CN117187428A - SNP molecular marker of rice blast resistance gene Pi2, KASP primer combination, kit and application of SNP molecular marker - Google Patents
SNP molecular marker of rice blast resistance gene Pi2, KASP primer combination, kit and application of SNP molecular marker Download PDFInfo
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- CN117187428A CN117187428A CN202310740875.4A CN202310740875A CN117187428A CN 117187428 A CN117187428 A CN 117187428A CN 202310740875 A CN202310740875 A CN 202310740875A CN 117187428 A CN117187428 A CN 117187428A
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 54
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- 241000209094 Oryza Species 0.000 claims abstract description 53
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Abstract
The invention discloses SNP molecular markers of rice blast resistance gene Pi2, KASP primer combinations, kits and application thereof, belonging to the fields of crop breeding and molecular markers. The SNP marker developed by the invention is developed based on the Pi2 gene core region, and after disease nursery material phenotype identification, the material containing the Pi2 gene is detected to show rice blast resistance, which indicates that the marker can rapidly and accurately detect the broad-spectrum rice blast resistance gene Pi2 in different germplasm resources. The KASP primer Pi2-C3 developed by the invention does not need electrophoresis, avoids the use of toxic chemicals such as nucleic acid dyes, and reduces the potential harm to human bodies and the environment. The result is more visual, different genotypes can be distinguished through the color, and the corresponding material can be directly positioned. High accuracy, and easy realization of high throughput and automation.
Description
Technical Field
The invention belongs to the fields of crop breeding and molecular markers, and particularly relates to SNP molecular markers of rice blast resistance gene Pi2, KASP primer combinations, kits and applications thereof.
Background
Rice is an important food crop in the world, and the population number of which rice is taken as a staple food exceeds half of the world. The rice blast is one of the most serious diseases which are harmful to rice production, the rice blast can cause more than 20% of yield loss every year, the yield of serious disease areas is reduced by 40% -50%, and even the particles are not harvested.
Cultivation of rice blast resistant varieties is the most environmentally friendly and effective method for controlling rice blast. However, since the physiological seeds of rice blast are rapidly replaced and the disease resistance of many disease-resistant varieties is gradually lost, the breeding of rice varieties with broad-spectrum disease-resistant genes of rice blast is an important way for solving the hazard of rice blast. There are 35 rice blast resistance (R) genes that have been identified and cloned so far, among which the Pi2 gene is derived from the Columbia indica variety 5173, which is highly resistant to rice blast, and is a dominant gene against rice blast in a broad spectrum. The gene showed high level resistance to 36 physiological races in 13 countries. Aiming at dominant physiological minispecies of rice blast in indica rice areas of south China, the Pi2 gene shows good resistance, and the Pi2 gene is utilized to breed rice varieties with broad-spectrum resistance, so that the method has important application value.
Selection by means of molecular Markers (MAS) improves the reliability of selection and can effectively improve breeding efficiency. Compared with other molecular markers, the appearance of Single Nucleotide Polymorphism (SNP) markers opens up a new way for rapidly detecting related genes. In recent years, competitive allele polymerase chain reaction technology has become a new method for efficiently and safely detecting SNPs. The detection process of the technology does not need electrophoresis, can effectively reduce the generation of harmful substances in the test process, and has the advantages of simple and convenient operation, low cost, short detection period, good marking stability and the like.
Disclosure of Invention
In order to solve the related problems, the primary aim of the invention is to provide a SNP molecular marker closely linked with a rice blast resistance gene Pi2.
Another object of the present invention is to provide a KASP primer combination and a kit for detecting the SNP molecular marker.
It is a further object of the present invention to provide the use of the above-mentioned KASP primer combination and kit.
In order to achieve the above object, the present invention adopts the following technical scheme:
a SNP molecular marker closely linked with a rice blast resistance gene Pi2, wherein the SNP molecular marker is positioned at a 10389007 base of a chromosome 6 of rice and is A/T polymorphism.
A KASP primer set for detecting the above SNP molecular markers, comprising:
(1) Two specific primers:
Pi2-C3-F1：5’-GAAGGTGACCAAGTTCATGCTCATGGGAGTATGTCCTGCAAAACT-3’；
Pi2-C3-F2：5’-GAAGGTCGGAGTCAACGGATTCATGGGAGTATGTCCTGCAAAACA-3’；
(2) A universal primer:
Pi2-C3-R：5’-CAAACTGCATGTGCTAGACTCTTGGGTC-3’。
a KASP kit for detecting the SNP molecular marker comprises the KASP primer combination.
The application of the KASP primer combination and the kit for detecting the SNP molecular marker is any one or more of the following applications:
I. the application in identifying the rice blast resistance gene Pi2 genotype;
II, application in identifying rice blast resistance;
III, application in cultivating high rice blast resistant rice.
Further, the application comprises the following steps: extracting genome DNA of a rice sample to be detected, carrying out KASP detection by taking the genome DNA as a template and adopting the KASP primer combination, and judging the Pi2 allele type of the rice variety to be detected according to a fluorescent signal result after the reaction is completed: if only the fluorescence signal corresponding to the fluorescence sequence connected with Pi2-C3-F1 is detected, the base of the detection site is T, namely the sample is of a pure type carrying a rice blast resistance gene Pi 2; if only the fluorescence signal corresponding to the fluorescence sequence connected with Pi2-C3-F2 is detected, the base of the detection site is A, namely the sample is of a pure type without the rice blast resistance gene Pi 2; if two fluorescence signals are detected simultaneously, the sample is heterozygous containing rice blast resistance gene Pi2.
Further, the system of the reaction comprises: 50 ng/. Mu.L of genomic DNA, 5. Mu.L of primer Mix, 0.4. Mu.L of 2 XKASP Master Mix, 4.6. Mu.L of mineral oil, 10. Mu.L;
further, the reaction conditions are as follows: pre-denaturation at 95 ℃ for 180s, and circulation for 1 time; denaturation at 95 ℃ for 10s, annealing/extension at 63.4-56.2 ℃ for 30s, -0.8 ℃/cycle, and 9 times; denaturation at 95℃for 10s, annealing/extension at 55℃for 30s, and cycling 29 times.
Further, the KASP primer combination includes:
(1) Two specific primers:
Pi2-C3-F1：5’-GAAGGTGACCAAGTTCATGCTCATGGGAGTATGTCCTGCAAAACT-3’；
Pi2-C3-F2：5’-GAAGGTCGGAGTCAACGGATTCATGGGAGTATGTCCTGCAAAACA-3’；
(2) A universal primer:
Pi2-C3-R：5’-CAAACTGCATGTGCTAGACTCTTGGGTC-3’。
compared with the prior art, the invention has the following advantages:
(1) The SNP marker developed by the invention is developed based on the Pi2 gene core region, and after disease nursery material phenotype identification, the material containing the Pi2 gene is detected to show rice blast resistance, which indicates that the marker can rapidly and accurately detect the broad-spectrum rice blast resistance gene Pi2 in different germplasm resources.
(2) The KASP primer Pi2-C3 developed by the invention does not need electrophoresis, avoids the use of toxic chemicals such as nucleic acid dyes, and reduces the potential harm to human bodies and the environment. The result is more visual, different genotypes can be distinguished through the color, and the corresponding material can be directly positioned. High accuracy, and easy realization of high throughput and automation.
Drawings
FIG. 1 is a molecular marker preparation and genotyping chart of example 1 of the present invention; wherein, A.Pi2-C1 (primer at 10388566 bp); pi2-C2 (10388841 bp primer); pi2-C3 (10389007 bp primer); pi2-C4 (10389256 bp primer); E.Pi2-C5 (10389341 bp primer); red dots are A/A genotypes, blue dots are T/T genotypes, and black dots are blank.
FIG. 2 is a graph showing the result of comparing the identification effect of KASP primers Pi2-C3 with the identification effect of SSR markers in example 2; wherein, A.84 field materials Pi 2KASP genotyping detection results; B. genotyping and SSR Marker alignment analysis, DNA Marker:2000bp.
FIG. 3 is a graph showing the results of detection of Pi2 gene and evaluation of resistance of 84 Yangjiang disease nursery materials.
Detailed Description
Further details will be described below with reference to the drawings in connection with embodiments. The experimental methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Example 1: development of molecular markers and preparation of KASP primers
Based on the haplotype analysis results of the material H-74 (R1179) containing the Pi2 gene and the material H-78 (Hua Hang No.) containing no Pi2 gene, and the background information of the Pi2 gene, the range of 10386510 ～ 10389466bp of chromosome 6 in the core region of the Pi2 gene resistance locus was determined. Referring to the genomic sequence information of Japanese sunny, 5 key SNP loci were finally selected, and KASP primers were designed at 10388566bp, 10388841bp, 10389007bp, 10389256bp and 10389341bp respectively using these 5 SNP loci (see Table 1). Next, KASP genotyping was performed on 3 sample materials known to contain the Pi2 gene (Longjing 12, huazhan, vietnam 2) and 3 sample materials known to not contain the Pi2 gene (Hua Hang, hua Hang and Nippon) using these primers, respectively, and the results showed (FIG. 1) that primers Pi2-C3 at the 10389007bp locus successfully separated the anti-susceptibility test sample materials. As can be seen from FIG. 1, the fluorescent signal of the amplified product when Pi2-C3 was used to detect a Pi2 positive sample is shown as blue dots (near the X-axis), indicating that the material contains the Pi2 gene, i.e., the T/T genotype; whereas fluorescence signal of amplification product of the detected Pi2 recessive sample is shown as red dot (near Y axis), it indicates that the material does not contain Pi2 gene, i.e. a/a genotype. The results show that Pi2-C3 can accurately type rice materials containing Pi2 genes and rice materials without Pi2 genes.
TABLE 1 KASP primer design sequence Listing
Example 2: identification of rice blast resistance gene Pi2 allele of rice variety by KASP primer Pi2-C3
To evaluate the detection effect of the KASP primer Pi2-C3, 6 materials with genotype T/T were selected as positive control, 4 materials with genotype A/A were selected as negative control, ddH based on the whole genome resequencing results 2 O was used as a blank control, and 84 parts of rice germplasm resources were P-treated with the designed KASP primers Pi2-C3 and reported SSR markers M-Pi2 (Pi 2-F:5'-CAGCGATGGTATGAGCACAA-3', pi2-R: 5'-CGTTCCTATACTGCCACATCG-3'), respectivelyi2 detection (Table 2). The genotyping results for Pi2-C3 showed 50 materials with blue (Pi 2 in) fluorescent signal spots, 38 materials with red (Pi 2 in absence) fluorescent signal spots, and 4 materials with green (Pi 2/Pi 2) fluorescent signal spots. The total of two tests are carried out, and the typing results are consistent, which shows that the KASP primer Pi2-C3 has better repeatability and stability. The result has 95% similarity with the result of SSR marker electrophoresis, and the detection results of 10 control materials are consistent with the result of early sequencing. It can be shown that KASP primer Pi2-C3 can successfully distinguish between the three genotypes of homozygous T/T, A/A and heterozygous T/A.
TABLE 2 Pi2 detection results of 84 rice germplasm
Example 3: disease nursery material Pi2 gene detection result and resistance evaluation
The presence of Pi2 gene was examined for 84 parts of the material tested in Yangjiang nursery (table 3) using primers Pi2-C3, while phenotypic investigation was performed to detect that 46 parts of the material containing Pi2 gene all exhibited rice blast resistance to varying degrees, again verifying the feasibility and accuracy of the invention (fig. 3).
TABLE 3 Pi2 Gene detection results and resistance evaluation of Guangdong Yangjiang disease nursery 84 material
The embodiments described above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made in the equivalent manner, and are included in the scope of the present invention.
Claims (8)
1. A SNP molecular marker closely linked to a rice blast resistance gene Pi2, characterized in that: the SNP molecular marker is positioned at the 10389007 base of the No. 6 chromosome of rice and is A/T polymorphism.
2. A KASP primer set for detecting the SNP molecular markers as set forth in claim 1, characterized in that: comprising the following steps:
(1) Two specific primers:
Pi2-C3-F1：5’-GAAGGTGACCAAGTTCATGCTCATGGGAGTATGTCCTGCAAAACT-3’；
Pi2-C3-F2：5’-GAAGGTCGGAGTCAACGGATTCATGGGAGTATGTCCTGCAAAACA-3’；
(2) A universal primer:
Pi2-C3-R：5’-CAAACTGCATGTGCTAGACTCTTGGGTC-3’。
3. a KASP kit for detecting the SNP molecular markers as set forth in claim 1, characterized in that: comprising a KASP primer combination as claimed in claim 2.
4. Use of a KASP primer combination and a kit for detecting the SNP molecular markers as set forth in claim 1, characterized in that: the application is any one or more of the following applications:
I. the application in identifying the rice blast resistance gene Pi2 genotype;
II, application in identifying rice blast resistance;
III, application in cultivating high rice blast resistant rice.
5. The use according to claim 4, characterized in that:
the application comprises the following steps: extracting genome DNA of a rice sample to be detected, carrying out KASP detection by taking the genome DNA as a template and adopting the KASP primer combination, and judging the Pi2 allele type of the rice variety to be detected according to a fluorescent signal result after the reaction is completed: if only the fluorescence signal corresponding to the fluorescence sequence connected with Pi2-C3-F1 is detected, the base of the detection site is T, namely the sample is of a pure type carrying a rice blast resistance gene Pi 2; if only the fluorescence signal corresponding to the fluorescence sequence connected with Pi2-C3-F2 is detected, the base of the detection site is A, namely the sample is of a pure type without the rice blast resistance gene Pi 2; if two fluorescence signals are detected simultaneously, the sample is heterozygous containing rice blast resistance gene Pi2.
6. The use according to claim 5, characterized in that:
the system of the reaction comprises: 50 ng/. Mu.L of genomic DNA, 5. Mu.L of primer Mix, 0.4. Mu.L of 2 XKASP Master Mix, 4.6. Mu.L of mineral oil, 10. Mu.L.
7. Use according to claim 5 or 6, characterized in that:
the reaction conditions are as follows: pre-denaturation at 95 ℃ for 180s, and circulation for 1 time; denaturation at 95 ℃ for 10s, annealing/extension at 63.4-56.2 ℃ for 30s, -0.8 ℃/cycle, and 9 times; denaturation at 95℃for 10s, annealing/extension at 55℃for 30s, and cycling 29 times.
8. The use according to claim 5, characterized in that:
the KASP primer combination comprises:
(1) Two specific primers:
Pi2-C3-F1：5’-GAAGGTGACCAAGTTCATGCTCATGGGAGTATGTCCTGCAAAACT-3’；
pi2-C3-F2:5'-GAAGGTCGGAGTCAACGGATTCATGGGAGTATGTCCTGCAAAACA-3'; (2) one universal primer:
Pi2-C3-R：5’-CAAACTGCATGTGCTAGACTCTTGGGTC-3’。
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