CN115948600B - Grape powdery mildew resistance dCAPS molecular marker and application thereof - Google Patents
Grape powdery mildew resistance dCAPS molecular marker and application thereof Download PDFInfo
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Abstract
The invention discloses a Chinese wild grape powdery mildew resistance closely linked SNP locus, a dCAPS molecular marker and application thereof, wherein the SNP locus is 1066968 th locus of Chinese wild grape chromosome 1, and polymorphism is T/C. Aiming at the sites, the invention also provides a corresponding dCAPS molecular marker, a specific primer pair and a method for identifying the powdery mildew resistant strain of the filial generation of the Chinese wild grape by using the dCAPS molecular marker.
Description
Technical Field
The invention relates to the fields of grape disease resistance breeding and molecular biology, in particular to screening and identifying of a Chinese wild grape powdery mildew resistance closely linked SNP locus, and development and application of dCAPS molecular markers thereof.
Background
One of the most common fungal diseases on grape production during powdery mildew seriously jeopardizes grape yield and quality, and can lead to yield reduction of more than 60% in epidemic years, thus causing huge economic loss. At present, chemical bactericides are mainly adopted to prevent and treat grape powdery mildew, so that the production cost is increased, the food safety is influenced, the ecological environment is polluted, the drug resistance of pathogenic bacteria is improved, and the prevention and treatment difficulty is increased (Gadoury et al 2012). Therefore, the new powdery mildew-resistant material is screened, new powdery mildew-resistant genes are excavated, and new powdery mildew-resistant varieties are cultivated, so that the damage of powdery mildew to the grape industry can be fundamentally solved.
The grapes produced at present are mainly European grapes, but most of the grapes are susceptible to powdery mildew. China has a large number of wild grape germplasm resources, most of which possess varying degrees of powdery mildew resistance. Since 1978, china began to collect wild grape germplasm resources on a large scale, and planted the wild grape germplasm resources in a wild grape resource nursery of northwest university of agriculture and forestry science and technology after unified numbering and asexual propagation. To determine the phenotype of the individual germplasm, researchers have performed years of continuous observation statistics and have used different methods to identify powdery mildew resistance of the Chinese wild grape germplasm (Wang et al, 1995; wan et al, 2007; gao et al, 2016). However, so far, the research on powdery mildew disease resistance breeding by utilizing Chinese wild grape germplasm is less.
Molecular markers generally refer to DNA molecular markers, which refer to differences in DNA sequences between individuals or species, that are close to or closely linked to a target gene expressing a trait, and that do not affect the expression of the target gene. DNA molecule markers fall into three categories, depending on the detection method and the history of development: the first generation molecular marking technology takes molecular hybridization technology as a core, such as restriction enzyme fragment length polymorphism (RFLP); the second generation molecular marker technology takes PCR technology as the core, such as random amplified polymorphic DNA markers (RAPD) and simple repeated sequences (SSR); third generation molecular marker techniques have centered on DNA sequences such as Single Nucleotide Polymorphisms (SNPs) (Amiteye et al, 2021). With the application of second-generation sequencing and third-generation sequencing technologies, research on SSR and SNP molecular markers gradually takes the main place in plant research. Molecular marker assisted selective breeding is one of the most important uses of molecular markers, and the genotype-to-phenotype selection based on the molecular markers can overcome the defects of time and labor consumption in traditional breeding.
A convenient method for detecting SNPs is the cleavage amplified polymorphic markers (CAPS) or the derived CAPS markers (dCAPS). Both methods combine PCR amplification with enzyme digestion reaction, utilize restriction enzyme to recognize target SNP site sequence and enzyme digestion, and then identify through electrophoresis typing. CAPS markers can be developed for SNP sequences with naturally occurring cleavage sites, while dCAPS can be developed after artificial introduction of mutant bases for SNPs with non-naturally occurring cleavage sites. The CAPS and dCAPS molecular markers have the characteristics of co-dominance, site specificity, simple operation, rapid detection, low cost, no dependence on precise instruments and equipment and the like, and can be used for rapidly carrying out genotyping, positioning, genetic diversity analysis, variety identification and the like on plants.
There is currently little research and report on dCAPS molecular markers related to grape resistance, particularly powdery mildew resistance, so there is an increasing need to develop dCAPS molecular markers related to grape powdery mildew and use them for the accurate and rapid identification of powdery mildew resistance in the identification of grape and its hybrid offspring.
Disclosure of Invention
The invention aims to provide a dCAPS molecular marker for identifying powdery mildew resistance of grapes;
Another object of the invention is to provide a method for identifying powdery mildew resistance of grapes by using dCAPS molecular markers.
The invention provides a dCAPS molecular marker for identifying powdery mildew resistance of grapes. The primer is used in the genome DNA of the grape to amplify a 168bp band, and after the amplified band is subjected to restriction enzyme Sma I digestion, if the digestion product contains two main bands of 168bp and 145bp, the phenotype of the grape is powdery mildew resistance, and if the digestion product contains only one main band of 145bp, the phenotype of the grape is powdery mildew resistance. This is because powdery mildew resistance is heterozygous for the T/C polymorphism at chromosome 1066968, while powdery mildew resistance is homozygous for the C/C polymorphism at chromosome 1066968.
The technical scheme of the patent application is as follows.
In a first aspect, the present invention provides a Chinese wild grape powdery mildew resistance closely linked SNP locus, wherein the SNP locus is 1066968 th locus on chromosome 1 of Chinese wild grape, and the polymorphism is T/C. In a second aspect, the present invention further provides a specific dCAPS molecular marker closely related to the resistance trait of grape powdery mildew, the nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1, and position 145Y of the sequence represents a polymorphism, which is T or C.
AGTTGTTATGCACCCTCTCACACACTTTGTGGACGAGAGTTGTCTTCCCCAATCCAGCCATCCCCACCACGAA
CATCACGTCAAGTCTCTTCTTTGGCTCCAACGCCCAAGAAACCAGCTTATTTCTTGGCCCCTCAATGCCCCYG GTGTCAGTAGTGTTGAACAAAG(SEQ ID NO:1)
In a third aspect, the present application further provides a primer pair for detecting the specific dCAPS molecular marker, wherein the sequence of the primer pair is shown as SEQ ID NO. 2 and SEQ ID NO. 3.
chr1_1066968-F：5’-AGTTGTTATGCACCCTCTCAC-3’(SEQ ID NO:2)
chr1_1066968-R：5’-CTTTGTTCAACACTACTGACCCC-3’(SEQ ID NO:3)
In a fourth aspect, the present application also provides a kit comprising the specific dCAPS molecular marker primer pair. Further, the kit also contains a restriction enzyme Sma I.
In a fifth aspect, the present invention further provides an application of the specific dCAPS molecular marker in screening powdery mildew resistant strains of grapes.
In a sixth aspect, the present application also relates to a method for rapid screening of a strain of dextran anti-powdery mildew based on dCAPS molecular markers, comprising the steps of:
1) Extracting genome DNA of grape plants;
2) Performing PCR amplification on the extracted genome DNA by using the primer pair for detecting dCAPS molecular markers;
3) The PCR product obtained by amplification is subjected to restriction endonuclease Sma I enzyme digestion and then polyacrylamide gel electrophoresis;
4) And (3) judging results: if the enzyme digestion product is electrophoresed to obtain two main bands of 168bp and 145bp, the genotype of the SNP locus of the grape is T to C, and the grape is powdery mildew resistant grape; if the electrophoresis of the enzyme digestion products is only 145bp of a main band, the genotype of the SNP locus of the grape is C:C, and the grape is powdery mildew-sensitive grape.
Preferably, in the method, the PCR amplification reaction system is: 1. Mu.L of genomic template (concentration: 100 ng/. Mu.L), 1. Mu.L of upstream primer at a concentration of 10. Mu.M/L, 1. Mu.L of downstream primer at a concentration of 10. Mu.M/L, 2X RAPID TAQ MASTER Mix 10. Mu.L, ddH 2 O were made up to 20. Mu.L.
Preferably, in the method, the PCR amplification conditions are: the pre-denaturation at 95 ℃ for 5min, the cycle parameters are denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 5s, 35 cycles are carried out, and the extension at 72 ℃ is fully carried out for 5min.
Preferably, in the method, the spotting volume in polyacrylamide gel electrophoresis is 1.5. Mu.L, and the electrophoresis conditions are 220V,400mA,45min.
The PCR amplification efficiency is high, the electrophoresis result has less impurity bands, and the system is optimized.
The dCAPS molecular marker of the invention can be used for rapidly and accurately detecting the powdery mildew-resistant strain of the grape, so that the detection accuracy is greatly improved and the detection time is shortened. 154 filial generations of Chinese wild grape with susceptibility or resistance to powdery mildew are detected, and the accuracy is close to 100 percent and reaches 99.4 percent (153/154). Therefore, the development of the dCAPS mark can accelerate the disease-resistant breeding process of grape powdery mildew.
Drawings
FIG. 1 is a Manhattan diagram and a Q-Q diagram of a Chinese wild grape hybrid offspring powdery mildew resistance whole genome association analysis.
FIG. 2 shows the result of agarose gel electrophoresis of 154 Chinese wild grape filial generation by PCR amplification using dCAPS primer pair. M is D2000 Marker, BS is disease-resistant parent 'white water-40', SB is disease-resistant parent 'Shaba pearl', other lanes are PCR amplification products of filial generation, R represents powdery mildew resistance, S represents powdery mildew resistance, and the length of the amplification products is 168bp.
FIG. 3 shows the genotypes of the two parents chr1_1066968 and SmaI cleavage results. A. Sequencing results of the two parent chr1_1066968 tandem pMD TM -T vectors were PCR amplified using dCAPS primers, BS-T and BS-C were both genotypes of `white water-40` and SB was the genotype of `Shaba pearl`. B, a polyacrylamide gel electrophoresis result of the PCR product after SmaI digestion, M is a D2000 Marker, BS-P is a PCR product of 'Shaba pearl', SB-P is a PCR product of 'Shaba pearl', BS-E is an electrophoresis result of the PCR product of 'Shaba pearl' after SmaI digestion by restriction endonuclease, and SB-E is an electrophoresis result of the PCR product of 'Shaba pearl'.
FIG. 4 shows the result of polyacrylamide gel electrophoresis of the PCR products of the two parents and the filial generation after restriction endonuclease SmaI cleavage. M is D2000 Marker, BS is parent 'white water-40', SB is parent 'Shaba pearl', other lanes are enzyme digestion results of PCR products of filial generation, R represents powdery mildew resistance, S represents powdery mildew susceptibility.
Detailed Description
China wild grape (Vitis piasezkii) is a Chinese-specific grape wild species, and the grape strain 'white water-40' of the grape strain stored in the wild grape germplasm resource nursery of northwest university of agriculture and forestry science and technology shows higher resistance to powdery mildew, but the specific genetic basis is not clear (Gao et al, 2016). Therefore, the invention uses the constructed hybrid population of Chinese wild compound leaf grape and European grape 'Shaba pearl', adopts whole genome association analysis to screen SNP obviously associated with powdery mildew resistance, and develops related dCAPS molecular markers
The method comprises the steps of utilizing 170 strain filial generations of Chinese wild compound leaf grape strain 'white water' -40 and European grape 'Shaba pearl', carrying out genome-wide association analysis based on powdery mildew resistance phenotype, screening SNP sites (chr1_ 1066968) which are located on Chinese wild compound leaf grape chromosome 1 and are obviously related to grape powdery mildew resistance, designing related dCAPS molecular markers and amplification primers thereof according to the SNP sites, and thus providing a specific dCAPS molecular marker closely linked to the grape powdery mildew resistance, wherein the specific dCAPS molecular marker can be used for rapidly identifying and screening the powdery mildew resistance strain of the grape filial generation, and has high accuracy rate which is close to 100%; the time is short and the results can be obtained in a few hours.
The invention is described in further detail below with reference to the examples and the attached drawings:
Example 1: acquisition of Chinese wild grape powdery mildew resistance closely linked SNP locus
Powdery mildew disease resistance identification is carried out on 218 filial generation of Chinese wild compound leaf grape strain 'white water-40' and European grape 'Shaba pearl' by taking powdery mildew physiological small species En.NAFU1 as pathogenic bacteria and adopting three methods of indoor isolated leaf disc inoculation, field spray inoculation and indoor isolated branch inoculation for three times in succession for three years (Hu Yang, 2021). Genome re-sequencing with depth of 10 x is carried out on the filial generation with good growth condition of 171 strains and the infectious parent 'Shaba pearl', a sequencing platform is Illumina, a joint, poly-N and low quality fragments are removed from original data to obtain CLEAN DATA, obtained CLEAN DATA is compared with China wild compound leaf grape 'white water-40' genome by Sentieon software, all SNP loci are obtained after sequencing, de-duplication and local re-comparison (Kendig et al, 2019), and SNP is filtered again by PLINK software with parameters of-indep 50 5-maf 0.05-geno 0.9-hwe 0.001.001.
And then, carrying out whole genome association analysis on the disease resistance of the 171 strain hybrid offspring powdery mildew and the SNP obtained by filtering by using PLINK software, wherein the detection method adopts chi-square detection. As a result, as shown in FIG. 1, there was a very significant correlation signal in the region of about 2.17Mb from chr1-670851 to chr1-2844373 on chromosome 1. Based on the gene annotation result of the genome of the Chinese wild compound grape, only one gene cluster capable of encoding NLR type immunoreceptor protein exists in the region, which is positioned between chr1_757595 and chr1_1170206 and is about 0.41 Mb. Verifying polymorphism of 15 SNP (Single nucleotide polymorphism) which are obviously related to powdery mildew resistance in the region by utilizing resequencing data, reducing a localization interval to 0.04Mb of chr1_1045489 to chr1_1089719 by screening of recombinant single plants, designing dCAPS primers to amplify the SNP which is obviously related to powdery mildew resistance in the region, and finally screening SNP suitable for developing dCAPS markers according to amplification results: the SNP locus chr1_1066968 is positioned at a physical position 1066968bp, a reference genome base is C, a mutant base is T, a P value is 1.862e-27, and the difference between the disease resistance and the disease-sensitive grape offspring is obvious, so that the SNP locus chr1_1066968 is predicted to be obviously related to powdery mildew resistance based on whole genome association analysis.
Example 2: development of dCAPS molecular markers closely linked to grape powdery mildew resistance
1. DCAPS primer design
Adjacent sequences were extracted from the genome of chinese wild multi-leaf grape according to the SNP locus provided in example 1, and dCAPS primer pairs were designed using DCAPS FINDER 2.0.0 (http:// helix. Edu/dCAPS /):
Forward primer: AGTTGTTATGCACCCTCTCAC (SEQ ID NO: 2);
reverse primer: CTTTGTTCAACACTACTGACCCC (SEQ ID NO: 3).
The PCR amplified product based on the primer pair is 168bp, the sequence of the PCR amplified product is shown as SEQ ID NO. 1, the 145 th bit of the sequence is T/C, and the PCR amplified product is a polymorphic site. The resequencing data analysis shows that the locus has two genotypes of T: C and C: C, wherein the genotype is T: C which represents that the locus is in a heterozygous state and can only be partially digested, and the genotype is C: C which can be completely digested.
2. Extraction and PCR amplification of genomic DNA from a sample
The method comprises the steps of selecting young leaves of two parents to extract genome DNA for subsequent experiments, wherein the DNA extraction method comprises the following steps: omega E.Z.N.A HP PLANT DNA MINI KIT. The extracted parental genomic DNA was diluted to a concentration of 100 ng/. Mu.L as a template, and a PCR reaction system (20. Mu.L) was constructed under the following conditions: 1. Mu.L of template, 1. Mu.L of upstream primer at a concentration of 10. Mu.M/L, 1. Mu.L of downstream primer at a concentration of 10. Mu.M/L, 2X RAPID TAQ MASTER Mix 10. Mu.L, ddH 2 O were made up to 20. Mu.L. The PCR amplification procedure was: the pre-denaturation at 95 ℃ for 5min, the cycle parameters are denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 5s, 35 cycles are carried out, and the extension at 72 ℃ is fully carried out for 5min.
After the amplification, the amplified product was stored at 4 ℃. The amplified products were subjected to 1.5% agarose gel electrophoresis and recorded by photographing under an ultraviolet gel imaging system. The electrophoresis results are shown in FIG. 2, and the amplified product of 168bp was obtained for both parents.
3. Identification of parent amplification products
The extracted genomic DNA of the parent is amplified by using Vazyme high-fidelity enzyme Phanta Max Super-FIDELITY DNA Polymerase, and the dCAPS primer pair is adopted as primers, and an amplification system (50 mu L) is as follows: 2X Phanta Max Buffer. Mu.L, 10mM/L dNTP Mix 1. Mu.L, template 1. Mu.L, 10. Mu.M/L upstream primer 1.5. Mu.L, 10. Mu.M/L downstream primer 1.5. Mu.L, phanta Max Super-FIDELITY DNA Polymerase 1. Mu.L, ddH 2 O make up to 50. Mu.L, and the amplification procedure was consistent with that of Taq enzyme described above.
The PCR amplified products were detected by 1.5% agarose gel electrophoresis, single bands were excised and the amplified bands were recovered by using the OMEGA gel recovery Kit E.Z.N.A.gel Extraction Kit, followed by addition of A tail and ligation to the pMDTM-T vector from TaKaRa, and then the plasmid was transformed into E.coli competent cells. After 16-18h of culture in a constant temperature incubator at 37 ℃, white monoclonal is selected, cultured for 3-6h at 180rpm of a constant temperature shaker at 37 ℃, and clones which are positive are identified by bacterial liquid PCR and are sent to Beijing Aoque Yang Lingce sequence for sequencing. The chr1066968 sequences of the two parents are shown in the figure 3 (A), the disease-resistant parent 'white water-40' has two genotypes T/C, and the disease-resistant parent 'Shaba pearl' has only one genotype C.
4. Enzyme cutting
The PCR product obtained in step 2 of this example was digested with restriction endonuclease Sma I of TaKaRa, and the digestion system (10. Mu.L) was as follows: 2. Mu.L of PCR product, 10X QuickCut Buffer. Mu.L, smaI0.5. Mu.L, ddH 2 O were made up to 10. Mu.L. The enzyme digestion conditions are as follows: the enzyme digestion time is 2 hours at 30 ℃.
8% Polyacrylamide gel electrophoresis was performed on the above digested product, the spotting volume was 1.5. Mu.L, and the electrophoresis conditions were 220V,400mA,45min. The PCR products of the two parents and the electrophoresis result after enzyme digestion are shown in the figure 3 (B), the PCR product (BS-P) of the disease-resistant parent 'white water-40' and the PCR product (SB-P) of the disease-resistant parent 'Shaba pearl' are 168bp, after SmaI enzyme digestion, the polypropylene gel electrophoresis result (BS-E) of the disease-resistant parent has two main bands which are 168bp and 145bp respectively, and the electrophoresis result (SB-E) of the disease-resistant parent only has one main band which is 145bp. The result shows that when the genotype is T-C, namely the disease-resistant genotype, sma I can only cut one haplotype by enzyme, and two main bands of 168bp and 145bp exist simultaneously after enzyme cutting; when the genotype is C-C, namely the genotype of the disease, the amplified product can be cut by all enzymes under the same condition, and only 145bp of main band and no 168bp of band are generated after the enzyme cutting. These results demonstrate that the chr1_1066968 genotypes of the two parents are consistent with genomic and resequenced data, and that the two genotypes can be accurately distinguished by amplification using the dCAPS primers of the present invention in combination with the cleavage results of Sma I.
Example 3: application of dCAPS molecular marker in screening powdery mildew resistance of Chinese wild grape hybrid offspring
And extracting genome DNA from 154 young leaves of the filial generation material, wherein 82 powdery mildew resistant offspring (genotype is T: C) and 72 susceptible offspring (genotype is C: C) are included. Genomic DNA of the hybridized offspring was diluted to a concentration of 100 ng/. Mu.L as a template and PCR amplified using Taq enzyme. The DNA extraction and PCR amplification method was the same as in example 2. The amplified products were detected by 1.5% agarose electrophoresis, and the result of the electrophoresis is shown in FIG. 2. The amplification result of 154 filial generations is the same as that of two parents, and a single band of 168bp is obtained.
The amplification products of the filial generation are subjected to enzyme digestion by utilizing SmaI, the enzyme digestion method is the same as that of the example 2, the enzyme digestion products are detected by polyacrylamide gel electrophoresis, the electrophoresis result is shown in figure 4, the electrophoresis result of 82 powdery mildew resistant offspring is the same as that of the disease resistant parent, and the bands of 168bp and 145bp exist at the same time, the genotype is T to C, and the genotype is consistent with the actual situation; in 72 powdery mildew offspring, the electrophoresis result of 71 materials is the same as that of the parent of the powdery mildew, and only 145bp bands exist, which indicates that the amplified product is completely cut, the genotype is C to C, and the amplified product is also consistent with the actual genotype and powdery mildew resistant phenotype.
Therefore, the dCAPS molecular marker designed for the SNP locus can be effectively used for molecular identification of Chinese wild grape filial generation with different powdery mildew resistance, the accuracy is 99.4 percent (153/154), the development of the dCAPS marker can obviously shorten the breeding period, reduce the breeding working cost, and has important significance in breeding powdery mildew resistant grape varieties by using Chinese wild resources.
The above examples only represent a few embodiments of the present patent application, which are described in more detail, but are not to be construed as limiting the scope of the present patent application. Based on the embodiments in the patent application, other embodiments obtained by a person skilled in the art without making creative efforts fall within the protection scope of the present invention. In the present patent application, the methods not specifically described are conventional techniques already known in the art.
Reference to the literature
Hu Yang (2021) research on genetic rule of powdery mildew resistance of Chinese wild grape and VpsCDPKs disease-resistant molecular mechanism
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Claims (9)
1. A dCAPS molecular marker for detecting the powdery mildew resistance of grape is provided, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and the 145 th Y of the sequence represents polymorphism, which is T or C.
2. A dCAPS molecular marker for detecting powdery mildew resistance traits in grapes, characterized in that a band of 168 bp is amplified in genomic DNA of grapes using a dCAPS molecular marker primer pair, wherein the amplified band of powdery mildew resistant grape variety is digested with restriction enzyme smal, yielding a main band of 168 bp and 145 bp; the amplified band of powdery mildew-sensitive grape variety is subjected to restriction enzyme cutting by a restriction enzyme Sma I to obtain a main band of 145 bp;
Wherein the upstream primer sequence of the dCAPS molecular marker primer pair is shown in SEQ ID NO:2, the sequence of the downstream primer is shown as SEQ ID NO: 3.
3. The specific primer pair for detecting the molecular marker as claimed in claim 1 or 2, wherein the upstream primer sequence of the specific primer pair is shown as SEQ ID N0:2, and the downstream primer sequence is shown as SEQ ID N0:3.
4. A kit comprising the specific primer pair according to claim 3.
5. The kit of claim 4, further comprising a restriction enzyme Sma I.
6. Use of a molecular marker according to claim 1 or 2 for identifying powdery mildew resistant strains of grapes.
7. Use of the molecular marker according to claim 1 or 2 in grape powdery mildew resistance molecular marker assisted breeding.
8. Use of a specific primer pair according to claim 3 or a kit according to claim 4 or 5 for identifying a powdery mildew resistant strain of grape.
9. A method for identifying a powdery mildew resistance trait or a powdery mildew resistance grape strain of a grape, the method comprising the steps of:
(1) Extracting genome DNA of the grape to be detected;
(2) Taking genomic DNA of grape to be detected as a template, and utilizing SEQ ID N0:2-3, performing PCR amplification reaction on the specific primer pair;
(3) Performing enzyme digestion on the PCR amplification product by using restriction enzyme Sma I, and performing polyacrylamide gel electrophoresis on the enzyme digestion product;
(4) And (3) judging results: if the enzyme cutting product contains 168 bp and 145 bp main bands, the grape is powdery mildew resistant grape; if the enzyme-digested product only contains 145 bp of a main band, the grape is powdery mildew-sensitive grape.
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