CN107988337B - Identification method of rice blast resistance rice, gene marking method and application thereof - Google Patents
Identification method of rice blast resistance rice, gene marking method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of crop molecular genetic breeding, and particularly relates to a rice blast resistance rice identification method, a gene marking method and application thereof, wherein the method comprises the following steps: detecting SNP sites at 5352 and 5353 bases from the 5 'end of the Pid2 gene sequence FJ915121.1 sequence and the homologous sequence thereof in the No. 6 chromosome of the rice to be detected, and identifying the SNP types at 5352 and 5353 bases from the 5' end of the Pid2 gene sequence FJ915121.1 sequence and the homologous sequence thereof in the No. 6 chromosome of the rice by PCR amplification and restriction enzyme digestion. The invention can accurately judge whether the selected rice plant contains the rice blast resistance gene Pid2, can effectively select the rice blast resistance gene Pid2 genotype, can be used for identifying and screening rice resources, and can also be used for molecular breeding of the rice blast resistance gene Pid 2.
Description
Technical Field
The invention belongs to the field of crop molecular genetic breeding, and relates to a rice blast resistance rice identification method, a gene marking method and application thereof.
Background
Rice is an important grain crop in the world, and rice blast is one of the most serious diseases in each rice area in the world, and the yield of the rice is seriously influenced. Due to the high variability and instability of rice blast germs, newly bred resistant varieties with single resistance genes become susceptible varieties after being popularized and planted for several years, which becomes an important obstacle for sustainable development of rice production, and the excavation of rice resistance gene resources is the basis of rice blast resistance breeding. Through extensive genetic analysis, 84 rice blast resistance genes have been identified and located, and at least 24 major genes have been isolated and cloned one after another. With the wide application of the rice DNA sequencing technology, the homologous sequence comparison of different resistant (phenotypic) variety sources becomes a method for determining new functional genes (Yudai Okuyama et al, 2011), and the development of the rice genome re-sequencing work can greatly promote the gene isolation and cloning process including rice blast resistant genes (Xu et al, 2011).
Pid2 is located in the near-centromere region of the 6 th chromosome of rice, is cloned in the 'ground grain' of indica rice variety in China as a main effective rice blast resistance gene, has higher leaf blast resistance to the physiological race of rice blast in China ZB15, and is an important resistance source for breeding rice blast in south China. Pi-d2 is different from the conventional "R" gene in terms of the structure of the cloned rice blast resistance gene. The main rice blast resistant gene Pi-d2 is derived from indica rice variety 'ground grain', and has high leaf blast resistance to physiological race of rice blast in China ZB15 (Chen, 2010). Pi-d2 is located in the juxtaglomerular region of rice chromosome 6 and is a single copy gene encoding a 825 amino acid transmembrane receptor protein kinase (RLK) containing a hydrophobic signal peptide at the amino terminus, a B-lectin domain, a PAN domain and a TM domain, and a serine/threonine kinase domain (STK) at the carboxy terminus. Pi-d2 belongs to a novel disease-resistant gene type because RLK has not been found to contain an extramembranous B-lectin structure in rice before.
The enzyme-cutting amplification Polymorphic Sequences (C1 improved Amplified Polymorphic Sequences, CAPS) are a co-dominant molecular marker based on PCR, and the basic principle is that a set of specific PCR primers (19-27 bp) are designed by using a DNA sequence of a known site, then the primers are used for amplifying a certain DNA fragment on the site, and then an Amplified band obtained by cutting with a specific restriction enzyme is subjected to RFLP analysis. The method discloses the information of restriction enzyme site variation in the DNA sequence of a specific PCR product, can detect the mode of DNA polymorphism, and has the advantages of rapidness, simplicity, high efficiency and the like.
The traditional conventional field phenotype identification has low selection efficiency and poor accuracy of rice blast resistance genes; an efficient breeding method of rice blast resistant rice is urgently needed.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for identifying rice blast resistance rice, a method for labeling a gene, and applications thereof. The method can effectively resist the selection of the rice blast gene Pid2 genotype, can be used for the identification and screening of rice resources, and can also be used for the molecular breeding of the rice blast resistance gene Pid 2.
The specific technical scheme is as follows:
a rice blast resistant rice identification method is realized by the following steps:
(1) carrying out PCR amplification and PCR product sequencing on alleles of a rice seed resource Pid2 coding region;
(2) two Pid2 specific SNP sites are obtained at 5352 th and 5353 th bases from the 5' end of a Pid2 gene sequence FJ915121.1 sequence and a homologous sequence thereof in a rice chromosome 6;
(3) there are three cases of the two Pid 2-specific SNP site bases: 1) the base type is "AT", 2) the base type is "GC", 3) the base type is "GT";
(4) if the bases of the 5352 and 5353 sites of the rice material to be detected are AT, the material is rice containing a rice blast resistance gene Pid2 or candidate rice containing a rice blast resistance gene Pid 2; if the base types at the 5352 and 5353 sites of the rice to be detected are GC or GT, the rice material is rice which does not contain the rice blast resistance gene Pid2 or rice which does not contain the rice blast resistance gene Pid2 as a candidate.
Preferably, the marking method of the rice blast resistance rice gene, namely the method for detecting the 5352 th and the 5353 th bases from the 5' end of the FJ915121.1 sequence of the rice Pid2 gene sequence and the homologous sequence thereof is as follows:
(1) carrying out PCR amplification by taking the genomic DNA of rice to be detected as a template and taking a molecular marker primer of a rice blast resistance gene Pid2 as a primer to obtain a PCR amplification product with the size of 700 bp;
(2) the PCR amplification product was digested with restriction enzymes Fau1 and Mlu1, respectively, as a result of which there were several cases:
1) after the PCR amplification product is cut by two restriction endonucleases, electrophoresis only shows 1 DNA strip, so that bases 5352 and 5353 from the 5 ' end of the gene sequence FJ915121.1 sequence of the rice to be detected and the homologous sequence thereof are homozygous ' AT ';
2) the PCR amplification product can be cut by restriction enzyme Fau1 and then electrophoresed to display two DNA bands, and after cut by restriction enzyme Mlu1 and then electrophoresed to display 1 DNA band, so that bases 5352 and 5353 from the 5' end of the rice gene sequence FJ915121.1 sequence and the homologous sequence thereof to be detected are homozygous GC;
3) the PCR amplification product can be cut by restriction enzyme Mlu1 and then electrophoresed to display two DNA bands, and after cut by restriction enzyme Fau1 and then electrophoresed to display 1 DNA band, the bases 5352 and 5353 from the 5 ' end of the rice gene sequence FJ915121.1 sequence and the homologous sequence thereof to be detected are homozygous ' GT ';
4) the PCR amplification product can be cut by restriction enzyme Fau1 and then electrophoresed to display three DNA bands, and after cut by restriction enzyme Mlu1 and then electrophoresed to display 1 DNA band, bases 5352 and 5353 from the 5 ' end of the homologous sequence of the rice gene sequence FJ915121.1 to be detected are heterozygous bases ' AT/GC ';
5) if the PCR amplification product can be cut by restriction enzyme Mlu1 and then electrophoresed to display 3 DNA bands, and after cut by restriction enzyme Fau1 and then electrophoresed to display 1 DNA band, the 5352 th and 5353 th bases from the 5 ' end of the homologous sequence of the rice gene sequence FJ915121.1 sequence to be detected are heterozygous bases ' AT/GT ';
6) after the PCR amplification product is cut by two restriction endonucleases, electrophoresis shows 3 DNA bands, and bases 5352 and 5353 from the 5 ' end of the homologous sequence of the rice gene sequence FJ915121.1 to be detected are heterozygous ' GC/GT ';
preferably, the primer is a primer capable of amplifying a molecular marker of the rice blast resistance gene Pid2, and the sequence of the primer is as follows:
DF：5’- AAGCTTGGTCAGGGAGGGTT-3’；
Pid2 DR：5’-TGTCATTGTACTTCA GCTTGGC-3’。
preferably, the nucleotide sequence of the molecular marker of the rice blast disease-resistant gene Pid2 is positioned in the sequence shown by nucleotides 4870 and 5570 from the 5' end of FJ915121.1 and homologous sequences thereof.
Preferably, the primers and the method are applied to identifying and screening rice blast resistant rice.
Preferably, the primers and the method are applied to rice blast resistance gene molecular breeding.
Compared with the prior art, the invention has the following advantages:
(1) different from the traditional method for identifying rice blast resistance alleles by utilizing physiological races of rice blast which are inoculated with different varieties, the molecular marker Pid2CAPS provided by the invention is positioned in the coding region of the Pid2 gene, is co-separated from Pid2 disease resistance genetically, has the selection efficiency of 100 percent, can judge whether a target material contains the Pid2 gene or not, can detect whether the Pid2 genotype of the target material is heterozygous or homozygous, and is more efficient compared with the existing method for detecting the Pid2 linkage marker by Uneven PCR.
(2) The molecular marker provided by the invention can be distinguished by restriction enzyme Fau1 and Mlu1 enzyme digestion, has three band types of 700bp, 425bp and 225bp in total, can be directly detected by using 1.5% agarose gel electrophoresis, and can be widely applied to populations with different genetic backgrounds. The existing method for detecting Pid2 by Uneven PCR is developed only aiming at the sequence polymorphism of two different parents in the same population, and the applicability of the markers in other populations is limited.
(3) The method is used for quickly identifying the allele type of the blast-resistant gene Pid2 in rice germplasm resources. The method of the invention does not need to repeatedly screen parent polymorphism, can accelerate the breeding progress of rice blast resistant rice varieties, and is suitable for transgenic breeding, gene polymerization and MAS technology-based resistance breeding of rice restorer lines and sterile line bone stock parent materials which are applied in large areas in China.
Drawings
FIG. 1 shows the sequence-specific SNP locus analysis of Pid2 allele of published Pid2 gene downloaded from public database and sequenced rice variety 93-11, Nipponbare and the rest 41 varieties, wherein the dark color blocks are the same parts of 3 types of gene fragments in base; the light color is that the two types of bases are the same;
FIG. 2 is a diagram showing the restriction enzyme digestion of the PCR product with restriction enzymes Mlu1 and Fau1 in sequence, and the agarose gel electrophoresis detection of the digestion product;
FIG. 3 shows the PCR amplification of genomic DNA of transgenic rice and Nipponbare, the resulting PCR products were digested with restriction enzyme Fau1, and the respective digested products were electrophoretically detected on agarose gel.
Detailed Description
A rice blast resistant rice identification method is realized by the following steps:
(1) carrying out PCR amplification and PCR product sequencing on alleles of a rice seed resource Pid2 coding region;
(2) two Pid2 specific SNP sites are obtained at 5352 th and 5353 th bases from the 5' end of a Pid2 gene sequence FJ915121.1 sequence and a homologous sequence thereof in a rice chromosome 6;
(3) there are three cases of the two Pid 2-specific SNP site bases: 1) the base type is "AT", 2) the base type is "GC", 3) the base type is "GT";
(4) if the bases of the 5352 and 5353 sites of the rice material to be detected are AT, the material is rice containing a rice blast resistance gene Pid2 or candidate rice containing a rice blast resistance gene Pid 2; if the base types at the 5352 and 5353 sites of the rice to be detected are GC or GT, the rice material is rice which does not contain the rice blast resistance gene Pid2 or rice which does not contain the rice blast resistance gene Pid2 as a candidate.
Preferably, the marking method of the rice blast resistance rice gene, namely the method for detecting the 5352 th and the 5353 th bases from the 5' end of the FJ915121.1 sequence of the rice Pid2 gene sequence and the homologous sequence thereof is as follows:
(1) carrying out PCR amplification by taking the genomic DNA of rice to be detected as a template and taking a molecular marker primer of a rice blast resistance gene Pid2 as a primer to obtain a PCR amplification product with the size of 700 bp;
(2) the PCR amplification product was digested with restriction enzymes Fau1 and Mlu1, respectively, as a result of which there were several cases:
1) after the PCR amplification product is cut by two restriction endonucleases, electrophoresis only shows 1 DNA strip, so that bases 5352 and 5353 from the 5 ' end of the gene sequence FJ915121.1 sequence of the rice to be detected and the homologous sequence thereof are homozygous ' AT ';
2) the PCR amplification product can be cut by restriction enzyme Fau1 and then electrophoresed to display two DNA bands, and after cut by restriction enzyme Mlu1 and then electrophoresed to display 1 DNA band, so that bases 5352 and 5353 from the 5' end of the rice gene sequence FJ915121.1 sequence and the homologous sequence thereof to be detected are homozygous GC;
3) the PCR amplification product can be cut by restriction enzyme Mlu1 and then electrophoresed to display two DNA bands, and after cut by restriction enzyme Fau1 and then electrophoresed to display 1 DNA band, the bases 5352 and 5353 from the 5 ' end of the rice gene sequence FJ915121.1 sequence and the homologous sequence thereof to be detected are homozygous ' GT ';
4) the PCR amplification product can be cut by restriction enzyme Fau1 and then electrophoresed to display three DNA bands, and after cut by restriction enzyme Mlu1 and then electrophoresed to display 1 DNA band, bases 5352 and 5353 from the 5 ' end of the homologous sequence of the rice gene sequence FJ915121.1 to be detected are heterozygous bases ' AT/GC ';
5) if the PCR amplification product can be cut by restriction enzyme Mlu1 and then electrophoresed to display 3 DNA bands, and after cut by restriction enzyme Fau1 and then electrophoresed to display 1 DNA band, the 5352 th and 5353 th bases from the 5 ' end of the homologous sequence of the rice gene sequence FJ915121.1 sequence to be detected are heterozygous bases ' AT/GT ';
6) after the PCR amplification product is cut by two restriction endonucleases, electrophoresis shows 3 DNA bands, and bases 5352 and 5353 from the 5 ' end of the homologous sequence of the rice gene sequence FJ915121.1 to be detected are heterozygous ' GC/GT ';
preferably, the primer is a primer capable of amplifying a molecular marker of the rice blast resistance gene Pid2, and the sequence of the primer is as follows:
DF：5’- AAGCTTGGTCAGGGAGGGTT-3’；
Pid2 DR：5’-TGTCATTGTACTTCA GCTTGGC-3’。
preferably, the nucleotide sequence of the molecular marker of the rice blast disease-resistant gene Pid2 is positioned in the sequence shown by nucleotides 4870 and 5570 from the 5' end of FJ915121.1 and homologous sequences thereof.
Preferably, the primers and the method are applied to identifying and screening rice blast resistant rice.
Preferably, the primers and the method are applied to rice blast resistance gene molecular breeding.
Example 1 primer design and detection of CAPS-labeled Pid2CAPS specific to the Rice blast resistance Gene Pid2
First, Pid2 gene sequence specificity SNP locus analysis
The published Pid2 gene and the Pid2 allele sequences of 93-11 sequenced rice varieties, Nipponbare and the rest 41 varieties are downloaded from a public database, and the sequences are compared to screen the SNP which is specific to the Pid2 and can be distinguished from the allele of the locus. As shown in FIG. 1, the bases located AT sites +2057 and +2058 after the initiation codon of the Pid2 gene are "AT", and the genotypes of all the other varieties have two types, one type is base "GC", and the other type is base "GT". And the SNP changes of the two sites just result in that the Pid2 allele of the type I can be recognized by the restriction enzyme Mlu1 at the position and cut at the position of +2051, the Pid2 allele of the type II can be recognized by the restriction enzyme Fau1 at the position and cut at the position of +2051, and the Pid2 gene cannot be recognized and cut by the two restriction enzymes. The sequence space of the two SNP sites and the remaining Mlu1 and Fau1 sites in the Pid2 and the allelic gene thereof is more than 700bp, thereby meeting the condition of CAPS marker development.
Second, primer design and synthesis
Designing paired primers at 480bp upstream and 219bp downstream of +2057bp site of Pid2 gene
Pid2 DF：5’- AAGCTTGGTCAGGGAGGGTT-3’；
Pid2 DR：5’-TGTCATTGTACTTCA GCTTGGC-3’。
Selecting rice material
The Pid2 gene sequence of the related rice seed resource is downloaded from the local rice seed in Yunnan and the public database.
(II) PCR amplification and enzyme digestion detection
Extracting the genome DNA of each rice material in the third step, and performing PCR amplification by taking the genome DNA as a template and the primer in the second step as an amplification primer to obtain a fragment with the size of 700bp, wherein the fragment is the Pid2caps mark. The Pid2caps labeled sequences obtained by amplification of the invention have three types, wherein bases of the first type AT 481 bp and 482bp are 'AT', bases of the second type AT 481 bp and 482bp are 'GC', and bases of the third type AT 481 bp and 482bp are 'GT'.
The PCR products were digested with restriction enzymes Mlu1 and Fau1, respectively, and the digested products were subjected to agarose gel electrophoresis, the results are shown in FIG. 1.
The PCR amplification system was 25. mu.L: ddH2O15.75 μ L, DNA1 μ L, 1 μ L each of 10mmol/L primers, 10 XPCRBuffer 2.5 μ L, 2 μ L of dNTP (2.5 mmol/L), Mgcl2(25 mmol/L) 1.5. mu.L, DNA polymerase (5U/. mu.l) 0.25. mu.L. The PCR amplification program is pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 57 ℃ (SFL) or 53 ℃ (Bar) for 45s, extension at 72 ℃ for 1min for 30 cycles; extension at 72 ℃ for 10 min.
The PCR products were cut by restriction enzymes Mlu1 and Fau1 in turn, and the cut products were subjected to agarose gel electrophoresis, the results are shown in FIG. 2.
In FIG. 2, materials No. 1, 2, 4, 7, and 9, which were sequenced to have bases "GC" at 481 and 482bp, were cleaved by Fau1 but not Mlu 1; 3. materials No. 5, 6, and 8, which were sequenced to have bases "GT" at 481 and 482bp, were cleaved by Mlu1 but not Fau 1; material 10 was not cleaved by Fau1 and Mlu1 and was sequenced to give "AT" bases AT 481 and 482 bp.
Example 2 verification and application of CAPS marker specific to blast-resistant Gene Pid2 in Rice transformed with Pid2 Gene coding region
Firstly, taking the rice variety Nipponbare without the rice blast resistance gene Pid2 as a transgenic receptor
The CDS region of the blast gene Pid2 is highly expressed in Nipponbare, and the rice with transferred Pid2 gene coding region is prepared.
And (II) extracting the genomic DNA of the transgenic rice and Nipponbare obtained in the step one, performing PCR amplification by using the primers of the embodiment 1 by using the genomic DNA of the transgenic rice and Nipponbare respectively used as templates, performing enzyme digestion on the obtained PCR products by using a restriction enzyme Fau1, and performing electrophoresis detection on the enzyme digestion products on agarose gel.
The result is shown in FIG. 3, lane 1 is a Nipponbare non-transgenic plant, which can be digested by Fau1 enzyme, and shows two bands of 255bp and 475 bp; lane 2 is the Pid2 transgenic plant, which has 3 bands after Fau1 enzyme digestion.
Sequence listing
<110> institute of biotechnology and germplasm resources of Yunnan academy of agricultural sciences
<120> identification method of rice blast resistance rice, gene marking method and application thereof
<160>2
<210>1
<211>20
<212>DNA
<213> Rice (Oryza sativa)
<220>
<223>
<400>1
AAGCTTGGTC AGGGAGGGTT 20
<210>2
<211>22
<212>DNA
<213> Rice (Oryza sativa)
<220>
<223>
<400>2
TGTCATTGTA CTTCAGCTTG GC 22
Claims (5)
1. The identification method of rice blast resistance rice is characterized by comprising the following steps of:
(1) carrying out PCR amplification and PCR product sequencing on alleles of a rice seed resource Pid2 coding region;
(2) two Pid2 specific SNP sites are obtained at 5352 th and 5353 th bases from the 5' end of a Pid2 gene sequence FJ915121.1 sequence and a homologous sequence thereof in a rice chromosome 6;
(3) there are three cases of the two Pid 2-specific SNP site bases: 1) the base type is "AT", 2) the base type is "GC", 3) the base type is "GT";
(4) if the bases of the 5352 and 5353 sites of the rice material to be detected are AT, the material is rice containing a rice blast resistance gene Pid2 or candidate rice containing a rice blast resistance gene Pid 2; if the base types at the 5352 and 5353 sites of the rice to be detected are GC or GT, the rice material is rice which does not contain the rice blast resistance gene Pid2 or rice which does not contain the rice blast resistance gene Pid2 as a candidate.
2. The method for identifying rice blast resistance rice as claimed in claim 1, wherein the method for labeling rice blast resistance rice gene comprises the following steps of detecting bases 5352 and 5353 from 5' end of FJ915121.1 sequence of rice Pid2 gene sequence and homologous sequence thereof:
(1) carrying out PCR amplification by taking the genomic DNA of rice to be detected as a template and taking a molecular marker primer of a rice blast resistance gene Pid2 as a primer to obtain a PCR amplification product with the size of 700 bp;
(2) the PCR amplification product was digested with restriction enzymes Fau1 and Mlu1, respectively, as a result of which there were several cases:
1) after the PCR amplification product is cut by two restriction endonucleases, electrophoresis only shows 1 DNA strip, so that bases 5352 and 5353 from the 5 ' end of the gene sequence FJ915121.1 sequence of the rice to be detected and the homologous sequence thereof are homozygous ' AT ';
2) the PCR amplification product can be cut by restriction enzyme Fau1 and then electrophoresed to display two DNA bands, and after cut by restriction enzyme Mlu1 and then electrophoresed to display 1 DNA band, so that bases 5352 and 5353 from the 5' end of the rice gene sequence FJ915121.1 sequence and the homologous sequence thereof to be detected are homozygous GC;
3) the PCR amplification product can be cut by restriction enzyme Mlu1 and then electrophoresed to display two DNA bands, and after cut by restriction enzyme Fau1 and then electrophoresed to display 1 DNA band, the bases 5352 and 5353 from the 5 ' end of the rice gene sequence FJ915121.1 sequence and the homologous sequence thereof to be detected are homozygous ' GT ';
4) the PCR amplification product can be cut by restriction enzyme Fau1 and then electrophoresed to display three DNA bands, and after cut by restriction enzyme Mlu1 and then electrophoresed to display 1 DNA band, bases 5352 and 5353 from the 5 ' end of the homologous sequence of the rice gene sequence FJ915121.1 to be detected are heterozygous bases ' AT/GC ';
5) if the PCR amplification product can be cut by restriction enzyme Mlu1 and then electrophoresed to display 3 DNA bands, and after cut by restriction enzyme Fau1 and then electrophoresed to display 1 DNA band, the 5352 th and 5353 th bases from the 5 ' end of the homologous sequence of the rice gene sequence FJ915121.1 sequence to be detected are heterozygous bases ' AT/GT ';
6) the electrophoresis of the PCR amplification product after being cut by two restriction endonucleases shows 3 DNA bands, and the bases 5352 and 5353 from the 5' end of the homologous sequence of the rice gene sequence FJ915121.1 to be detected are heterozygous GC/GT.
3. The method for identifying rice blast resistance rice as claimed in claim 2, wherein the primer is a primer capable of amplifying a molecular marker of the rice blast resistance gene Pid2, and the sequence of the primer is as follows:
DF：5’- AAGCTTGGTCAGGGAGGGTT-3’；
Pid2 DR：5’-TGTCATTGTACTTCA GCTTGGC-3’。
4. the method for identifying rice blast resistance rice as claimed in claim 2, wherein the nucleotide sequence of the molecular marker of the rice blast resistance gene Pid2 is located in the sequence shown by nucleotides 4870 and 5570 from the 5' end of FJ915121.1 and its homologous sequence.
5. The primer capable of amplifying the molecular marker of the rice blast resistance gene Pid2 as claimed in claim 3, wherein the primer and the method are applied to identify and screen rice blast resistance rice.
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