CN112725518A - PARMS marker based on SNP mutation of coding region of rice blast resistance gene Pid2 and application - Google Patents
PARMS marker based on SNP mutation of coding region of rice blast resistance gene Pid2 and application Download PDFInfo
- Publication number
- CN112725518A CN112725518A CN202110225819.8A CN202110225819A CN112725518A CN 112725518 A CN112725518 A CN 112725518A CN 202110225819 A CN202110225819 A CN 202110225819A CN 112725518 A CN112725518 A CN 112725518A
- Authority
- CN
- China
- Prior art keywords
- pid2
- rice blast
- gene
- rice
- parms
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/172—Haplotypes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a PARMS marker and application based on SNP mutation of a rice blast resistance gene Pid2 coding region, wherein a first generation sequencing is utilized to detect the difference of Pid2 in a high rice blast resistant material Y127 and a susceptible variety Nipponbare, the A → G mutation is found at the 554bp site of the gene coding region, the difference of two basic groups is introduced into a designed forward primer, two different fluorescent marker universal primers are added on the basis, and a fluorescent functional molecular marker of the rice blast resistance gene Pid2 based on the PARMS technology is developed, so that the Pid2 gene can be conveniently applied to rice resistance molecular breeding.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of genetic engineering, and relates to a PARMS marker based on SNP mutation of a rice blast resistance gene Pid2 coding region and application thereof.
[ background of the invention ]
The rice blast is a destructive fungal disease of rice caused by rice blast fungi, which seriously affects the yield and quality of rice planting areas in the world, thereby threatening the global food safety, while the rice is one of the main food crops on which human beings live and is the staple food of more than half of the world population. Rice blast is one of the most devastating fungal diseases in world rice production, and the occurrence of rice blast has been reported in over 80 countries to date, with Asia and Africa being the most severe. It is estimated that rice lost to rice blast is sufficient to live 6000 million people each year.
Chemical control is used as a main and traditional pest control means, and long-term use of the chemical control not only pollutes the environment, but also increases the economic cost. The most effective and economic strategy is to cultivate disease-resistant rice varieties. Molecular breeding practices show that the gene structure variation of important agronomic character genes of rice is analyzed, a target gene function marker is developed, direct selection and effective polymerization of the genes can be realized, the breeding efficiency is greatly improved, and the breeding time is shortened. Related researches exist, for example, Chinese patent application CN201711080527 discloses a rice blast resistance rice identification method and a gene marking method and application thereof, wherein SNP sites at bases 5352 and 5353 from the 5 'end of a Pid2 gene sequence and a homologous sequence thereof in a No. 6 chromosome of rice are detected, and then the types of SNPs at bases 5352 and 5353 from the 5' end of the Pid2 gene sequence and the homologous sequence thereof in the No. 6 chromosome of the rice are identified through PCR amplification and restriction endonuclease digestion, so that whether the selected rice plant contains a rice blast resistance gene Pid2 or not is accurately judged, the rice blast resistance gene Pid2 genotype selection is effectively resisted, and the method can be used for identification and screening of rice resources and molecular breeding of the rice blast resistance gene Pid 2. For example, Chinese patent application CN201711396038 also discloses a molecular marker for identifying rice blast resistance, an identification method and application thereof, wherein the molecular marker is Pid2-TPAP, and comprises Pid2-TPAP-O-F with a nucleotide sequence shown as SEQ ID No.1, Pid2-TPAP-O-R with a nucleotide sequence shown as SEQ ID No.2, Pid 2-TPAP-I-F with a nucleotide sequence shown as SEQ ID No.3 and Pid 2-TPAP-I-R with a nucleotide sequence shown as SEQ ID No. 4. The molecular marker can be used for directly identifying two types of alleles of the resistance of the Pyricularia oryzae of the Pid2 gene, is based on a PCR technology, does not need sequencing or enzyme digestion, can accurately identify the two types of alleles of the resistance by PCR only once, and greatly improves the detection efficiency of the gene locus.
At present, commonly used molecular markers RFLP, RAPD, CAPS, AFLP, SSR, ISSR, STS, SRAP and IRAP can be used for linkage analysis and detection of target genes, but the markers are long in detection time, complicated in operation, low in efficiency, low in automation degree, toxic substances and the like, and are not suitable for large-scale genotype analysis. In the past, a large number of molecular markers linked with a rice blast resistance gene, such as RFLP markers, SSLP markers and RAPD markers, are developed, an unknown rice blast resistance gene, STS markers, SNP markers and the like are positioned by using the molecular markers, but the markers are only closely linked markers and can be used only by constructing a corresponding F2 group, so that the utilization efficiency of the markers is influenced.
A single nucleotide polymorphism SNP refers to a variation of a single base caused at the genomic level, which occurs at a frequency of not less than 1% in a population. The SNP can occur at any position of a genome, is used as a new generation of molecular marker, and has the advantages of large quantity, uniform distribution, rich polymorphism, high precision and the like. The PARMS technology, namely a five-primer amplification hindered mutation system, is a newly developed SNP genotyping method based on fluorescence detection, utilizes five primers (a pair of general fluorescent primers, a pair of allele specific primers and a reverse common primer) to carry out allele specific amplification on SNP or short Indel sites, carries out genotyping through fluorescence scanning, and has the advantages of simple and convenient operation, short time consumption and low cost. Based on the fluorescent molecular marker developed by the PARMS technology, a detection sample is amplified only by one-time PCR without electrophoresis detection, and amplification data is directly obtained on an original plate by using a fluorescent scanner, and a corresponding genotype result is quickly obtained through software analysis.
Therefore, in order to improve the efficiency of selection, it is necessary to develop a novel molecular marker.
[ summary of the invention ]
Aiming at the defects that marker detection for preventing and treating rice blast in the prior art is long in time consumption, tedious in operation, low in efficiency, low in automation degree, toxic substances are used, and the marker is not suitable for large-scale genotype analysis, the PARMS marker and the application based on the SNP mutation of the rice blast resistance gene Pid2 coding region are provided, the invention utilizes first-generation sequencing to detect the difference of Pid2 in a high rice blast resistance material Y127 and a susceptible variety Nipponbare, discovers that A → G mutation occurs at the 554bp site of the gene coding region, introduces the difference of the two basic groups into a designed forward primer, adds two different fluorescence marker universal primers on the basis, develops a fluorescence functional molecular marker of the rice blast resistance gene Pid2 based on the PARMS technology, and is convenient for the application of the Pid2 gene on rice blast resistance molecules.
The PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 and the application thereof comprise the following steps:
1) identification of rice blast resistance of rice materials: taking Nipponbare as a contrast, artificially inoculating and identifying the rice blast resistance of a plurality of rice materials in the seedling stage, and screening physiological races ZA9 and ZA13 resistant to rice blast as material Y127;
2) structural analysis of Pid2 gene: extracting DNA of Y127 and Nipponbare fresh leaves, carrying out segmented amplification, purifying, then sending to a biological company for sequencing, analyzing the difference of the Pid2 gene in two rice materials by using a software Vector NTI 11, and finding that A → G mutation occurs at a 555bp site of a coding region of the gene;
3) design and synthesis of functional marker of Pid2 gene: according to the 555-site G → A mutation of the coding region of the Pid2, 1 PARMS molecular marker PARMS-Pid2 is designed, the marker is composed of 3 specific primers of the Pid2 gene, and a difference of two bases is introduced into the designed forward primer:
forward primer Pid 2-Ra: CTGACCAGACAGAAGAGTGTCTGTCGAAGGTGACCAAGTTCATGCT；
Forward primer Pid 2-Rg: CTGACCAGACAGAAGAGTGTCTGTTGAAGGTCGGAGTCAACGGATT；
Reverse primer Pid 2-F: GTGCTTGGGAAAGATGCCTC, respectively;
two universal primers are also included, which are identical to the underlined parts of the two forward primers, respectively, and the tails of the two universal primers are labeled with different fluorescent labels:
#1：GAAGGTGACCAAGTTCATGCT；
#2：GAAGGTCGGAGTCAACGGATT；
4) use of PARMS-Pid 2: the marked 3 primers designed according to the sequence of the Pid2 gene and two general fluorescent primers are added into a PCR reaction system for amplification, the Pid2 allele sequence is matched with a forward primer Pid2-Fg according to SNP difference to obtain an FAM fluorescent signal value through amplification, the FAM fluorescent signal value is matched with a forward primer Pid2-Fa to obtain an HEX fluorescent signal value through amplification, and if a rice sample is in a heterozygous state at the site, the two forward primers are amplified simultaneously;
5) PCR amplification and genotyping of the Pid2 gene: after identifying the rice blast resistance of multiple rice materials polymerizing multiple resistance genes, the rice blast gene Pid2 is typed by PARMS-Pid2, and 3 designed primers Pid2-Fg, Pid2-Fa, Pid2-R and two general primers of #1 and #2 are simultaneously added into a PCR reaction system, wherein the PCR reaction system is 10 mu L: mu.L of 2 XPAMS master mix, 0.15. mu.L of 10 mM Pid2-Fg labeled primer, 0.15. mu.L of 10 mM Pid2-Fa labeled primer, 0.4. mu.L of 10 mM Pid2-R universal reverse primer, 1. mu.L of template DNA, 3.3. mu.L of ddH2O；
The PCR product is rapidly detected in an enzyme labeling instrument comprising three fluorescence detection channels of FAM, HEX and ROX, the fluorescence intensity signal value is read, then a fluorescence signal value file is analyzed through SNP decoder (http:// www.snpway.com/snpdecoder01/) software to obtain the amplified FAM and HEX fluorescence signal intensity of each sample, each signal point is output in a graphic mode, and finally, genotyping is automatically carried out according to the fluorescence signal intensity to obtain a genotype result;
performing analysis according to the fluorescence signal value, wherein FAM fluorescence signals (blue) obtained by fluorescence scanning are rice blast susceptible Pid2 allele (G) type materials, HEX fluorescence signals (green) obtained are rice blast resistant Pid2(A) type materials, and gray is negative control;
6) PARMS-Pid2 effectiveness evaluation: rice material with multiple polymerized resistance genes is analyzed to determine phenotype and genotype.
In the invention:
the multiple parts in the step 1) are 200 parts and 250 parts.
The DNA extraction of the Y127 and the Nipponbare fresh leaves in the step 2) is carried out by adopting a CTAB method to extract the DNA of the Y127 and the Nipponbare fresh leaves.
The multiple parts of rice materials identified and polymerized by the step 5) are 70-80 parts of rice materials identified and polymerized by multiple resistance genes.
The PCR reaction program in the PCR reaction system in the step 5) is as follows: 95 ℃ for 5 min; then 10 cycles of 95 deg.C, 20s, 65 deg.C (-0.8 deg.C/cycle), 1 min; then 32 cycles of 95 deg.C, 20s, 57 deg.C, 1 min.
The phenotype in the step 6) is the feeling or infection of rice blast, resistance or high resistance to rice blast; the genotype identification results are GG type and AA type.
Compared with the prior art, the invention has the following advantages:
1. the PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 and the application of the marker only need one-time PCR amplification, the operation process is simple and easy, the marker is convenient to master, and the DNA fragment does not need to be separated by gel electrophoresis, so that the marker is prevented from contacting toxic chemical substances.
2. The PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 and the application thereof have the advantages of excellent polymorphism, clear gene typing, convenient data reading of software, difficult error and reliable preparation of the detection result of PARMS-Pid 2.
[ description of the drawings ]
FIG. 1 is a sequence alignment chart of Pid2 gene in Y127 and Nipponbare rice material in example 1 of the present invention.
FIG. 2 is a fluorescence image of Pid2 gene in Y127 and Nipponbare rice materials in example 1 of the present invention.
[ detailed description ] embodiments
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1:
the PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 and the application thereof comprise the following steps:
1) identification of rice blast resistance of rice materials: taking Nipponbare as a control, artificially inoculating and identifying the rice blast resistance of 226 rice materials in a seedling stage, and screening physiological races ZA9 and ZA13 resistant to rice blast as material Y127;
2) structural analysis of Pid2 gene: extracting DNA of Y127 and Nipponbare fresh leaves by a CTAB method, carrying out segmented amplification, purifying, then sending to a biological company for sequencing, analyzing the difference of the Pid2 gene in two rice materials by using a software Vector NTI 11, and finding that A → G mutation occurs at a 555bp site of a coding region of the gene (see attached figure 1);
3) design and synthesis of functional marker of Pid2 gene: according to the 555-site G → A mutation of the coding region of the Pid2, 1 PARMS molecular marker PARMS-Pid2 is designed, the marker is composed of 3 specific primers of the Pid2 gene, and a difference of two bases is introduced into the designed forward primer:
forward primer Pid 2-Ra: CTGACCAGACAGAAGAGTGTCTGTCGAAGGTGACCAAGTTCATGCT；
Forward primer Pid 2-Rg: CTGACCAGACAGAAGAGTGTCTGTTGAAGGTCGGAGTCAACGGATT；
Reverse primer Pid 2-F: GTGCTTGGGAAAGATGCCTC, respectively;
two universal primers are also included, which are identical to the underlined parts of the two forward primers, respectively, and the tails of the two universal primers are labeled with different fluorescent labels:
#1：GAAGGTGACCAAGTTCATGCT；
#2：GAAGGTCGGAGTCAACGGATT；
4) use of PARMS-Pid 2: the marked 3 primers designed according to the sequence of the Pid2 gene and two general fluorescent primers are added into a PCR reaction system for amplification, the Pid2 allele sequence is matched with a forward primer Pid2-Fg according to SNP difference to obtain an FAM fluorescent signal value through amplification, the FAM fluorescent signal value is matched with a forward primer Pid2-Fa to obtain an HEX fluorescent signal value through amplification, and if a rice sample is in a heterozygous state at the site, the two forward primers are amplified simultaneously;
5) PCR amplification and genotyping of the Pid2 gene: after identifying the rice blast resistance of 75 parts of rice materials aggregating a plurality of resistance genes, the rice blast gene Pid2 is typed by PARMS-Pid2, and 3 designed primers Pid2-Fg, Pid2-Fa, Pid2-R and two universal primers of #1 and #2 are simultaneously added into a PCR reaction system, wherein the PCR reaction system is 10 mu L: mu.L of 2 XPAMS master mix, 0.15. mu.L of 10 mM Pid2-Fg labeled primer, 0.15. mu.L of 10 mM Pid2-Fa labeled primer, 0.4. mu.L of 10 mM Pid2-R universal reverse primer, 1. mu.L of template DNA, 3.3. mu.L of ddH2O; the PCR reaction program in the PCR reaction system is as follows: 95 ℃ for 5 min; then 10 cycles of 95 deg.C, 20s, 65 deg.C (-0.8 deg.C/cycle), 1 min; then 32 cycles of 95 ℃, 20s, 57 ℃ and 1 min;
the PCR product is rapidly detected in an enzyme labeling instrument comprising three fluorescence detection channels of FAM, HEX and ROX, the fluorescence intensity signal value is read, then a fluorescence signal value file is analyzed through SNP decoder (http:// www.snpway.com/snpdecoder01/) software to obtain the amplified FAM and HEX fluorescence signal intensity of each sample, each signal point is output in a graphic mode, and finally, genotyping is automatically carried out according to the fluorescence signal intensity to obtain a genotype result;
performing analysis according to the fluorescence signal value, wherein FAM fluorescence signals (blue) obtained by fluorescence scanning are rice blast susceptible Pid2 allele (G) type materials, HEX fluorescence signals (green) obtained are rice blast resistant Pid2(A) type materials, and gray is negative control;
6) PARMS-Pid2 effectiveness evaluation: analyzing rice materials with a plurality of polymerized resistance genes, wherein in 75 materials, 23 phenotypes are susceptible or rice blast, and the genotype identification result is GG (see figure 2); 52 parts of materials are resistant or highly resistant to rice blast, genotype AA, the effectiveness of the marker is 100%, and the marker can be used for molecular breeding of rice blast resistance.
Calculating the average resistance grade of the rice blast of each strain, wherein the formula is as follows:
average resistance ═ Σ (number of each resistant strain × corresponding resistance level)/total number of strains.
The average resistance rating was:
1.0-1.9 is High Resistance (HR),
2.0-3.9 is anti (R),
4.0-5.9 is medium-resistant (MR),
(iii) a neutral feeling (MS) of 6.0-7.9,
high Sensitivity (HS) is 8.0-9.0.
Example 2:
the PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 and the application thereof comprise the following steps:
1) identification of rice blast resistance of rice materials: taking Nipponbare as a control, artificially inoculating 200 parts of rice materials in a seedling stage to identify the rice blast resistance, and screening physiological races ZA9 and ZA13 resistant to rice blast as material Y127;
2) structural analysis of Pid2 gene: extracting DNA of Y127 and Nipponbare fresh leaves by adopting a CTAB method, carrying out segmented amplification, purifying and then sending the DNA to a biological company for sequencing, analyzing the difference of the Pid2 gene in two rice materials by utilizing a software Vector NTI 11, and finding that A → G mutation occurs at a 555bp site of a coding region of the gene;
3) design and synthesis of functional marker of Pid2 gene: according to the 555-site G → A mutation of the coding region of the Pid2, 1 PARMS molecular marker PARMS-Pid2 is designed, the marker is composed of 3 specific primers of the Pid2 gene, and a difference of two bases is introduced into the designed forward primer:
forward primer Pid 2-Ra: CTGACCAGACAGAAGAGTGTCTGTCGAAGGTGACCAAGTTCATGCT；
Forward primer Pid 2-Rg: CTGACCAGACAGAAGAGTGTCTGTTGAAGGTCGGAGTCAACGGATT；
Reverse primer Pid 2-F: GTGCTTGGGAAAGATGCCTC, respectively;
two universal primers are also included, which are identical to the underlined parts of the two forward primers, respectively, and the tails of the two universal primers are labeled with different fluorescent labels:
#1：GAAGGTGACCAAGTTCATGCT；
#2：GAAGGTCGGAGTCAACGGATT；
4) use of PARMS-Pid 2: the marked 3 primers designed according to the sequence of the Pid2 gene and two general fluorescent primers are added into a PCR reaction system for amplification, the Pid2 allele sequence is matched with a forward primer Pid2-Fg according to SNP difference to obtain an FAM fluorescent signal value through amplification, the FAM fluorescent signal value is matched with a forward primer Pid2-Fa to obtain an HEX fluorescent signal value through amplification, and if a rice sample is in a heterozygous state at the site, the two forward primers are amplified simultaneously;
5) PCR amplification and genotyping of the Pid2 gene: after identifying the rice blast resistance of 70 parts of rice materials aggregating a plurality of resistance genes, the rice blast gene Pid2 is typed by PARMS-Pid2, and 3 designed primers Pid2-Fg, Pid2-Fa, Pid2-R and two general primers of #1 and #2 are simultaneously added into a PCR reaction system, wherein the PCR reaction system is 10 mu L: mu.L of 2 XPAMS master mix, 0.15. mu.L of 10 mM Pid2-Fg labeled primer, 0.15. mu.L of 10 mM Pid2-Fa labeled primer, 0.4. mu.L of 10 mM Pid2-R universal reverse primer, 1. mu.L of template DNA, 3.3. mu.L of ddH2O; the PCR reaction program in the PCR reaction system is as follows: 95 ℃ for 5 min; then 10 cycles of 95 deg.C, 20s, 65 deg.C (-0.8 deg.C/cycle), 1 min; then 32 cycles of 95 ℃, 20s, 57 ℃ and 1 min;
the PCR product is rapidly detected in an enzyme labeling instrument comprising three fluorescence detection channels of FAM, HEX and ROX, the fluorescence intensity signal value is read, then a fluorescence signal value file is analyzed through SNP decoder (http:// www.snpway.com/snpdecoder01/) software to obtain the amplified FAM and HEX fluorescence signal intensity of each sample, each signal point is output in a graphic mode, and finally, genotyping is automatically carried out according to the fluorescence signal intensity to obtain a genotype result;
performing analysis according to the fluorescence signal value, wherein FAM fluorescence signals (blue) obtained by fluorescence scanning are rice blast susceptible Pid2 allele (G) type materials, HEX fluorescence signals (green) obtained are rice blast resistant Pid2(A) type materials, and gray is negative control;
6) PARMS-Pid2 effectiveness evaluation: analyzing rice materials polymerized with a plurality of resistance genes, wherein in 75 materials, 23 phenotypes are susceptible or rice blast, and the genotype identification result is GG type; 52 parts of materials are resistant or highly resistant to rice blast, genotype AA, the effectiveness of the marker is 100%, and the marker can be used for molecular breeding of rice blast resistance.
Example 3:
the PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 and the application thereof comprise the following steps:
1) identification of rice blast resistance of rice materials: taking Nipponbare as a control, artificially inoculating 250 parts of rice materials in a seedling stage to identify the rice blast resistance, and screening physiological races ZA9 and ZA13 resistant to rice blast as material Y127;
2) structural analysis of Pid2 gene: extracting DNA of Y127 and Nipponbare fresh leaves by adopting a CTAB method, carrying out segmented amplification, purifying and then sending the DNA to a biological company for sequencing, analyzing the difference of the Pid2 gene in two rice materials by utilizing a software Vector NTI 11, and finding that A → G mutation occurs at a 555bp site of a coding region of the gene;
3) design and synthesis of functional marker of Pid2 gene: according to the 555-site G → A mutation of the coding region of the Pid2, 1 PARMS molecular marker PARMS-Pid2 is designed, the marker is composed of 3 specific primers of the Pid2 gene, and a difference of two bases is introduced into the designed forward primer:
forward primer Pid 2-Ra: CTGACCAGACAGAAGAGTGTCTGTCGAAGGTGACCAAGTTCATGCT；
Forward primer Pid 2-Rg: CTGACCAGACAGAAGAGTGTCTGTTGAAGGTCGGAGTCAACGGATT；
Reverse primer Pid 2-F: GTGCTTGGGAAAGATGCCTC, respectively;
two universal primers are also included, which are identical to the underlined parts of the two forward primers, respectively, and the tails of the two universal primers are labeled with different fluorescent labels:
#1：GAAGGTGACCAAGTTCATGCT；
#2：GAAGGTCGGAGTCAACGGATT；
4) use of PARMS-Pid 2: the marked 3 primers designed according to the sequence of the Pid2 gene and two general fluorescent primers are added into a PCR reaction system for amplification, the Pid2 allele sequence is matched with a forward primer Pid2-Fg according to SNP difference to obtain an FAM fluorescent signal value through amplification, the FAM fluorescent signal value is matched with a forward primer Pid2-Fa to obtain an HEX fluorescent signal value through amplification, and if a rice sample is in a heterozygous state at the site, the two forward primers are amplified simultaneously;
5) PCR amplification and genotyping of the Pid2 gene: after identifying the rice blast resistance of 80 parts of rice materials aggregating a plurality of resistance genes, the rice blast gene Pid2 is typed by PARMS-Pid2, and 3 designed primers Pid2-Fg, Pid2-Fa, Pid2-R and two general primers of #1 and #2 are simultaneously added into a PCR reaction system, wherein the PCR reaction system is 10 mu L: mu.L of 2 XPAMS master mix, 0.15. mu.L of 10 mM Pid2-Fg labeled primer, 0.15. mu.L of 10 mM Pid2-Fa labeled primer, 0.4. mu.L of 10 mM Pid2-R universal reverse primer, 1. mu.L of template DNA, 3.3. mu.L of ddH2O; the PCR reaction program in the PCR reaction system is as follows: 95 ℃ for 5 min; then 10 cycles of 95 deg.C, 20s, 65 deg.C (-0.8 deg.C/cycle), 1 min; then 32 cycles of 95 ℃, 20s, 57 ℃ and 1 min;
the PCR product is rapidly detected in an enzyme labeling instrument comprising three fluorescence detection channels of FAM, HEX and ROX, the fluorescence intensity signal value is read, then a fluorescence signal value file is analyzed through SNP decoder (http:// www.snpway.com/snpdecoder01/) software to obtain the amplified FAM and HEX fluorescence signal intensity of each sample, each signal point is output in a graphic mode, and finally, genotyping is automatically carried out according to the fluorescence signal intensity to obtain a genotype result;
performing analysis according to the fluorescence signal value, wherein FAM fluorescence signals (blue) obtained by fluorescence scanning are rice blast susceptible Pid2 allele (G) type materials, HEX fluorescence signals (green) obtained are rice blast resistant Pid2(A) type materials, and gray is negative control;
6) PARMS-Pid2 effectiveness evaluation: analyzing rice materials polymerized with a plurality of resistance genes, wherein in 75 materials, 23 phenotypes are susceptible or rice blast, and the genotype identification result is GG type; 52 parts of materials are resistant or highly resistant to rice blast, genotype AA, the effectiveness of the marker is 100%, and the marker can be used for molecular breeding of rice blast resistance.
To summarize:
1. in the prior art, the conventional DNA marker detection methods mainly comprise polyacrylamide electrophoresis and agarose gel electrophoresis, and the detection methods have the defects of long time consumption, complex operation, low efficiency, low automation degree, toxic substance use and the like; capillary electrophoresis has poor preparation capability due to small sample injection amount; the small diameter of the capillary tube leads to too short light path, and the sensitivity is lower when some detection methods (such as ultraviolet absorption spectroscopy) are used; electroosmosis varies depending on the sample composition, and further affects the reproducibility of separation. Through the examples 1-3, the PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 and the application of the marker are shown, only one-time PCR amplification is needed, the operation process is simple and easy, the mastering is convenient, the DNA fragment is separated without gel electrophoresis, and the toxic chemical substances are avoided being contacted.
2. The PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 and the application thereof have the advantages of excellent polymorphism, clear gene typing, convenient data reading of software, difficult error and reliable preparation of the detection result of PARMS-Pid 2.
The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention.
Sequence listing
<110> Guangxi Zhuang nationality college of autonomous region agro-sciences
<120> PARMS marker based on SNP mutation of coding region of rice blast resistance gene Pid2 and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ctgaccagac agaagagtgt ctgtcgaagg tgaccaagtt catgct 46
<210> 2
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ctgaccagac agaagagtgt ctgttgaagg tcggagtcaa cggatt 46
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gtgcttggga aagatgcctc 20
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gaaggtgacc aagttcatgc t 21
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gaaggtcgga gtcaacggat t 21
Claims (6)
1. PARMS marker and application based on rice blast resistance gene Pid2 coding region SNP mutation are characterized in that: the method comprises the following steps:
1) identification of rice blast resistance of rice materials: taking Nipponbare as a contrast, artificially inoculating and identifying the rice blast resistance of a plurality of rice materials in the seedling stage, and screening physiological races ZA9 and ZA13 resistant to rice blast as material Y127;
2) structural analysis of Pid2 gene: extracting DNA of Y127 and Nipponbare fresh leaves, carrying out segmented amplification, purifying, then sending to a biological company for sequencing, analyzing the difference of the Pid2 gene in two rice materials by using a software Vector NTI 11, and finding that A → G mutation occurs at a 555bp site of a coding region of the gene;
3) design and synthesis of functional marker of Pid2 gene: according to the 555-site G → A mutation of the coding region of the Pid2, 1 PARMS molecular marker PARMS-Pid2 is designed, the marker is composed of 3 specific primers of the Pid2 gene, and a difference of two bases is introduced into the designed forward primer:
forward primer Pid 2-Ra: CTGACCAGACAGAAGAGTGTCTGTCGAAGGTGACCAAGTTCATGCT；
Forward primer Pid 2-Rg: CTGACCAGACAGAAGAGTGTCTGTTGAAGGTCGGAGTCAACGGATT；
Reverse primer Pid 2-F: GTGCTTGGGAAAGATGCCTC, respectively;
two universal primers are also included, which are identical to the underlined parts of the two forward primers, respectively, and the tails of the two universal primers are labeled with different fluorescent labels:
#1：GAAGGTGACCAAGTTCATGCT；
#2：GAAGGTCGGAGTCAACGGATT；
4) use of PARMS-Pid 2: the marked 3 primers designed according to the sequence of the Pid2 gene and two general fluorescent primers are added into a PCR reaction system for amplification, the Pid2 allele sequence is matched with a forward primer Pid2-Fg according to SNP difference to obtain an FAM fluorescent signal value through amplification, the FAM fluorescent signal value is matched with a forward primer Pid2-Fa to obtain an HEX fluorescent signal value through amplification, and if a rice sample is in a heterozygous state at the site, the two forward primers are amplified simultaneously;
5) PCR amplification and genotyping of the Pid2 gene: after identifying the rice blast resistance of multiple rice materials polymerizing multiple resistance genes, the rice blast gene Pid2 is typed by PARMS-Pid2, and 3 designed primers Pid2-Fg, Pid2-Fa, Pid2-R and two general primers of #1 and #2 are simultaneously added into a PCR reaction system, wherein the PCR reaction system is 10 mu L: mu.L of 2 XPAMS master mix, 0.15. mu.L of 10 mM Pid2-Fg labeled primer, 0.15. mu.L of 10 mM Pid2-Fa labeled primer, 0.4. mu.L of 10 mM Pid2-R universal reverse primer, 1. mu.L of template DNA, 3.3. mu.L of ddH2O；
The PCR product is rapidly detected in an enzyme-labeling instrument containing three fluorescence detection channels of FAM, HEX and ROX, the fluorescence intensity signal value is read, and then the fluorescence signal value file passes through an SNP decoderhttp://www.snpway.com/ snpdecoder01/Analyzing by software to obtain the amplified FAM and HEX fluorescence signal intensity of each sample, outputting each signal point in a graphic mode, and finally automatically carrying out genotyping according to the fluorescence signal intensity to obtain a genotype result;
analyzing according to the fluorescence signal value, obtaining an FAM fluorescence signal by fluorescence scanning, obtaining an HEX fluorescence signal by blue rice blast susceptible Pid2 allele G type materials, obtaining green rice blast resistant Pid 2A type materials and taking grey as negative control;
6) PARMS-Pid2 effectiveness evaluation: rice material with multiple polymerized resistance genes is analyzed to determine phenotype and genotype.
2. The PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 as claimed in claim 1 and the application thereof are characterized in that: the multiple parts in the step 1) are 200 parts and 250 parts.
3. The PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 as claimed in claim 1 and the application thereof are characterized in that: the DNA extraction of the Y127 and the Nipponbare fresh leaves in the step 2) is carried out by adopting a CTAB method to extract the DNA of the Y127 and the Nipponbare fresh leaves.
4. The PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 as claimed in claim 1 and the application thereof are characterized in that: the multiple parts of rice materials identified and polymerized by the step 5) are 70-80 parts of rice materials identified and polymerized by multiple resistance genes.
5. The PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 as claimed in claim 1 and the application thereof are characterized in that: the PCR reaction program in the PCR reaction system in the step 5) is as follows: 95 ℃ for 5 min; then 10 cycles of 95 ℃, 20s, 65 ℃, minus 0.8 ℃/cycle for 1 min; then 32 cycles of 95 deg.C, 20s, 57 deg.C, 1 min.
6. The PARMS marker based on the SNP mutation of the coding region of the rice blast resistance gene Pid2 as claimed in claim 1 and the application thereof are characterized in that: the phenotype in the step 6) is the feeling or infection of rice blast, resistance or high resistance to rice blast; the genotype identification results are GG type and AA type.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110225819.8A CN112725518A (en) | 2021-03-01 | 2021-03-01 | PARMS marker based on SNP mutation of coding region of rice blast resistance gene Pid2 and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110225819.8A CN112725518A (en) | 2021-03-01 | 2021-03-01 | PARMS marker based on SNP mutation of coding region of rice blast resistance gene Pid2 and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112725518A true CN112725518A (en) | 2021-04-30 |
Family
ID=75595580
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110225819.8A Pending CN112725518A (en) | 2021-03-01 | 2021-03-01 | PARMS marker based on SNP mutation of coding region of rice blast resistance gene Pid2 and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112725518A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117701755A (en) * | 2023-12-21 | 2024-03-15 | 辽宁省水稻研究所 | SNP molecular marker of rice blast resistance gene Ptr, primer set and application |
| CN117821636A (en) * | 2023-12-20 | 2024-04-05 | 辽宁省水稻研究所 | SNP molecular marker of rice blast resistance gene Pib, primer set and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009077611A2 (en) * | 2007-12-19 | 2009-06-25 | Basf Plant Science Gmbh | Plants with increased yield and/or increased tolerance to environmental stress (iy-bm) |
| CN103409417A (en) * | 2013-08-14 | 2013-11-27 | 浙江大学 | Rice blast resistance gene Pi-d2 functional molecular marker, exclusive primer sequence thereof, and application thereof |
| CN107858446A (en) * | 2017-12-21 | 2018-03-30 | 辽宁省盐碱地利用研究所 | A kind of molecular labeling, authentication method and application for identifying Rice Resistance To Rice Blast |
| CN107988337A (en) * | 2017-11-06 | 2018-05-04 | 云南省农业科学院生物技术与种质资源研究所 | A kind of labeling method and its application of rice blast resistant rice identification method and gene |
| CN112746120A (en) * | 2020-12-30 | 2021-05-04 | 袁隆平农业高科技股份有限公司 | KaSP technology-based rice blast-resistant gene marker system and application thereof |
-
2021
- 2021-03-01 CN CN202110225819.8A patent/CN112725518A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009077611A2 (en) * | 2007-12-19 | 2009-06-25 | Basf Plant Science Gmbh | Plants with increased yield and/or increased tolerance to environmental stress (iy-bm) |
| CN101952305A (en) * | 2007-12-19 | 2011-01-19 | 巴斯夫植物科学有限公司 | Plants with increased yield and/or increased tolerance to environmental stress (IV-BM) |
| CN103409417A (en) * | 2013-08-14 | 2013-11-27 | 浙江大学 | Rice blast resistance gene Pi-d2 functional molecular marker, exclusive primer sequence thereof, and application thereof |
| CN107988337A (en) * | 2017-11-06 | 2018-05-04 | 云南省农业科学院生物技术与种质资源研究所 | A kind of labeling method and its application of rice blast resistant rice identification method and gene |
| CN107858446A (en) * | 2017-12-21 | 2018-03-30 | 辽宁省盐碱地利用研究所 | A kind of molecular labeling, authentication method and application for identifying Rice Resistance To Rice Blast |
| CN112746120A (en) * | 2020-12-30 | 2021-05-04 | 袁隆平农业高科技股份有限公司 | KaSP technology-based rice blast-resistant gene marker system and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| DBSNP: "rs19986658", 《ENSEMBL》 * |
| J.B. LI 等: "Natural variation of rice blast resistance gene", 《GENETICS AND MOLECULAR RESEARCH》 * |
| JU GAO等: "Development of the PARMS marker of the TAC1 gene and its utilization in rice plant architecture breeding", 《EUPHYTICA》 * |
| NCBI: "Oryza sativa Indica Group cultivar Yixiang1B PID2 gene, complete cds", 《GENBANK》 * |
| NCBI: "PREDICTED: Oryza sativa Japonica Group G-type lectin S-receptor-like serine/threonine- protein kinase SD2-5 (LOC4341092), transcript variant X2, mRNA", 《GENBANK》 * |
| 高利军等: "4个抗稻瘟病基因分子标记的建立及在水稻亲本中的分布", 《杂交水稻》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117821636A (en) * | 2023-12-20 | 2024-04-05 | 辽宁省水稻研究所 | SNP molecular marker of rice blast resistance gene Pib, primer set and application |
| CN117701755A (en) * | 2023-12-21 | 2024-03-15 | 辽宁省水稻研究所 | SNP molecular marker of rice blast resistance gene Ptr, primer set and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112481275B (en) | Wheat stripe rust resistant gene yrZ15-1370 and molecular marker and application thereof | |
| CN112080582A (en) | KASP molecular marker closely linked with spike length major QTL locus and application thereof | |
| CN110295251A (en) | Chain SNP marker and its application with wheat available tillering QTL | |
| CN109295179B (en) | Method for screening wheat with different zinc content and iron content and special kit thereof | |
| CN112725518A (en) | PARMS marker based on SNP mutation of coding region of rice blast resistance gene Pid2 and application | |
| CN116875736A (en) | SNP molecular marker of rice blast resistance gene pizz, primer set and application | |
| CN112522433B (en) | SNP marker closely linked with tomato root knot nematode resistant gene Mi1 and application thereof | |
| AU2020103706A4 (en) | Ssr molecular marker primer related to walnut black spot disease and application thereof | |
| CN113584215A (en) | Development and application of KASP marker of wheat powdery mildew resistance gene pmCH7015 | |
| CN113528703A (en) | Development and application of KASP molecular marker of rice blast resistance gene Pid3-A4 | |
| CN116622877B (en) | SNP molecular marker related to lotus rhizome internode shape and application thereof | |
| CN110283929B (en) | SNP (single nucleotide polymorphism) marker 5-160 related to pepper phytophthora blight resistance gene as well as specific primer and application thereof | |
| CN112725519A (en) | PARMS marker based on brown planthopper resistance gene Bph14 of rice and application | |
| CN108517374A (en) | A kind of SNP marker and its application | |
| CN115094158A (en) | KASP marker development of rice blast resistance gene Pid4 and application thereof | |
| CN115141893A (en) | Molecular marker group containing 7 molecular markers and used for predicting dry matter content of kiwi fruit, application and kit thereof | |
| CN111560460B (en) | SNP (Single nucleotide polymorphism) markers related to soybean gray spot disease resistant No. 7 physiological race traits and application thereof | |
| CN114606335A (en) | Development and application of KASP molecular marker of sugarcane mosaic virus disease resistance gene of corn | |
| CN112725517A (en) | Functional PARMS marker based on insertion mutation of second exon of rice blast resistance gene RGA4 and application | |
| CN112813187A (en) | PARMS marker based on SNP mutation of rice blast resistance gene Pita coding region and application | |
| CN112813188A (en) | PARMS marker based on brown planthopper resistance gene Bph26 and application | |
| CN114561488B (en) | Functional molecular marker of gene OsNPF3.1 with rice nitrogen fertilizer utilization efficiency and application | |
| CN113637790B (en) | KASP molecular marker of stripe rust resistance gene YrAS2388R, primer, kit and application | |
| CN114836556B (en) | Molecular marker closely linked with wheat stripe rust resistance QTL QYr.sicau-6B and application | |
| CN116200528B (en) | SNP molecular marker linked with wheat stripe rust resistance gene QYr.sicau. -2BL and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |