A molecular marker for identifying barley powdery mildew susceptibility and use thereof

Abstract

The application provides a molecular marker for identifying barley powdery mildew susceptibility and application, and belongs to the technical field of molecular biology and plant molecular breeding, wherein a SNP marker is present on a BPM1 gene, the SNP marker is located at the 1688th position of the BPM1 gene, and the polymorphic site is C / T. Specific amplification primers are designed according to the molecular marker, and genotypes are identified through PCR amplification and high-resolution melting curve analysis. The molecular marker and the amplification primers thereof can be used for predicting barley powdery mildew resistance, breeding barley germplasm resources resistant to powdery mildew, and have the advantages of simple operation, high identification accuracy, cost saving and the like, and can significantly shorten the breeding period of the barley resistant to powdery mildew and save manpower and material resources.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and plant molecular breeding, and in particular relates to a molecular marker for detecting susceptibility to barley powdery mildew and its application. Background Art

[0002] Barley (Hordeum vulgare L.) is an important cereal crop, widely used in the food, feed and brewing industries. However, barley is susceptible to a variety of diseases during its growth process, among which powdery mildew is a common fungal disease caused by the fungus Erysiphegraminis f.sp.hordei. Powdery mildew not only reduces the yield and quality of barley, but may also affect food safety and human health. The importance of studying barley resistance to powdery mildew lies in reducing yield losses, ensuring food security, and reducing the use of chemical pesticides to protect the environment. At the same time, the exploration and utilization of disease-resistant gene resources can promote barley variety improvement and industrial development. At present, research on barley powdery mildew genes is mostly focused on the MLO gene, and research on other genes is not in-depth enough. It is necessary to discover new molecular markers for genes in the powdery mildew resistance segment to develop suitable for efficient screening of powdery mildew-resistant barley and shorten breeding time. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a molecular marker for detecting susceptibility to powdery mildew in barley and its application, which can efficiently screen powdery mildew-resistant barley and shorten breeding time.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a molecular marker for identifying barley powdery mildew susceptibility, wherein the molecular marker detects barley powdery mildew susceptibility by detecting SNP markers;

[0006] The SNP marker is located at position 1688 of the BPM1 gene on barley chromosome 4, where the base is C or T. If the base is C, it is a powdery mildew resistance allele, and if the base is T, it is a powdery mildew susceptible allele.

[0007] The molecular markers include primers for detecting SNP markers: HvBg01-F and HvBg01-R, and the nucleotide sequences thereof are shown in SEQ ID No: 3 and SEQ ID No: 4, respectively.

[0008] The present invention provides a method for identifying barley powdery mildew susceptibility genotypes, comprising the following steps:

[0009] 1) Extracting genomic DNA from the barley to be tested, using it as a template, and performing PCR amplification using primers HvBg01-F and HvBg01-R to obtain an amplified product;

[0010] 2) Analyze barley genotype based on the melting curve of the amplified product;

[0011] When the melting curve of the amplified product is a single peak and the melting temperature is 87.2-87.6°C, the genotype of the barley BPM1 gene at position 1688 is determined to be CC, which is a homozygous genotype for powdery mildew resistance.

[0012] When the melting curve of the amplified product showed a single peak and the melting temperature was 86.6-86.9°C, the genotype of the barley BPM1 gene at position 1688 was determined to be TT, which was confirmed to be a homozygous genotype susceptible to powdery mildew.

[0013] When the melting curve of the amplified product is a double peak and the melting temperature of the main peak is between the two peaks, the genotype of the barley BPM1 gene at position 1688 is determined to be CT, which is determined to be a powdery mildew resistance heterozygous genotype.

[0014] The present invention provides application of the molecular marker in identifying barley powdery mildew susceptibility genotypes.

[0015] The present invention provides application of the molecular marker in identifying susceptibility to barley powdery mildew.

[0016] The present invention provides application of the molecular marker in identifying and screening powdery mildew-resistant barley germplasm resources.

[0017] The present invention provides application of the molecular marker in molecular marker-assisted breeding of barley varieties resistant to powdery mildew.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a molecular marker method, distinct from the MLO gene, for identifying barley powdery mildew resistance. By identifying a single-nucleotide polymorphism (SNP) marker in the BPM1 gene, specific amplification primers are designed for the SNP marker, and genotype identification is performed through PCR amplification and high-resolution melting curve (HRM) analysis. The amplification products of the molecular markers provided by the present invention have significantly different annealing temperatures, and the haplotype of the barley material to be tested can be determined through HRM analysis. This method is low-cost, requires no enzyme digestion, and does not require gel electrophoresis. The detection procedure is convenient and fast, and can shorten the breeding cycle of powdery mildew-resistant barley and reduce breeding costs. The method has the advantages of simple operation, high identification accuracy, and cost savings. It can significantly shorten the breeding cycle of powdery mildew-resistant barley and save manpower and material resources for breeding. When the molecular markers provided by the present invention are used for assisted breeding, accurate, simple, and efficient detection of the BPM1 genotype of progeny can be achieved, while also enabling non-destructive detection in the early stages of barley growth. This method is suitable for large-scale screening of BPM1 genotypes in commercial large-scale breeding and identification of BPM1 alleles in barley germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a sequence alignment diagram of the BPM1 gene between the powdery mildew-resistant parent Feng7 and the powdery mildew-susceptible parent Vlamingh;

[0021] Figure 2 These are the HRM curves of the powdery mildew-resistant variety Feng7, the powdery mildew-susceptible variety Vlamingh, and the high-quality and high-yield barley variety Hua30; where F is Feng7, V is Vlamingh, and H is Hua30;

[0022] Figure 3 This is the HRM curve of the F2 heterozygous type constructed from the powdery mildew-resistant parent Feng7 and the powdery mildew-susceptible parent Vlamingh. Among them, A is the individual with the same genotype as the powdery mildew-susceptible parent Vlamingh, B is the individual with the same genotype as the powdery mildew-resistant parent Feng7, and Z is the heterozygous type.

[0023] Figure 4 These are the sequencing results of homozygous and heterozygous types of barley parents and F2 populations; among them, A is the individual in the powdery mildew-susceptible parent or F2, B is the individual in the powdery mildew-resistant parent or F2 population, and H is the heterozygous individual. DETAILED DESCRIPTION

[0024] The present invention provides a molecular marker for identifying barley powdery mildew susceptibility, wherein the molecular marker detects barley powdery mildew susceptibility by detecting SNP markers;

[0025] The SNP marker is located at position 1688 of the BPM1 gene on barley chromosome 4. The base at this position is C or T. If the base is C, it is a powdery mildew resistance allele, and if the base is T, it is a powdery mildew susceptible allele. The powdery mildew resistance sequence of the BPM1 gene is shown in SEQ ID No: 1, and the powdery mildew susceptible sequence is shown in SEQ ID No: 2. The sequences of SEQ ID No: 1 and SEQ ID No: 2 are shown in Figure 1 .

[0026] The molecular markers include primers for detecting SNP markers: HvBg01-F and HvBg01-R, and the nucleotide sequences thereof are shown in SEQ ID No: 3 and SEQ ID No: 4, respectively.

[0027] The present invention provides a method for identifying barley powdery mildew susceptibility genotypes, comprising the following steps:

[0028] 1) Extracting genomic DNA from the barley to be tested, using it as a template, and performing PCR amplification using primers HvBg01-F and HvBg01-R to obtain an amplified product;

[0029] 2) Analyze barley genotype based on the melting curve of the amplified product;

[0030] When the melting curve of the amplified product is a single peak and the melting temperature is 87.2-87.6°C, the genotype of the barley BPM1 gene at position 1688 is determined to be CC, which is a homozygous genotype for powdery mildew resistance.

[0031] When the melting curve of the amplified product showed a single peak and the melting temperature was 86.6-86.9°C, the genotype of the barley BPM1 gene at position 1688 was determined to be TT, which was confirmed to be a homozygous genotype susceptible to powdery mildew.

[0032] When the melting curve of the amplified product is a double peak and the melting temperature of the main peak is between the two peaks, the genotype of the barley BPM1 gene at position 1688 is determined to be CT, which is determined to be a powdery mildew resistance heterozygous genotype.

[0033] In the present invention, the method for extracting the genomic DNA of the barley to be tested is preferably to use the TianGen high-efficiency plant genomic DNA extraction kit for extraction, as follows:

[0034] Sample preparation: Grind the plant tissue and mix with Buffer FGA and RNase A, vortex and let it stand.

[0035] Lysis: Add buffer LP2 and mix thoroughly, then centrifuge and collect the supernatant.

[0036] Adsorption: Add buffer LP3 (containing anhydrous ethanol) and mix well. Add the mixture to adsorption column CB3, centrifuge and discard the waste liquid.

[0037] Cleaning: Wash the adsorption column twice with rinse solution PW containing anhydrous ethanol, centrifuge and discard the waste liquid.

[0038] Drying: Centrifuge the column to remove residual rinse solution and allow to dry.

[0039] Elution: Transfer the adsorption column to a new centrifuge tube, add elution buffer TB dropwise, and centrifuge to collect the DNA.

[0040] Please refer to the instruction manual for more detailed steps.

[0041] After obtaining the genomic DNA of the barley, the present invention uses primers HvBg01-F and HvBg01-R to perform PCR amplification to obtain an amplified product; the PCR reaction system preferably includes the following components in 10 μL: 1 μL of EasyTaq reaction buffer, 0.8 μL of 2.5 mM dNTP, primers HvBg01-F and HvBg01-R are preferably used at a concentration of 10 μM and in an amount of 0.2 μL, 0.125 μL of 20×EvaGreen fluorescent nucleic acid dye, 0.1 μL of EasyTaq DNA polymerase (5 U / μL), 1 μL of DNA template, and ultrapure water to make up the remainder of the PCR reaction system.

[0042] The PCR reaction procedure is preferably: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 53°C for 30 seconds, and extension at 72°C for 10 seconds, for a total of 35 cycles: the last extension is at 72°C for 5 minutes.

[0043] The present invention provides application of the molecular marker in identifying barley powdery mildew susceptibility genotypes.

[0044] The present invention provides application of the molecular marker in identifying susceptibility to barley powdery mildew.

[0045] The present invention provides application of the molecular marker in identifying and screening powdery mildew-resistant barley germplasm resources.

[0046] The present invention provides application of the molecular marker in molecular marker-assisted breeding of barley varieties resistant to powdery mildew.

[0047] The DNA extraction kit used was the TianGen High-Efficiency Plant Genomic DNA Extraction Kit (Cat. No. DP350).

[0048] Fengdamai No. 7 (Feng7) comes from the Dali Agricultural Science Institute and is a hybrid of Fengdamai No. 6 and S500;

[0049] Vlamingh is a major barley variety grown in Australia and was provided by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences;

[0050] Hua 30, formerly known as Hua 94-30, is a two-ribbed barley variety bred by the Shanghai Academy of Agricultural Sciences and the Jiaxing Academy of Agricultural Sciences using anther culture technology on the F3 hybrid material of 82164 and Xiumai No. 1.

[0051] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1 Development of SNP Molecular Markers in the Barley Powdery Mildew Resistance Region

[0053] By performing BSA analysis on the F2 hybrid population of the powdery mildew-resistant parent Feng7 and the powdery mildew-susceptible parent Vlamingh, data quality control, variant detection, and association analysis were performed, and the powdery mildew resistance interval was located in the 623165594-623744888 segment of chromosome 4. Local and online sequence alignment analysis was performed using software such as DNAMAN and sequence analysis tools such as the NCBI database, and several SNP sites were obtained. PCR amplification primers were designed for these sites, and their reliability was confirmed by gel electrophoresis and HRM experiments. The available primers were combined with the phenotype of the F2 segregating population to determine the linkage relationship, and sites that can reflect powdery mildew resistance were screened. Among them, a SNP site was obtained in the BPM1 gene, where the powdery mildew resistance allele is C or heterozygous, and the powdery mildew sensitivity allele is T (such as Figure 1 ). Molecular markers developed for this specific nucleic acid sequence can be used to identify the haplotype of this segment.

[0054] Example 2 Design of Detection Primers (PCR-HRM Analysis Primers) for SNP Molecular Markers in the Barley Powdery Mildew Resistance Region

[0055] PCR-HRM analysis primers were designed based on the molecular markers developed in Example 1. The primer design principles are as follows:

[0056] Primers are designed on both sides of the SNP so that the length of the amplified product is between 100-200bp, and the only difference in the target SNP site exists in this sequence of the susceptible parent. A fluorescent nucleic acid dye is added to the PCR amplification system. The dye will emit fluorescence when the DNA is double-stranded and lose fluorescence when the DNA is single-stranded. After PCR amplification, the dye is distributed in the double-stranded DNA of the amplified product. When performing HRM analysis, the PCR product is slowly heated and the fluorescence value is recorded to obtain a curve with a fluorescence value ranging from high to low. The derivative of this curve is the melting curve. The melting curve of the homozygous type presents a single peak, and the horizontal coordinate of the highest point of the curve is the melting temperature (T mSince there is only one SNP difference in the amplified product, when the SNP is C, there are three hydrogen bonds in the base pair, T m The value is higher; when the SNP is T, there are 2 hydrogen bonds in the base pair, T m The value is low; when the site is heterozygous, T m The value is between the two homozygous types, and there is a secondary peak. For the haplotype in the present invention, if the melting curve of the amplified product is a single peak, the melting temperature (T m If the melting curve of the amplified product is a single peak, the melting temperature (T m If the melting curve of the amplified product is a double peak, and the melting temperature of the main peak (T m If the value is between the two, the barley genotype is determined to be heterozygous. The primers are shown in Table 1.

[0057] Table 1 Specific molecular marker primer sequences and related parameters

[0058]

[0059] Example 3 Establishment of a SNP Molecular Marker Detection Method in the Powdery Mildew Resistance Region of Barley

[0060] According to the detection primers of the molecular marker designed in Example 2, the PCR reaction program and reaction system were designed:

[0061] PCR reaction system (10 μL): 1 μL of 10× EasyTaq reaction buffer, 0.8 μL of 2.5 mM dNTPs, primers HvBg01-F and HvBg01-R at a concentration of 10 μM, each used in 0.2 μL, 20×0.125 μL of EvaGreen fluorescent nucleic acid dye, 0.1 μL of EasyTaq DNA polymerase (5 U / μL), 1 μL of DNA template, and ultrapure water to make up the excess. The PCR instrument used was the Dongsheng gene thermal cycler (model: ETC811), and the HRM instrument used was the Thermo Fisher Applied Biosystems instrument. TM 7500Fast Real-Time PCR System.

[0062] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 53°C for 30 seconds, and extension at 72°C for 10 seconds, for a total of 35 cycles; and extension at 72°C for 5 minutes.

[0063] The HRM analysis parameters were as follows: pre-denaturation at 94°C for 30 seconds; annealing at 68°C for 1 minute; heating to 94°C at a rate of 0.5% and recording data, maintaining at 94°C for 30 seconds and then cooling to room temperature.

[0064] Example 4 Application of SNP Molecular Markers in the Barley Powdery Mildew Resistance Region in Detecting Barley Germplasm Parent Resources

[0065] 1. Barley varieties

[0066] The powdery mildew-resistant variety Feng7 (F), the powdery mildew-susceptible variety Vilamg (V), and the high-quality and high-yield barley variety Hua 30 (H).

[0067] 2. Barley genomic DNA extraction and primer synthesis

[0068] The genomic DNA of the above barley materials was extracted by the CTAB method or using a plant genomic DNA extraction kit and the primer sequences shown in Table 1 were synthesized.

[0069] 3. PCR amplification

[0070] The PCR reaction system and reaction procedure are as described in Example 3.

[0071] 4. HRM analysis

[0072] The HRM analysis parameters were as described in Example 3.

[0073] 5. Results Analysis

[0074] HRM results are as follows Figure 2 As shown, the flower 30 curve is classified with Vlamingh, and both are not resistant to powdery mildew. In order to verify the accuracy of the HRM test results, the three parental materials were sequenced and then sequenced and compared. The results showed that the molecular marker detection results were consistent with the sequencing results, as shown in Figure 2. Figure 4 , proving that the primers provided by the present invention can accurately identify barley powdery mildew resistance and can realize accurate and efficient screening of barley variety resources.

[0075] Example 5 Detection of Single Gene Segregation of Barley Powdery Mildew Resistance Gene BPM1 in BCF Population

[0076] 1. Barley material

[0077] The F2 population constructed from the powdery mildew-resistant parent Feng7 and the powdery mildew-susceptible parent Vlamingh, as well as the two parents

[0078] 2. Extraction and PCR detection of barley genomic DNA

[0079] The primers, reaction procedures and systems for barley genomic DNA extraction, PCR amplification and HRM analysis were as described in Example 3.

[0080] 3. Results Analysis

[0081] The HRM typing results of 50 strains of the F2 population constructed from the powdery mildew resistant parent Feng7 and the powdery mildew susceptible parent Vlamingh are shown in the figure below. Figure 3 As shown, the melting temperature (T m If the melting curve of the amplified product is a single peak, the melting temperature (T m If the melting curve of the amplified product is a double peak, and the melting temperature of the main peak (T m If the value is between the two, the barley is considered to be a heterozygous genotype and phenotypically powdery mildew resistant. The results showed that 50 F2 strains were tested, with A being the same genotype as the powdery mildew-susceptible parent, Vlamingh; B being the same genotype as the powdery mildew-resistant Feng7; and H being a heterozygous genotype. The segregation ratio of the three different genotypes was A:B:H = 11:13:26. This marker can distinguish between two different homozygous and heterozygous genotypes, enabling efficient and accurate identification of the BPM1 genotype in the powdery mildew resistance region. It can be used for screening and identification of barley resources and for molecular genetic breeding of barley for powdery mildew resistance.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A molecular marker for identifying susceptibility to barley powdery mildew, characterized in that: The molecular marker detects the susceptibility of barley powdery mildew by detecting SNP markers; The SNP marker is located at position 1688 of the BPM1 gene on barley chromosome 4, where the base is C or T. If the base is C, it is a powdery mildew resistance allele, and if the base is T, it is a powdery mildew susceptible allele. The molecular markers include primers for detecting SNP markers: HvBg01-F and HvBg01-R, the nucleotide sequence of HvBg01-F is as SEQ ID No: 3; The nucleotide sequence of HvBg01-R is shown in SEQ ID No: 4; The powdery mildew resistance sequence of the BPM1 gene is shown in SEQ ID No: 1, and the powdery mildew susceptible sequence is shown in SEQ ID No:

2.

2. A method for identifying barley powdery mildew susceptibility genotypes, characterized in that: The following steps are involved: 1) Extracting genomic DNA from the barley to be tested, using it as a template, and performing PCR amplification using primers HvBg01-F and HvBg01-R to obtain an amplified product; 2) Analyze barley genotype based on the melting curve of the amplified product; When the melting curve of the amplified product is a single peak and the melting temperature is 87.2-87.6°C, the genotype of the barley BPM1 gene at position 1688 is determined to be CC, which is a homozygous genotype for powdery mildew resistance. When the melting curve of the amplified product showed a single peak and the melting temperature was 86.6-86.9°C, the genotype of the barley BPM1 gene at position 1688 was determined to be TT, which was confirmed to be a homozygous genotype susceptible to powdery mildew. When the melting curve of the amplified product shows a double peak and the melting temperature of the main peak is between the two peaks, the genotype of the barley BPM1 gene at position 1688 is determined to be CT, which is determined to be a powdery mildew resistance heterozygous genotype; The powdery mildew resistance sequence of the BPM1 gene is shown in SEQ ID No: 1, and the powdery mildew susceptible sequence is shown in SEQ ID No: 2; The nucleotide sequence of HvBg01-F is shown in SEQ ID No: 3; The nucleotide sequence of HvBg01-R is shown in SEQ ID No:

4.

3. Use of the molecular marker according to claim 1 in identifying barley powdery mildew susceptibility genotypes.

4. Use of the molecular marker according to claim 1 in identifying susceptibility to powdery mildew in barley.

5. Use of the molecular marker according to claim 1 in identifying and screening barley germplasm resources resistant to powdery mildew.

6. Use of the molecular marker according to claim 1 in molecular marker-assisted breeding of barley varieties resistant to powdery mildew.

Images