii), the product is 375 bp; if it is a susceptible type (genotype of Chinese spring), the product is 480 bp. Through this difference, the genotype can be directly identified, and whether the plant has the ability to resist powdery mildew can be determined, which is suitable for gene screening of Aegilops tauschii and common wheat varieties.

[0034] The WTK4-Indel molecular marker of the application has important application value in molecular marker-assisted selection breeding. It can be used for early screening of disease-resistant wheat varieties, avoiding the complexity and timeliness problems of traditional disease resistance testing. By PCR amplification and electrophoresis detection of breeding materials, the disease-resistant genotype of the plant can be quickly determined, and disease-resistant materials containing the WTK4 gene are selected, which speeds up the breeding process and improves the breeding efficiency.

[0035] In addition, the marker can also be extended to other related plant materials in specific applications, providing a general method for disease resistance gene detection. By adapting the WTK4-Indel marker, researchers can quickly obtain the disease-resistant genotype information of plants in a variety of wheat lines and different growth stages, effectively assist the transmission and stability evaluation of disease-resistant genes, and realize the early selection and promotion of disease-resistant varieties.

[0036] The embodiment of the application provides a functional molecular marker WTK4-Indel for detecting a wheat powdery mildew resistance gene WTK4. The marker is based on the difference between the WTK4 gene sequences in Aegilops tauschii and Chinese spring, and detects a 105 bp Indel site in the third intron region. For Aegilops tauschii, there is no 105 bp insertion at the 855th to 856th nucleotide of the third intron of the WTK4 gene, and the nucleotide sequence is shown as SEQ ID NO. 1. The homologous gene of WTK4 in Chinese spring has a 105 bp insertion, and the sequence is shown as SEQ ID NO. 2 (Table 1; Figure 1 A).

[0037] Example 1: Obtaining of the functional molecular marker WTK4-Indel of the wheat powdery mildew resistance gene WTK4

[0038] 1. Development of the functional molecular marker WTK4-Indel of WTK4

[0039] This invention first identifies a wheat powdery mildew resistance gene in the jointed wheat variety CIae8, finding that its location overlaps with the cloned wheat powdery mildew resistance gene WTK4. Functional verification confirms that the powdery mildew resistance locus on the 7DS of CIae8 is indeed WTK4. Using the WheatOmics 1.0 website, a genome-wide alignment of WTK4 was performed in the Triticeae reference genome database. Homologous genes of WTK4 were found only on the short arm 4A of *Triticeae chinensis*. and Besides containing a large amount Aside from the locus, the main difference lies in the presence of two [unclear] in the third intron region. site ( Figure 1 B). Both The sequence similarity was 98.8%. TraesCS4A02G481300 in Chinese spring contains 20 SNP sites and one 6 bp insertion. The amino acid sequence similarity between the two is 98.34%, containing 10 amino acid differences and 2 amino acid insertions. Figure 1 C).

[0040] Based on the differences in the WTK4 gene sequence between Chinese Spring and CIae8, this invention uses Primer 5.0 software to design an Indel marker WTK4-Indel at the 105 bp Indel site in the third intron region.

[0041] Table 1. Genotyping information of the developed functional molecular markers

[0042] Primer sequences for developed functional molecular markers

[0043] The forward primer WTK4-Indel-F sequence is as follows:

[0044] 5'-GTGGGTTTTTGGAGCAGTGG-3'

[0045] The reverse primer WTK4-Indel-R sequence is as follows:

[0046] 5'-AATAACGCACAACCATAGGCA-3'.

[0047] 2. Validation of the functional molecular marker WTK4-Indel for WTK4

[0048] The CTAB method is used to extract the genomic DNA of the sample to be detected, including Aegilops tauschii materials: CIae8, PI574467, AL8 / 78, AY61, and common wheat materials: Fielder, Chinese Spring (CS), Chinese Spring nullisomic-tetrasomic materials (N7AT7B, N7BT7A, N7DT7B and N4AT4B).

[0049] The PCR amplification system is as follows: 10 μL of 2×PCR MasterMix, 1 μL of 10 μmol / L primer, 1 μL of 10 ng / μL template DNA, 7 μL of double-distilled water, and 1 μL of Taq DNA polymerase. L, including: 50 ng / μL of genomic DNA, 1 μL of 10 μmol / L primer, 1 μL of 10 ng / μL template DNA, 7 μL of double-distilled water, and 1 μL of Taq DNA polymerase. L of genomic DNA, 1.0 μL of 10 μmol / L primer, 1 μL of 10 ng / μL template DNA, 7 μL of double-distilled water, and 1 μL of Taq DNA polymerase. L, 5 μL of 2×PCR MasterMix, L of 2×PCR MasterMix, 1 μL of 10 μmol / L primer, 1 μL of 10 ng / μL template DNA, 7 μL of double-distilled water, and 1 μL of Taq DNA polymerase.

[0050] The PCR amplification conditions are as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 15 s, 32 cycles; 72℃ extension for 5 min; and 4℃ preservation.

[0051] The PCR product is subjected to electrophoresis separation by using 1% agarose gel, and the band is read by using a gel imaging instrument.

[0052] The marker detection result shows that the amplified band size in CIae8 is 375 bp, the amplified band size in Chinese Spring is 480 bp, and there is no amplified band in the sequencing varieties PI574467, AL8 / 78, AY61 and Fielder which do not contain the WTK4 gene. Figure 2 The amplification result of the marker in the nullisomic-tetrasomic of the 7th homologous group of Chinese Spring shows that there are amplified bands in N7AT7B, N7BT7A and N7DT7B, and the band size is consistent with that of Chinese Spring, which indicates that the WTK4 does not exist on the 7A, 7B and 7D of Chinese Spring. Figure 2

[0053] The molecular marker designed in the embodiment of the present application can specifically track the WTK4 gene, that is, the plant DNA is detected by using the primer group for PCR amplification, if the amplification product is a 375 bp specific band, the WTK4 carried in the material is of the Aegilops tauschii type, if the amplification product is a 480 bp specific band, the WTK4 carried in the material is of the Chinese Spring type, and if there is no amplification product, the WTK4 gene is not carried in the wheat.

[0054] Example 2: Application of the functional molecular marker of the wheat powdery mildew resistance gene WTK4 to the powdery mildew resistance of the Aegilops tauschii / wheat natural population.

[0055] ​In order to evaluate the effect of WTK4 gene on powdery mildew resistance, the presence of WTK4 gene in a natural population consisting of 385 wheat varieties and 94 Aegilops lines is detected, the powdery mildew resistance of the material containing WTK4 gene is identified at the seedling stage, and the WTK4 gene sequence of the material containing WTK4 gene is cloned and compared and analyzed.

[0056] 1. Identification of WTK4 functional molecular marker in a natural population

[0057] The DNA of 385 wheat varieties and 94 Aegilops lines is extracted by using the CTAB method, and the molecular marker WTK4-Indel developed in the application is used for identification.

[0058] The PCR reaction system and product detection are the same as those in Example 1.

[0059] If the amplification product is a 375bp specific band, the WTK4 carried in the material is of the Aegilops genotype, if the amplification product is a 480bp specific band, the WTK4 carried in the material is of the Chinese Spring genotype, and if there is no amplification product, the WTK4 gene is not carried in the wheat.

[0060] The results show that in the amplification results of 94 Aegilops lines, 12 Aegilops lines amplify a specific band of 375bp, and the size of the amplification band is consistent with that of CIae8 (A). Figure 2 B). In the 385 wheat natural population, only 7 wheat varieties amplify a specific band of 480bp, and the size of the amplification band is consistent with that of Chinese Spring (B). Figure 2

[0061] 2. Cloning and sequence analysis of WTK4 gene

[0062] The WTK4 gene is cloned in the 7 common wheat varieties containing the amplification band with the size consistent with that of Chinese Spring and the 12 Aegilops lines containing the amplification band with the size consistent with that of CIae8, and sequence comparison analysis is carried out by using the DNAMAN software.

[0063] The sequence analysis results show that the WTK4 of the 12 Aegilops lines containing WTK4 has a sequence similarity of 100% with the WTK4 in CIae8 (A), and all belong to the string bead variety of Aegilops. Figure 3 The sequence of WTK4 in the 7 common wheat varieties has a sequence similarity of 100% with the homologous gene in Chinese Spring (A), and most of the common wheat containing WTK4 belongs to the local variety of wheat (A). Figure 3

[0064] ​​​The results show that the developed functional molecular marker of the gene can not only specifically detect the germplasm resources containing the WTK4 gene in a natural population, but also can distinguish the materials containing the WTK4 gene into the Aegilops genotypes and the Chinese Spring genotypes according to the size of the amplified bands.

[0065] 3. Powdery mildew resistance identification of the natural population

[0066] The powdery mildew resistance of a natural population consisting of 385 wheat varieties and 94 Aegilops lines is identified. The test materials are planted in a sterile greenhouse with humidity of 65%-75%, light of 14 h / 22°C, and darkness of 10 h / 20°C, and when they grow to two-leaf-one-heart, they are inoculated with the physiological race E26 in vivo, and when the spores of the powdery mildew on the susceptible control are fully diseased (about 7 days after inoculation), the phenotypes of the powdery mildew are investigated. The disease level is divided according to the 0-4 grade standard at the seedling stage, that is:

[0067] IT=0: immune, no hypersensitive spot and spore;

[0068] 0: highly resistant, with hypersensitive spot on the leaf;

[0069] 1: highly resistant, with a small number of obvious powdery mildew spores on the leaf;

[0070] 2: moderately resistant, with powdery mildew conidia on the leaf;

[0071] 3: moderately susceptible, with a large number of conidia on the leaf;

[0072] 4: highly susceptible, with a large number of conidia on the leaf.

[0073] The results show that the eight common wheat varieties containing the WTK4 gene including Chinese Spring are all highly susceptible to the powdery mildew, and the thirteen Aegilops lines including CIae8 are all highly resistant to the wheat powdery mildew. Figure 3 B). The results show that the developed functional molecular marker of the WTK4 gene can not only specifically screen the materials containing the WTK4 gene, but also can distinguish them into the resistant Aegilops genotypes and the susceptible Chinese Spring genotypes. The molecular marker can be applied to the molecular marker-assisted selection breeding, and accelerate the transfer of the WTK4 gene to the wheat varieties.

[0074] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited to this. Any modification, equivalent replacement and improvement made by any person skilled in the art within the technical range disclosed by the present application, as long as it is within the spirit and principle of the present application, should be covered within the protection scope of the present application.

Claims

1. Use of a primer of a gene functional molecular marker for detecting wheat powdery mildew resistance gene WTK4 in identifying the powdery mildew resistance of natural populations of Aegilops tauschii and wheat, which can specifically track the WTK4 gene and distinguish the WTK4 genotype into the Chinese spring susceptible genotype and the Aegilops tauschii resistant genotype; the primer comprises an upstream primer WTK4-Indel-F and a downstream primer WTK4-Indel-R, and the nucleotide sequences of the upstream and downstream primers are as follows: WTK4-Indel-F: 5'-GTGGGTTTTTGGAGCAGTGG-3' WTK4-Indel-R: 5'-AATAACGCACAACCATAGGCA-3' The DNA of the Aegilops tauschii or wheat to be tested is detected by using the primer, if a specific band of 375 bp appears in the PCR product, it indicates that the sample to be tested contains the WTK4 gene of the Aegilops tauschii genotype and has the powdery mildew resistance; if a specific band of 480 bp appears in the PCR product, it indicates that the sample to be tested contains the WTK4 gene of the Chinese spring genotype and does not have the powdery mildew resistance.

2. The primer of claim 1, wherein the primer is a pair of primers, and the pair of primers comprises the upstream primer WTK4-Indel-F and the downstream primer WTK4-Indel-R.

3. The primer of claim 1, wherein the primer is a pair of primers, and the pair of primers comprises the upstream primer WTK4-Indel-F and the downstream primer WTK4-Indel-R. ​ ​

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