CN111662997B - Primer group for identifying rice blast germs as well as screening method and application thereof - Google Patents
Primer group for identifying rice blast germs as well as screening method and application thereof Download PDFInfo
- Publication number
- CN111662997B CN111662997B CN202010405191.5A CN202010405191A CN111662997B CN 111662997 B CN111662997 B CN 111662997B CN 202010405191 A CN202010405191 A CN 202010405191A CN 111662997 B CN111662997 B CN 111662997B
- Authority
- CN
- China
- Prior art keywords
- artificial sequence
- dna
- pyricularia oryzae
- primer
- sequence
- 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.)
- Active
Links
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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B20/30—Detection of binding sites or motifs
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/20—Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medical Informatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Evolutionary Biology (AREA)
- Biochemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Theoretical Computer Science (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a primer group for identifying rice blast germs, a screening method and application thereof. The primer group comprises a forward primer and a reverse primer, and the sequences of a plurality of primers in the primer group are shown as SEQ ID NO 1-SEQ ID NO 200 in a sequence table. The screening method of the primer group comprises the following steps: 1) taking the genome sequence of the rice blast germs as a query sequence, comparing the query sequence with a genome sequence database, and aligning all genome sequences; 2) translating along the aligned genomic sequence; every translation is carried out once, pairwise comparison is carried out on the genome segments of the rice blast germs in the window, and the gene segments with the discrimination are left to serve as candidate marker sites; 3) carrying out primer design on a conserved region of each candidate marker locus among a plurality of gene groups; 4) and (4) screening out a primer group. The primer group can accurately identify the rice blast germs at low cost. The invention also comprises the application of the primer group.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a primer group for identifying rice blast germs, a screening method and application thereof.
Background
The rice blast is one of the most important diseases of rice, a large amount of grain loss is caused every year, and the pathogen of the rice blast is rice blast. The traditional rice blast bacteria identification method is field inoculation, which is time-consuming and labor-consuming. The DNA molecule identification method mainly comprises the identification of a whole gene re-sequencing method, a SSR (simple Sequence repeat) marking method and an SNP (single Nucleotide polymorphism) marking method. The rice blast germs can be identified by a field inoculation method, wherein the rice blast germs need to be separated by whole-gene re-sequencing, time and labor are wasted, and meanwhile, the sequencing depth is insufficient due to overlarge sequencing amount, so that the accuracy is reduced; the SSR marker method has the phenomenon of slippage and the like in the PCR (Polymerase Chain Reaction) amplification process, which causes the detection accuracy and sensitivity to be very low, and the SSR marker also needs to be amplified one by one and detected one by one, and has limited flux, thereby causing heavy workload; the SNP marker in the SNP marker method has low polymorphism, and the SNP marker is mostly detected by adopting a full-genotype re-sequencing method, so the defect of full-genotype re-sequencing also exists in the SNP marker method, and some SNP chips are also used for detection, which has the problem that the chip technology error is more than 1 percent, the chip design is complicated, and the unit cost can be reduced only by synthesizing and detecting a large amount of chips at one time, so that the whole cost of each use is high, and any marker site needing to be detected cannot be flexibly adjusted.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to screen out a primer group capable of accurately identifying rice blast germs at low cost.
In order to solve the technical problems, the invention provides a primer group for identifying rice blast germs and a screening method and application thereof.
The invention provides a primer group for identifying Pyricularia oryzae, which comprises a forward primer and a reverse primer, wherein sequences of a plurality of primers in the primer group are shown as SEQ ID NO 1-SEQ ID NO 200 in a sequence table.
The invention also provides a screening method of the primer group, which comprises the following steps:
1) taking the genome sequence of the rice blast germs as a query sequence, comparing the query sequence with a genome sequence database, and aligning all genome sequences in the genome sequence database;
2) translating the aligned genomic sequences along a window of a certain length; every translation is carried out once, pairwise comparison is carried out on the genomic fragments of the rice blast germs in the window, and the gene fragments with the discrimination are left to serve as candidate marker sites;
3) designing a primer for each candidate marker locus, wherein the length of a region amplified by the primer is between 200bp and 300bp, and the annealing temperature between the loci is between 60 and 70; all primer sequences are designed in conserved regions among multiple genomes;
4) similar primers are abandoned, and the primer group is screened out.
Further, in step 1), the query sequence is aligned with the genome sequence database using BLASTN software, the output format is-outfmt 1, and the maximum output sequence number is set to 300.
Further, in step 2), the window 125bp long is translated along the aligned genomic sequence by 5bp each time.
Further, in step 2), if the discrimination of the gene segments is greater than 0.2, the gene segments are used as the candidate marker sites; and D/c, wherein D is the number of comparisons of difference between two blast fungus genome fragments in the genome sequence database, and c is the total number of comparisons.
Further, in step 4), the similar primers mean that the similarity between the primers and other primers is greater than 60%, and the alignment length is greater than 10 bp.
In addition, the invention also provides application of the primer group or the primer group obtained by screening the primer group by the screening method in identifying rice blast germs.
The application comprises the following steps:
s1, amplifying the detection sites of the genomic DNA of the Pyricularia oryzae to be detected and the genomic DNA of the Pyricularia oryzae to be controlled by using the primer group through multiple PCR to obtain multiple PCR amplification products;
s2, carrying out high-throughput sequencing on the multiple PCR amplification product to obtain a sequencing fragment;
s3, obtaining the genotype of the detection site according to the sequencing fragment;
s4, judging whether the Pyricularia oryzae to be detected and the Pyricularia oryzae contrast are the same or not according to the genotype of the detection site.
Further, in step S4, if the genotype of the Pyricularia oryzae to be tested and the gene of the Pyricularia oryzae to be controlled are the same, determining that the Pyricularia oryzae to be tested and the Pyricularia oryzae to be controlled are the same; and if the genotype of the to-be-detected Pyricularia oryzae is not completely the same as the genotype of the reference Pyricularia oryzae, judging that the to-be-detected Pyricularia oryzae is different from the reference Pyricularia oryzae.
In addition, the invention also provides application of the primer group obtained by screening the primer group or the screening method of the primer group in preparation of a kit for screening rice blast germs.
Compared with the prior art, the invention has the advantages that: taking the genome sequence of the rice blast germs as a query sequence, comparing the query sequence with a genome sequence database, and aligning all genome sequences in the genome sequence database; translating the aligned genomic sequences along a window of a certain length; comparing every two of the genomic fragments of the rice blast germs in the window once in translation, and leaving gene fragments with discrimination as candidate marker sites to ensure the proportion occupied by the combination with difference in the primer amplification region; any one primer is not matched with any other selected primer, and a primer dimer is not formed; designing a primer for each candidate marker locus, wherein the length of a region amplified by the primer is between 200bp and 300bp so as to avoid preferential amplification of short fragments, and the annealing temperature between each locus is between 60 and 70; all primer sequences are designed in conserved regions among multiple genomes; similar primers are abandoned, and the primer groups are screened out, so that the method ensures that any different rice blast germs can be distinguished by the screened primer groups, 100 pairs of positive and negative primer groups are screened out by the method, and the primer groups can accurately identify the rice blast germs at low cost.
Detailed Description
The present invention will be described in detail with reference to the following embodiments in order to make the aforementioned objects, features and advantages of the invention more comprehensible. 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.
The specific embodiment provides a primer group for identifying Pyricularia oryzae, which comprises a forward primer and a reverse primer, wherein the sequences of a plurality of primers in the primer group are shown as SEQ ID NO. 1-SEQ ID NO. 200 in a sequence table.
The specific embodiment further comprises a screening method of the primer group, which comprises the following steps:
1) taking the genome sequence of the rice blast germs as a query sequence, comparing the query sequence with a genome sequence database by using BLASTN software, outputting the sequence in a format of-outfmt 1, setting the maximum output sequence number to be 300, and aligning all genome sequences in the genome sequence database.
2) Translating a 125bp long window along the aligned genome sequence, moving by 5bp every time, comparing every two of the genome fragments of the rice blast germs in the window every time of translation, and taking the gene fragments as candidate marker sites if the discrimination of the gene fragments is more than 0.2; and D/c, wherein D is the number of comparisons of difference between two blast fungus genome fragments in the genome sequence database, and c is the total number of comparisons.
3) Designing a primer for each candidate marker locus, wherein the length of a region amplified by the primer is between 200bp and 300bp, and the annealing temperature between the loci is between 60 and 70 ℃; all primer sequences were designed to be conserved between 300 genomes;
4) discarding similar primers, and screening the primer group; the similar primers mean that the similarity between the primers and other primers is more than 60 percent, and the comparison length is more than 10 bp.
The specific embodiment also comprises an application of the primer group in identifying Pyricularia oryzae.
The application comprises the following steps:
s1, amplifying the detection sites of the genomic DNA of the Pyricularia oryzae to be detected and the genomic DNA of the Pyricularia oryzae to be controlled by using the primer group through multiple PCR to obtain multiple PCR amplification products;
s2, carrying out high-throughput sequencing on the multiple PCR amplification product to obtain a sequencing fragment;
s3, obtaining the genotype of the detection site according to the sequencing fragment;
s4, judging whether the Pyricularia oryzae to be detected and the Pyricularia oryzae contrast are the same or not according to the genotype of the detection site; if the genotype of the to-be-detected Pyricularia oryzae is the same as the genotype of the reference Pyricularia oryzae, judging that the to-be-detected Pyricularia oryzae is the same as the reference Pyricularia oryzae; and if the genotype of the to-be-detected Pyricularia oryzae is not completely the same as the genotype of the reference Pyricularia oryzae, judging that the to-be-detected Pyricularia oryzae is different from the reference Pyricularia oryzae.
The specific embodiment also comprises an application of the primer group in preparation of a kit for screening Pyricularia oryzae.
The following examples are given to illustrate the present invention in detail.
Example 1
The screening method of the primer group comprises the following steps:
1) an index file was constructed on a database of genome sequences of 300 rice BLAST germs using the formatdb command in the BLAST toolkit provided by the National Center for Biotechnology Information (NCBI). Selecting a genome sequence which is most completely assembled as a query sequence, comparing the query sequence with a genome sequence database of 300 rice blast germs by using BLASTN software (the version number is 2.9.0), outputting the sequence in the format of-outfmt 1, setting the maximum output sequence number to be 300, and aligning all genome sequences in the genome sequence database.
2) And translating a window with the length of 125bp along the aligned genome sequence, moving 5bp every time, comparing every two of the genome fragments of 300 rice blast germs in the window every time of translation, and if the difference between the two fragments exceeds 2 SNP, determining that the difference is different, otherwise, determining that the difference is not. If the discrimination of the gene segments is more than 0.2, the gene segments are used as candidate marker loci; and D/c, wherein D is the number of comparisons of difference between two blast fungus genome fragments in the genome sequence database, and c is the total number of comparisons.
3) For each candidate site we performed primer design with primer3 (version number 2.3.6), which amplifies a region between 200bp and 300bp in length and anneals between sites at 60-70 ℃; all primer sequences are designed into conserved regions among 300 gene groups so as to ensure the universality of the primers;
4) we compared the similarity between any two pairs of primers using BLASTN (version number 2.9.0) with the parameters set to-W7, -e1e-3, -max _ target _ SEQs 1, discarded similar primers, and screened a primer set comprising a forward primer and a reverse primer and the sequences of the primers in the primer set are shown in Table 1 and SEQ ID NO: 1-SEQ ID NO:200 in the sequence Listing, and the corresponding detection site (i.e., the candidate marker site retained). If the similarity between one primer and the other primer is > 60%, the alignment length is >10bp, then the primer is discarded. The corresponding candidate marker site was also discarded as a null site.
The start and end points in Table 1 refer to the base positions on the reference genome of Pyricularia oryzae with the version number magnaporthe _ oryzae _ 70-15.
TABLE 1 detection sites, chromosomes and corresponding primer sets
Example 2
Two different rice blast germs with monoclonal sources are separated from two different rice areas in Hunan, and the numbers of the rice blast germs are respectively 1 and 2, wherein the number 1 rice blast germs are divided into two parts, and the numbers of the two parts are respectively 1-1 and 1-2. Thus, Pyricularia oryzae nos. 1-1 and 2 are different, Pyricularia oryzae nos. 1-2 and 2 are different, and Pyricularia oryzae nos. 1-1 and 1-2 are the same.
The application of the primer group in identifying the rice blast germs comprises the following steps:
s1, amplifying the detection sites of the genomic DNA of the rice blast germs to be detected and the control rice blast germs by using the primer group through multiple PCR to obtain multiple PCR amplification products.
In this example, all primer sets from site 1 to site 100 in Table 1 were synthesized and mixed by the United states thermal company, the primer sets included a forward primer and a reverse primer, and the sequences of the primer sets were shown in SEQ ID NO. 1 to SEQ ID NO. 200 in the sequence Listing. And amplifying the detection sites of the genomic DNA of the rice blast germs to be detected and the control rice blast germs by utilizing the primer group through multiple PCR, wherein the detection sites comprise a plurality of base sites with single nucleotide polymorphism. Specifically, genomic DNA of rice leaf numbers 1-1, 1-2 and 2 was multiply amplified 3 times in total according to an amplicon kit (cat # 4475345) produced by U.S. thermal power corporation, and the procedure was performed according to the instructions of the kit to obtain multiplex PCR amplification products.
S2, carrying out high-throughput sequencing on the multiple PCR amplification product to obtain a sequencing fragment.
In this example, a high-throughput sequencing library was constructed using an amplicon construction kit (cat 4475345) produced by thermoelectric corporation and the obtained multiplex PCR amplification products, and the procedures were described in the specification of the kit. 3 constructed libraries are mixed together to form a high-throughput sequencing library, and the constructed high-throughput sequencing library is subjected to high-throughput sequencing by using an S5 high-throughput sequencer produced by the United states of America, wherein the operation steps are carried out according to the specification of the kit, each Pyricularia oryzae sample is set as a 1M (1M is 1000000) sequencing fragment in the depth of the high-throughput sequencing, and the length of the high-throughput sequencing is set as 300 bp. The number of sequencing fragments obtained for samples 1-1, 1-2 and 2 was 1.1M, 0.9M and 0.9M, respectively. Thus, the average number of sequencing per detection site per sample was close to 10000 on average.
S3, obtaining the genotype of the detection site according to the sequencing fragment.
In this example, the obtained sequencing fragments were compared with the reference genome of Pyricularia oryzae with a version number of magnaporthe _ oryzae _70-15 using Bowtie2 software (version number: 2.1.0) and its default parameters, and only the sequencing fragments covering the complete sequence between the amplification starting point and the end point listed in Table 1 in the reference genome of Pyricularia oryzae were retained; the combination of the allelic type of the SNP in the reserved sequencing fragment, which is different from that of the reference genome, forms the MNP genotype of the sequencing fragment; the MNP genotype that has the greatest number of sequencing fragments in a detection site is referred to as the genotype of that detection site. For example, in the detection site AMPL3589924 of sample 1-1, the starting point 242345 of the amplicon in chromosome Supercontig _8_1 is set as position 0, the 35 th and 187 th bases are different from the reference genome and are both A, so the MNP genotype is marked as A (35) A (187), and the MNP genotype of total 9898 sequencing fragments is A (35) A (187), which is the largest in number, so A (35) A (187) is the genotype of the detection site AMPL 3589924. In the same manner, the genotypes of the detection sites AMPL3589924 of samples 1-2 and 2 were obtained as A (35) A (187) and C (35) C (187), respectively. Therefore, in the detection site AMPL3589924, the genotypes of samples 1-1 and 1-2 are the same, the genotypes of samples 1-1 and 2 are different, and the genotypes of samples 1-2 and 2 are different. In the same manner, it was judged whether the genotypes of each of the test sites were the same among the samples 1-1, 1-2 and 2.
S4, judging whether the Pyricularia oryzae to be detected and the Pyricularia oryzae contrast are the same or not according to the genotype of the detection site; if the genotype of the to-be-detected Pyricularia oryzae is the same as the genotype of the reference Pyricularia oryzae, judging that the to-be-detected Pyricularia oryzae is the same as the reference Pyricularia oryzae; and if the genotype of the to-be-detected Pyricularia oryzae is not completely the same as the genotype of the reference Pyricularia oryzae, judging that the to-be-detected Pyricularia oryzae is different from the reference Pyricularia oryzae.
In this example, the genotypes of all the commonly detected 90 detection sites of samples 1-1 and 1-2 were the same; 35 detection sites in 95 detection sites detected together in the samples 1-1 and 2 have different genotypes; of the 92 detection sites detected together in samples 1-2 and 2, 35 genotypes were not identical. Therefore, it is determined that the Pyricularia oryzae 1-1 to be tested is the same as the Pyricularia oryzae 1-2 of the control, the Pyricularia oryzae 1-1 to be tested is different from the Pyricularia oryzae 2 of the control, and the Pyricularia oryzae 1-2 to be tested is different from the Pyricularia oryzae 2 of the control. The result is consistent with the real situation, which shows that the method for identifying the rice blast germs is accurate.
By utilizing the method, the genome DNA is directly extracted from the rice leaves containing the rice blast germs to identify the rice blast germs, so that the separation work of the rice blast germs is avoided, and compared with the methods of whole genotype group sequencing and field inoculation identification, the method greatly saves manpower and material resources; the number of detection sites reaches 100, so the detection fineness is far greater than that of a method based on 1 or a few detection sites, such as an ITS (alternate transcription region) marker and other methods, for example, the ITS marker method can only identify the rice blast fungus, while the method can distinguish 1-1 and 2 and 1-2 and 2 different strains below the rice blast fungus, so the species identification distinguishing capability is stronger; the invention adopts a sequencing method, can know the composition of each base of each locus, and further enhances the fineness of species identification; the sequencing method is the 'gold standard' of any gene detection, so the accuracy of the method is superior to the SSR marker detection method or the SNP marker detection method based on electrophoresis and chip hybridization; because the method only detects 100 sites on the genome, but not every site on the whole genome, the average sequencing time of every detection site is close to 1 ten thousand times, which is far higher than that of the whole genome under the same sequencing cost, and the accuracy of detection is further remarkably improved; the invention mixes all 100 primers together, therefore, the working efficiency is improved by 100 times compared with a method based on single-site or few-site amplification, such as an SSR labeling method or an SNP labeling method. In fact, in this embodiment, only 3 times of multiplex amplification and one time of high-throughput sequencing are performed to detect 3 rice blast pathogen samples, and the working efficiency is greatly improved.
The application of the primer group in identifying Pyricularia oryzae provided by the invention is characterized in that multiple PCR amplification is carried out on multiple regions of genomic DNAs of Pyricularia oryzae to be detected and Pyricularia oryzae to be controlled by adopting the primer group to obtain an amplification product, the amplification product is detected by high-throughput sequencing, multiple (25-96) SNP sites are obtained in each division and are constructed into MNP (multiple Nucleotide polymorphism) genotypes, and whether the Pyricularia oryzae to be detected and the Pyricularia oryzae to be controlled are the same or not is identified by utilizing the MNP genotypes. The invention solves the defects of the existing rice blast germ identification method, and comprises the following steps: by utilizing multiple amplification and high-throughput sequencing, hundreds of marker sites can be detected at one time, and each marker site is repeatedly sequenced hundreds of times, so that the detection accuracy is improved, and the detection workload is greatly reduced; the multiplex amplification primers can be added and reduced at any time, the cost of single sample detection cannot be increased, and the identification flexibility is greatly increased; the detection cost of a single sample is also obviously reduced compared with the whole genome sequencing and chip technology.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
Sequence listing
<110> research center for hybrid rice in Hunan province
<120> primer group for identifying Pyricularia oryzae, and screening method and application thereof
<130> 2020.04.30
<160> 200
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ttttccctct caggctgcat ag 22
<210> 2
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
caggtggaaa tagtcaactg gtactc 26
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcgaaattgg ttttgttgaa ccga 24
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gatctcctat cgcactgatc agtc 24
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ctaccgcaac gacatcaaac tc 22
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gcaaacaagg agtcgtacag gtt 23
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tagcattggc tcaactttgg tattatctag 30
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gtcacccagt ttctccgttc tat 23
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caataataac gaccgtggtt gcaa 24
<210> 10
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cgtcatgaag acagcgaagg taat 24
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
catacgggtg ttgcttacca ct 22
<210> 12
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tcgatgagat cggtaataat accagct 27
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
cgcttcgtca tgtcacgttt ag 22
<210> 14
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cccaggaggt atctttccag ga 22
<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gcctgaattg tcgccgtaat tc 22
<210> 16
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
caagaacatc gattctaaca caagtctg 28
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
cccgtcccaa tgtcggttac 20
<210> 18
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
attttctgga tccaattcct tggagt 26
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
cccaaaatca accaccgaac tt 22
<210> 20
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
atcgtcctgg tagtgaccgt at 22
<210> 21
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tttttagttc aacacgtacc cagtctt 27
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
atacctgcga aggcactcat tt 22
<210> 23
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
cgattctgag aagagtaaag ccgat 25
<210> 24
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ttaccacaat ttcgacgata gccat 25
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
cgagacgcta ttggctctgt ac 22
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ctcgagccac gatgacttca at 22
<210> 27
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ctggctctcg agccactt 18
<210> 28
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
cctcgcgaac ctgctgtt 18
<210> 29
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ctgaagtacg agcatcacca gt 22
<210> 30
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ttgcttgacg aaaaacaaag taccataaat 30
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tgagcgccat tatttcgagg tt 22
<210> 32
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gagcttctcg gctacgttag ac 22
<210> 33
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gcaaagcagg gattagatat agttctagag 30
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ggtggctgca tatctgcatg 20
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
aaaactactt gctctccaac acct 24
<210> 36
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gcttcgacta ctccaacagc atc 23
<210> 37
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ttgcgattgc aattcatata cttcagc 27
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
accggctcaa atatttgggc tat 23
<210> 39
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
caagatatcg atggcatttg atgctg 26
<210> 40
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gcaaatacaa gaaaccattg gattgaatga 30
<210> 41
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
aggattccac gtcctgaaat aatagc 26
<210> 42
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
tggcgaagaa gactttagat gagac 25
<210> 43
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
agactgagtt atcgtggtga gcta 24
<210> 44
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
aaaaagttgg ttgacgtgac attgt 25
<210> 45
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ggttcgcctt gtcttgtctg at 22
<210> 46
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ccctgctact gaacagacta agc 23
<210> 47
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
cccagaatat acggtataaa tagtttctcc 30
<210> 48
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
tacaaccaac tgtccctgca ttt 23
<210> 49
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ctttacatgt gattgtatct ctgccatttt 30
<210> 50
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
gcttttgcga gaaataaatg tttattgacg 30
<210> 51
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
ggcagggcac ttttctaaat aaagtg 26
<210> 52
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gttagcgaaa cgcttaacgt gataatag 28
<210> 53
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
aataccgtca ccaattcact gct 23
<210> 54
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
ggccgtggga gtaagtaaat tcat 24
<210> 55
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
taagggatat gtaaatacct ctggccttt 29
<210> 56
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gcataaaaag cagggtggat ttgg 24
<210> 57
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
gctcaagctg gcaaggctaa 20
<210> 58
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
tgggcatcga cctcatcaac 20
<210> 59
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
acgtcagggt ggagggaga 19
<210> 60
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
cgacaagtcc ctggcgat 18
<210> 61
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
cctgaaaata tcaacattca tgctacgt 28
<210> 62
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
atggcggacg tgaaaggaaa at 22
<210> 63
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
cactgccaag attacatcct acaga 25
<210> 64
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
aggcgatttc tacgttggtt ctc 23
<210> 65
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
aagatgcaaa atctcatcta aggatggt 28
<210> 66
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ttgttgcttt gttcataaca tgcgt 25
<210> 67
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
ctcggtaata tattcagctt tgacagct 28
<210> 68
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
tcaaagagat gccaaataac tctgca 26
<210> 69
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
gcgggactgg acaatgattg aa 22
<210> 70
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
ttggtttgcc tctatcttct tgttgt 26
<210> 71
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
ggtatccgat gagaggtaat atccaca 27
<210> 72
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
gttcgaaagt ctggattaaa cggttc 26
<210> 73
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
gcaaagacgg gtgtttgttg ac 22
<210> 74
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
aaaaaggtat ctaagcgaaa atttggca 28
<210> 75
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
aagaaatgct gatcgtcgaa ggtta 25
<210> 76
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
aacatcaccc aattgatgaa attcttcac 29
<210> 77
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
gcttgggatt cggtcatgat tatga 25
<210> 78
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
gatgggtcga ctgattacta cttgaaa 27
<210> 79
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
aatgcggtgg gaatgttgta gattat 26
<210> 80
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
ggtggttgaa gcggtccaat aa 22
<210> 81
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
gggtacctcg actttgtaaa gaca 24
<210> 82
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
ctcaaccgca actttactta ccc 23
<210> 83
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
gtgcaaatgg gttgcaaaat aacc 24
<210> 84
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
cgacggagtt tcaattcctt caaaaa 26
<210> 85
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
ctgactggtg cattattatt tagcaaatgt 30
<210> 86
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gtactaattg ggctcttcta gagggta 27
<210> 87
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
ccaaggtcct atctcggcct at 22
<210> 88
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
tcccttgtac tagttgggaa acca 24
<210> 89
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
attgcaaggg tgactcagtg at 22
<210> 90
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
cagcagggtt aattggaata gatgc 25
<210> 91
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
acgcgactct cacttaaatc aaagt 25
<210> 92
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
catacattgc gccaagaaga aacc 24
<210> 93
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
agggccactc tatttgactg aga 23
<210> 94
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
gattatgtaa acgtttcttc tcattcgcat 30
<210> 95
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
cgtaacaaca tttagttgat cgatcgatc 29
<210> 96
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
ggctttacac tttttacaaa ttggagttga 30
<210> 97
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
gccatcctgt ccgagattct tg 22
<210> 98
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
ctcaacaccc agagcgacgt tt 22
<210> 99
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
caccgtaaat cacgctctac taagt 25
<210> 100
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
ggctttgata tccaccagcc att 23
<210> 101
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
cgatcgattg gagattcggg aa 22
<210> 102
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
cgactttgta cccttgatgg tca 23
<210> 103
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
tgcaaatgtt aaattttggc ttccagt 27
<210> 104
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
gagagatggt cgatttgcca gt 22
<210> 105
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
gcgacggttg gcaacctaat 20
<210> 106
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
ccgtgtccag gaccaatgac 20
<210> 107
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
aaagaaacac gagtcgtgaa atgc 24
<210> 108
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
aagcattccg ctttattcgt tcc 23
<210> 109
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
cgttcaacaa aaaggagcac atgt 24
<210> 110
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
gtaatgctga ttgaagaatg tagctagaga 30
<210> 111
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
cgcctttgtc tgttactata ttttgtgt 28
<210> 112
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
ttcagaaaca caagagaata gaaacgagca 30
<210> 113
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
ccgattctcc gattctcgga gta 23
<210> 114
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
ggcggtcttg tctgacattt tg 22
<210> 115
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
cctggaaaat gtgctaatgt atggc 25
<210> 116
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
gactgtcgcc caaaactgaa tc 22
<210> 117
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
gcttgccagc atcatgacag at 22
<210> 118
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
cgttcctccg atgcatacag ac 22
<210> 119
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
cgcctgctat ccaccttttt ac 22
<210> 120
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
ctcatgattt tctctgtatg tcactttctg 30
<210> 121
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
gccaactgat cccattcttt gc 22
<210> 122
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
gctggttgac aattgcttca agg 23
<210> 123
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
agagagtatc cctactcctc aacaag 26
<210> 124
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
actctaccaa cagaaacgtc attacac 27
<210> 125
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
tgcgggacgt ggctcat 17
<210> 126
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
gtgcagcgcc agattgc 17
<210> 127
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
caattcaggg cttcaggcct at 22
<210> 128
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
caaaacatat ttgacacaat gcttgtcag 29
<210> 129
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
attgaaagtt tggtcaagca tgca 24
<210> 130
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
gctctcctgg gtagctaaaa agc 23
<210> 131
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
gactggccaa cgccattata att 23
<210> 132
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
tgatgtcttc gtcaacgaaa ctagttc 27
<210> 133
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
gcatacgctc ggaacaaatt atcc 24
<210> 134
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
cttggttgta catcagatgt ataggca 27
<210> 135
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
caaccatgtc atagatgagg aattgc 26
<210> 136
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
catgaaacaa tcgcccttct tacc 24
<210> 137
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
gcgtgccgta cagtagtagt ta 22
<210> 138
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
ggcacgacaa tactatgtat gggt 24
<210> 139
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
cttttagggt tgcgcaacga at 22
<210> 140
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
ttaatctcgt tcgttctttg caacttg 27
<210> 141
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
gatggaacgg gcaacctata ca 22
<210> 142
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
gttggaactg acagtcaaac tgtc 24
<210> 143
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
acttaatctt ttggctggct gtagaa 26
<210> 144
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
cctgagagaa aagaaaacta ttgttatggc 30
<210> 145
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
caaccttatg atctccgctg tatagt 26
<210> 146
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
cagcttgatc gtctaatgac tatgtcc 27
<210> 147
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
caagtctact tcggcattgc attc 24
<210> 148
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
cctgatcgca gactgaatgt gt 22
<210> 149
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
gggatgttga tagaacgaag tatgaaca 28
<210> 150
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
actccacaac ccattgcaat atcat 25
<210> 151
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
gatcaacgag tcgatcgacc ta 22
<210> 152
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
aatactgccc agtttgttaa gtgatga 27
<210> 153
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
tagaaatgca tttcaagagt cagtccat 28
<210> 154
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
gcccttttca gtctcgaaat cct 23
<210> 155
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
gactgatctt gtacagggca caa 23
<210> 156
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
cataactgcc ttctaatcga aatacgc 27
<210> 157
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
tctccttgtt gctacgatat aaagagc 27
<210> 158
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
tcagagtcgt gatctctgcc tat 23
<210> 159
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
catcgagtcc tgtgcttgac t 21
<210> 160
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
caattaagtc gtcgttcaaa cattcaaatc 30
<210> 161
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
caacggctac atactataga gaatcgattc 30
<210> 162
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
catagtcttg catacgccca gat 23
<210> 163
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
cgactccgac atgtgtttaa attatcg 27
<210> 164
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
acgacatact aaccatactc aatttgctg 29
<210> 165
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
cgaaattcaa attccacatc tgaagaact 29
<210> 166
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
attagcaggg tttcttttgc aatgtc 26
<210> 167
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
cccaatcagc actgacattt gac 23
<210> 168
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
caggtcttct gggatgatat gtgg 24
<210> 169
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
acggagaggc tgttctacga 20
<210> 170
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
ctcgccttct ccttcttctc tt 22
<210> 171
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
gcatccaggg ttacaagatt atagagg 27
<210> 172
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
tactgcccac gtatcacaaa gg 22
<210> 173
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
ccctttcaag ttacgtattt ttgtggt 27
<210> 174
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
ctaaattacg gaatcccgac caagt 25
<210> 175
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
gtgcattgaa ctgggtcgaa ag 22
<210> 176
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
tgatcaatca ggagcaggca aat 23
<210> 177
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
ctgccaacga ccaaagttaa cg 22
<210> 178
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
tctttgtcag tcatttgatt ctcatgct 28
<210> 179
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
cgaaaaacaa agggatcaca tcca 24
<210> 180
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
taacaaatag tagacagcca aatgatgacc 30
<210> 181
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
ctcttctcca ggacccacat tt 22
<210> 182
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
gttccacttt gacactctac acgta 25
<210> 183
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
agggagtatg gatgcacctt ga 22
<210> 184
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
caatcaatcg agccaaagat gcaa 24
<210> 185
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 185
agacaaaacc tcgagctttg gt 22
<210> 186
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 186
gccacttgaa gtatcccaat tgtcat 26
<210> 187
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 187
ctgagggata tcccgttgac aa 22
<210> 188
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 188
gaacagatac aagagcggaa aaattcc 27
<210> 189
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 189
cgatgacaag agtgcggata aca 23
<210> 190
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 190
agacattcac aacacctcaa acca 24
<210> 191
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 191
accaagtctg caagtggact tt 22
<210> 192
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 192
ggtaatcctt gccacaggga ttg 23
<210> 193
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 193
agttcatggc agtatctcag tttcc 25
<210> 194
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 194
tgatgatggc aacaaaaatg ctgatc 26
<210> 195
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 195
atcaccaaac acagatggca ggat 24
<210> 196
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 196
gcgattgagc caaaactcta gaaatagata 30
<210> 197
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 197
cggcaggaac tgtcgataac ag 22
<210> 198
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 198
ctcgtcgtgc gagtgttatg at 22
<210> 199
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 199
tgtttggcga gcaatatact tttatgc 27
<210> 200
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 200
tcgcttctat gcccttgtat tcc 23
Claims (5)
1. A primer group for identifying Pyricularia oryzae is characterized by comprising a forward primer and a reverse primer, wherein sequences of a plurality of primers in the primer group are shown as SEQ ID NO 1-SEQ ID NO 200 in a sequence table.
2. Use of the primer set according to claim 1 for identifying Pyricularia oryzae.
3. Use according to claim 2, characterized in that it comprises the following steps:
s1, amplifying the detection sites of the genomic DNA of the Pyricularia oryzae to be detected and the genomic DNA of the Pyricularia oryzae to be controlled by using the primer group through multiple PCR to obtain multiple PCR amplification products;
s2, carrying out high-throughput sequencing on the multiple PCR amplification product to obtain a sequencing fragment;
s3, obtaining the genotype of the detection site according to the sequencing fragment;
s4, judging whether the Pyricularia oryzae to be detected and the Pyricularia oryzae contrast are the same or not according to the genotype of the detection site.
4. The use according to claim 3, wherein in step S4, if the genotype of the Pyricularia oryzae to be tested and the gene of the Pyricularia oryzae to be controlled are both the same, it is determined that the Pyricularia oryzae to be tested and the Pyricularia oryzae to be controlled are the same; and if the genotype of the to-be-detected Pyricularia oryzae is not completely the same as the genotype of the reference Pyricularia oryzae, judging that the to-be-detected Pyricularia oryzae is different from the reference Pyricularia oryzae.
5. An application of the primer set of claim 1 in the preparation of a kit for screening Pyricularia oryzae.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010405191.5A CN111662997B (en) | 2020-05-14 | 2020-05-14 | Primer group for identifying rice blast germs as well as screening method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010405191.5A CN111662997B (en) | 2020-05-14 | 2020-05-14 | Primer group for identifying rice blast germs as well as screening method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111662997A CN111662997A (en) | 2020-09-15 |
| CN111662997B true CN111662997B (en) | 2022-02-25 |
Family
ID=72383588
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010405191.5A Active CN111662997B (en) | 2020-05-14 | 2020-05-14 | Primer group for identifying rice blast germs as well as screening method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111662997B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114277183B (en) * | 2021-11-06 | 2023-09-08 | 江汉大学 | MNP (MNP-associated protein) marker combination, primer pair combination, kit and application of MNP marker combination and primer pair combination of 5 human enteroviruses |
| CN114107525B (en) * | 2021-11-10 | 2023-05-05 | 江汉大学 | MNP (MNP) marking site of pseudomonas aeruginosa, primer composition, kit and application of MNP marking site |
| CN116904620B (en) * | 2023-03-23 | 2024-04-30 | 三亚中国检科院生物安全中心 | Primer group, kit and method for rapidly detecting rice pathogenic bacteria based on MNP |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010109463A2 (en) * | 2009-03-24 | 2010-09-30 | Yeda Research And Development Co. Ltd. | Methods of predicting pairability and secondary structures of rna molecules |
| CN105603082A (en) * | 2016-01-29 | 2016-05-25 | 中国科学院遗传与发育生物学研究所 | Qualitative and quantitative detection method for paddy microorganisms |
| CN105603075A (en) * | 2016-01-29 | 2016-05-25 | 江汉大学 | Qualitative and quantitative wheat microorganism detecting method |
| CN108048591A (en) * | 2017-12-08 | 2018-05-18 | 江汉大学 | A kind of Pyricularia grisea Race isolation and identification method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190214109A1 (en) * | 2018-01-08 | 2019-07-11 | Rutgers, The State University Of New Jersey | Methods For Finding Genome Rearrangments From Sequencing Data |
-
2020
- 2020-05-14 CN CN202010405191.5A patent/CN111662997B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010109463A2 (en) * | 2009-03-24 | 2010-09-30 | Yeda Research And Development Co. Ltd. | Methods of predicting pairability and secondary structures of rna molecules |
| CN105603082A (en) * | 2016-01-29 | 2016-05-25 | 中国科学院遗传与发育生物学研究所 | Qualitative and quantitative detection method for paddy microorganisms |
| CN105603075A (en) * | 2016-01-29 | 2016-05-25 | 江汉大学 | Qualitative and quantitative wheat microorganism detecting method |
| CN108048591A (en) * | 2017-12-08 | 2018-05-18 | 江汉大学 | A kind of Pyricularia grisea Race isolation and identification method |
Non-Patent Citations (3)
| Title |
|---|
| An accurate and efficient method for large-scale SSR genotyping and applications;Lun Li 等;《Nucleic Acids Research》;20170210;第45卷(第10期);第1-12页 * |
| characterization of molecular identity and pathogenicity of rice blast fungus in Hunan province of China;Junjie Xing 等;《plant disease》;20170430;第101卷(第4期);第557-561页 * |
| 杂交水稻稻瘟病抗性育种体系研究进展;邢俊杰 等;《杂交水稻》;20140331;第29卷(第2期);第1-5页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111662997A (en) | 2020-09-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111662997B (en) | Primer group for identifying rice blast germs as well as screening method and application thereof | |
| EP0620862B1 (en) | Method for the identification of microorganisms by the utilization of directed and arbitrary dna amplification | |
| KR101403494B1 (en) | SSR Primer for the Identification of Sweet Potatoes and Method for Identifying Sweet Potatoes | |
| CN107619870B (en) | Molecular marker capable of indicating and identifying length of sheep wool and specific primer pair and application thereof | |
| CN108374054B (en) | Rice SSR molecular markers suitable for capillary electrophoresis detection technology and application thereof | |
| CN109880913B (en) | Composite amplification kit for 38 human Y chromosome loci and application thereof | |
| CN102559868A (en) | Method for qualitative and quantitative detection of multiple target nucleotide sequences with single tube | |
| Trtkova et al. | Molecular-genetic approaches to identification and typing of pathogenic Candida yeasts | |
| KR101961653B1 (en) | SNP molecular marker for selecting cultivars of sweet potatoes and uses thereof | |
| CN106319079B (en) | Method for detecting 22q11.2 copy number loss | |
| Chen et al. | Multiplex PCR with the blunt hairpin primers for next generation sequencing | |
| CN107937493B (en) | Hairpin modified primer for allele PCR | |
| CN108384884B (en) | Corn SSR molecular markers and application thereof | |
| CN116334280A (en) | Identification method, primer pair and kit for cryptococcus | |
| CN106676176B (en) | Method for performing SSR (simple sequence repeat) analysis on tetraploid alfalfa by utilizing multiple PCR (polymerase chain reaction) | |
| KR101955074B1 (en) | Snp markers for discrimination of raphanus sativus | |
| KR102174274B1 (en) | Molecular marker for discriminating Zizyphus jujuba 'Boeun' and 'Chuseok' cultivar and uses thereof | |
| Zhu et al. | High resolution melting curve analysis: an efficient method for fingerprinting of hybrid rice cultivars and their parental lines | |
| CN108642190B (en) | Forensic medicine composite detection kit based on 14 autosomal SNP genetic markers | |
| CN109504744B (en) | Multiplex loop-mediated isothermal nucleic acid amplification detection method and kit based on high-resolution fusion | |
| CN111321139A (en) | Composition for cat genotyping and application thereof | |
| CN113444823B (en) | Primer group and method for identifying streptomyces albidoflavus W68 by using primer group | |
| CN108277295B (en) | Corn SSR molecular markers suitable for capillary electrophoresis detection technology and application thereof | |
| KR102461752B1 (en) | Primer Set for Detecting Peronospora Destructor Berk, Diagnostic kit using the same | |
| CN113444824A (en) | Primer for identifying 4 staphylococci in environment based on isothermal amplification technology, detection kit and application |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |