Molecular marker for tracking powdery mildew-resistant gene PmL180 of Teimofei wheat and application of molecular marker

Abstract

The invention discloses a molecular marker for tracking a powdery mildew-resistant gene PmL180 of Teimofei wheat and application of the molecular marker, and relates to the technical field of agricultural biological genes. The molecular marker is obtained through amplification of a ZLN2 primer, the ZLN2 primer comprises an upstream primer ZLN2-F and a downstream primer ZLN2-R, and nucleotide sequences of the upstream primer ZLN2-F and the downstream primer ZLN2-R are provided. According to the invention, the wheat powdery mildew resistance gene PmL180 is detected by using the primer amplification molecular marker, and parents of wheat are purposefully selected in wheat breeding, so that a guidance basis is provided for breeding a new variety of powdery mildew resistance type wheat.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biological gene technology, and in particular to a molecular marker for tracking a powdery mildew resistance gene PmL180 of wheat timofeiwei and an application thereof. Background Art

[0002] Wheat is an important food crop in the world, and its production is threatened by various diseases. Among them, wheat powdery mildew is an airborne fungal disease caused by Blumeria graminis f.sp. tritici of the Poaceae family. The disease has the characteristics of diverse physiological subspecies and rapid mutation. In the field of disease-resistant breeding, although effective powdery mildew resistance genes such as Pm2, Pm4a, Pm4b and Pm21 are still playing a role in the current wheat promotion varieties, the varieties frequently lose resistance, and the discovery and use of new disease-resistant genes is particularly urgent.

[0003] As a close relative of common wheat, Triticum timopheevii (2n=4x=28, AtAtGG) has rich genetic diversity and is an important gene source for discovering excellent disease resistance genes and broadening the genetic basis of wheat. T.timopheevii is resistant to fungal diseases such as leaf rust, powdery mildew and stem rust. In addition, Triticumtimopheevii has also been proven to be resistant to abiotic stresses such as salt tolerance and traits that affect grain quality. Using the rich genetic resources of Triticum timopheevii, several excellent powdery mildew resistance genes (Pm2, Pm6, Pm27, Pm37) have been successfully discovered, providing new impetus for wheat genetic improvement and the improvement of variety resistance. The application of molecular breeding technology has greatly improved breeding efficiency. Through molecular marker-assisted selection, the target gene can be accurately integrated into the new variety, while reducing the introduction of unnecessary genes and accelerating the breeding process. Therefore, the development of molecular markers for the powdery mildew resistance gene PmL180 and its application in molecular marker-assisted selection breeding based on the PmL180 gene is of great significance for the selection of powdery mildew resistant wheat varieties and the effective prevention and control of wheat powdery mildew. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a molecular marker for tracking the powdery mildew resistance gene PmL180 of Timofeiwei wheat and its application, use primers to amplify the molecular marker to detect the wheat powdery mildew resistance gene PmL180, and purposefully select its parents in wheat breeding, so as to provide guidance for breeding new wheat varieties with resistance to powdery mildew.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: a molecular marker for tracking the powdery mildew resistance gene PmL180 of wheat timofeiwei is provided, and the molecular marker is obtained by amplification of ZLN2 primers, and ZLN2 primers include upstream primer ZLN2-F and downstream primer ZLN2-R. The nucleotide sequence of the upstream primer ZLN2-F is shown in SEQ ID NO.1, specifically GGTTGTCCAGGCGGTTCAC, and the nucleotide sequence of the downstream primer ZLN2-R is shown in SEQ ID NO.2, specifically GCTTGTATGGCACGACTCTTCT.

[0006] Furthermore, molecular markers were amplified from wheat genomic DNA using the ZLN2 primer pair.

[0007] Furthermore, the CDS region sequence of the powdery mildew resistance gene PmL180 of wheat Timofeiwei is shown in SEQ ID NO.3.

[0008] Furthermore, the molecular markers of the powdery mildew resistance gene PmL180 of Timofeiwei wheat are tracked for their application in the detection, identification and auxiliary identification of wheat powdery mildew resistance traits or molecular marker-assisted breeding.

[0009] A method for detecting a powdery mildew resistance gene PmL180 in a wheat sample of Timofeed, comprising the following steps: (1) Extracting genomic DNA from the wheat sample to be tested; (2) using the above-mentioned ZLN2 primer to perform PCR amplification on the genomic DNA of the wheat sample to be tested to obtain amplification primers; (3) Perform electrophoresis and detection on the amplification primers; if a specific band of 231 bp can be amplified, it indicates that the tested wheat sample carries the powdery mildew resistance gene PmL180; otherwise, the tested wheat sample does not carry the powdery mildew resistance gene PmL180.

[0010] Furthermore, in step (2), during PCR amplification, the PCR amplification system is 10ul, including: 1.0 μL of 50 ng / μL Timofeiwei wheat genomic DNA, 5 μL of PCR Master Mix, 0.3 μL of upstream primer, 0.3 μL of downstream primer, and 3.4 μL of sterile deionized water.

[0011] Furthermore, in step (2), during PCR amplification, the PCR amplification program is: pre-denaturation at 94°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 55°C for 25 seconds, extension at 72°C for 5 seconds, 30 cycles; extension at 72°C for 10 minutes; and storage at 4°C.

[0012] The present invention has the following beneficial effects: 1. The specific ZLN2 primer provided by the present invention, combined with PCR amplification and electrophoresis detection technology, can quickly and accurately identify whether wheat carries the powdery mildew resistance gene PmL180 in a short time (such as through a 231 bp specific band). Compared with traditional phenotypic identification methods, it avoids the disadvantages of environmental interference and long time consumption, and significantly improves detection efficiency and reliability.

[0013] 2. The molecular markers of the present invention can be directly applied to early screening in disease-resistant breeding. Through molecular marker-assisted selection (MAS), plants carrying the PmL180 gene can be accurately screened, reducing the workload of blind backcrossing and field trials, shortening the breeding cycle, and reducing breeding costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Some phenotypic identification results of 234 wheat populations infected by Bgt1 strain; Figure 2 This is a schematic diagram of the mixed pool positioning results; Figure 3 A schematic diagram of the haplotype results of the common introgression interval of 234 materials; Figure 4 The p-value, expression level and annotation information of 18 genes; Figure 5 The p-value, expression level and annotation information of 5 disease resistance-related genes; Figure 6 This is a schematic diagram of the wheat material testing results. DETAILED DESCRIPTION

[0015] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0016] Example 1 Screening and molecular markers of the powdery mildew resistance gene PmL180, the specific process is as follows: (1) Phenotypic identification of 234 wheat populations was performed using the Bgt1 strain (collected from Jinzhong City, Shanxi Province and containing AvrPmL180) (part of the phenotypic identification results are shown in Figure 1 As shown in the figure), through GWAS and haplotype analysis, a disease-resistant material 23B130 was screened out. This material comes from the American Germplasm Resources Information Network, with the provenance number PI170911, and is a common hexaploid wheat.

[0017] (2) The resistant material was hybridized with the susceptible material Fielder to obtain F1 that showed resistance. The F1 was self-pollinated to obtain F2, and the extreme resistance-susceptibility segregation ratio of F2 was 41:115, which was consistent with the resistance-susceptibility segregation ratio of 1:3 (χ2 = 0.137, P = 0.71). The disease-resistant pool and the disease-susceptible pool were constructed according to the segregation grouping population analysis method, and transcriptome sequencing was performed. Using the Fielder reference genome, the SNP difference frequency of the offspring was calculated and the candidate region was analyzed to be on chromosome 2B, such as Figure 2 shown.

[0018] (3) The 23B130 material was resequenced and aligned to the combined reference genome of CS+ Triticum timopheevii. By calculating similarity and deep comparison, it was found that chromosome 2B was introgressed from chromosome 2G on Triticum timopheevii. Among the 234 wheat populations, there were 11 materials with a similar resistance spectrum to 23B130. After comparison and deep statistics, these 11 materials all had Triticum timopheevii introgression on chromosome 2B, and the common introgression interval was Chr2B:691-733M (CSv1.1). The phenotypes of the 12 materials containing the introgressed fragments were all resistant, and the haplotypes in this region were clearly distinguished from the other 229 wheat materials, such as Figure 3 As shown. Among them, Figure 3 In the figure, the bar graph on the left is the phenotype of each wheat material to the Bgt1 strain, with a total of 0-4 levels. The lighter the color of the heat map, the weaker the virulence, and the darker the color, the stronger the virulence.

[0019] (4) Due to the existence of recombination suppression on wheat chromosomes, the gene could not be found through traditional genetic positioning. Therefore, the TWAS method was used to find the gene. The 234 wheat materials were sampled before inoculation (0d) and after inoculation (8d), and then transcriptome sequencing was performed. The Triticum timopheevi genome was used for samlon quantification. The strain Bgt1 (known to contain Avr-PmN2) was used as the phenotype. Different models (MLM, CMLM, MMLM) were used. At 0d, a common association peak was found at the end of the long arm of Chr2G, and its significant pick was in the interval Chr2G:693-725M, which also proved that there was indeed an introgression fragment in this region. The same operation was performed on the 8d data, and the significant pick was located in the same interval. There were 1659 genes in the interval, and the cutoff threshold was set to be greater than 6 (B The Ferroni correction threshold line was set to 6.5 (-log10(0.05 / number of genes)), and 18 genes exceeded the threshold line. Combined with the relevant functional annotation information, it was found that only 2 of the 18 genes were related to the disease resistance type. The average expression levels of the materials containing the introgression fragments (12) and the average expression levels of the materials without the introgression fragments (229) were statistically analyzed. It was found that the only gene with a significant difference in the average expression level of these 12 wheat materials at 8 days from the average expression level of the remaining 229 was gene1 (12wheat_TPM:10.56; 229 wheat_TPM:0.03). The expression level of another gene related to disease resistance (12wheat_TPM:2.65; 229 wheat_TPM:0.06) did not vary much, and the p-value of gene1 was greater than that of another gene (such as Figure 4 If the threshold line is not set, the 1659 genes are arranged in ascending order according to the p-value, ending with gene1. It is found that there are 3 genes related to disease resistance, but only the expression level of gene1 in 12 wheat materials is significantly greater than the average expression level of the remaining 229 materials, and the expression levels of the other two genes do not change much.

[0020] (5) To reduce the interference of noise, the CS+Triticum timopheevi combined genome was used for samlon quantification. The same operation was performed as above, without setting the threshold line. The p-values ​​of the genes in the interval were arranged from small to large at 8 days, ending with gene1. It was found that there were 5 genes related to disease resistance. Only the expression level of gene1 in 12 wheat materials (12wheat_TPM: 15.25) was significantly greater than the average expression level of the remaining 229 materials (229 wheat_TPM: 0.02). The expression levels of the remaining genes did not change much (such as Figure 5Therefore, gene1 was temporarily named as the candidate gene of PmL180.

[0021] (6) Based on the cds region of gene1, the primer5.0 software was used to design molecular markers. By detecting the ZLN2 molecular marker, it is possible to specifically detect whether the material carries the gene. If a specific band of 231 bp can be amplified, it means that the sample to be tested carries the PmL180 gene; otherwise, the sample to be tested does not carry the PmL180 gene. For example, the gel image below (as shown in Figure 6 ), 1-5 are wheat materials containing the introgressed fragment (5 materials were selected from the above 12 materials, carrying the PmL180 gene), 6 is the susceptible variety Fielder, which has no band because it does not contain the disease-resistant gene PmL180, and 7 is the susceptible variety CS, which has no band because it does not contain the disease-resistant gene PmL180.

[0022] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A molecular marker for tracking the powdery mildew resistance gene PmL180 in wheat timofeiwei, characterized in that: The molecular marker is amplified by ZLN2 primers, wherein the ZLN2 primers include an upstream primer ZLN2-F and a downstream primer ZLN2-R, wherein the nucleotide sequence of the upstream primer ZLN2-F is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer ZLN2-R is shown in SEQ ID NO.

2.

2. The use of the molecular marker for tracking the powdery mildew resistance gene PmL180 of wheat Timofeiwei according to claim 1 in the detection, identification and auxiliary identification of the powdery mildew resistance trait of wheat or molecular marker-assisted breeding.

3. A method for detecting the powdery mildew resistance gene PmL180 in a wheat sample of Timofeed, characterized in that: The following steps are involved: (1) Extracting genomic DNA from the wheat sample to be tested; (2) using the ZLN2 primer described in claim 1 to perform PCR amplification on the genomic DNA of the wheat sample to be tested to obtain amplification primers; (3) Perform electrophoresis and detection on the amplification primers; if a specific band of 231 bp can be amplified, it indicates that the tested wheat sample carries the powdery mildew resistance gene PmL180; otherwise, the tested wheat sample does not carry the powdery mildew resistance gene PmL180.

4. The method for detecting the powdery mildew resistance gene PmL180 in a wheat sample of Timofeed according to claim 3, characterized in that: In step (2), during PCR amplification, the PCR amplification system is 10ul, including: 1.0 μL of 50 ng / μL Timofeiwei wheat genomic DNA, 5 μL PCR Master Mix, 0.3 μL of upstream primer, 0.3 μL of downstream primer, and 3.4 μL of sterile deionized water.

5. The method for detecting the powdery mildew resistance gene PmL180 in a wheat sample of Timofeed according to claim 3, characterized in that: In step (2), during PCR amplification, the PCR amplification program is: pre-denaturation at 94°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 55°C for 25 seconds, extension at 72°C for 5 seconds, 30 cycles; extension at 72°C for 10 minutes; and storage at 4°C.

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