CN113789404B - Functional specificity molecular marker of rice blast resistance gene Pik-zh and application - Google Patents

Functional specificity molecular marker of rice blast resistance gene Pik-zh and application Download PDF

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CN113789404B
CN113789404B CN202111045221.7A CN202111045221A CN113789404B CN 113789404 B CN113789404 B CN 113789404B CN 202111045221 A CN202111045221 A CN 202111045221A CN 113789404 B CN113789404 B CN 113789404B
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pik
rice blast
resistance gene
rice
blast resistance
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CN113789404A (en
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杨德卫
何旎清
郑向华
黄凤凰
程朝平
叶宁
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Rice Research Institute Fujian Academy Of Agricultural Sciences
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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention discloses a functional specific molecular marker of a rice blast resistance gene Pik-zh and application thereof, wherein the functional specific molecular marker of the rice blast resistance gene Pik-zh is Pik-zh-InDel, and a molecular marker which is in a specific band type with the rice blast resistance gene Pik-zh is amplified from genome DNA of a rice variety carrying the rice blast resistance gene Pik-zh through primer pairs SEQ ID NO.1 and SEQ ID NO. 2; wherein SEQ ID NO.1 is 5'-AGCAGTGGGACGAGTGGGA-3'; SEQ ID NO.2 is 5'-GGTAGGACACCTGCCATCA-3'; the functional specificity molecule of the rice blast disease resistance gene Pik-zh can be applied to the identification of the rice blast resistance gene in rice varieties.

Description

Functional specificity molecular marker of rice blast resistance gene Pik-zh and application
Technical Field
The invention belongs to the technical field of biological molecular markers, and particularly relates to a functional specificity molecule of a rice blast resistance gene Pik-zh and application thereof.
Background
Rice is one of the main food crops in the world, and rice blast caused by rice blast bacteria is a worldwide disease and has been in a trend of increasing in recent years. The rice blast not only causes the great yield reduction (10% -35%) of the rice, but also can affect the rice quality even if the particles are not harvested in severe cases. From the aspects of grain safety and rice disease-resistant breeding, cultivation of disease-resistant varieties is one of the most economical and effective methods for preventing and treating rice blast. However, the traditional disease-resistant breeding strategy has the characteristics of large workload, long period and the like. Therefore, strategies to alter traditional disease-resistant breeding are urgently needed.
The DNA molecular marker is a genetic marker developed based on polymorphism of biological macromolecules, and has been applied to genome mapping, gene positioning, genetic diversity evaluation, functional genomics research, transgenic plant identification, molecular marker assisted selection breeding and other aspects at present. The gene function specific molecular marker is developed by utilizing the specificity and the difference of the DNA sequence in the target gene. The gene characteristic molecular marker is a functional molecular marker developed from functional SNP loci or InDels in a target functional gene sequence, can accurately track and position functional alleles, has higher accuracy in the identification of crop phenotypic traits, and can efficiently screen out favorable genes required by breeding.
In the 60 th century, the study of rice blast resistance gene analysis of rice varieties was first carried out in japan, 14 genes at the first 8 resistance sites were identified, and a set of identification system for rice blast resistance gene analysis was established, and in recent decades, with the rapid development of molecular genetics and biotechnology, we have gradually carried out systematic study of rice blast resistance genetics in our country, many rice blast resistance genes were located or cloned, and development of rice disease resistance polymerization breeding was greatly promoted. However, some disease resistance genes are located at the same site, and the complex alleles and the functional sequences and the nonfunctional sequences are highly homologous, so that the traditional method is difficult to distinguish. Therefore, the target gene is selected by analyzing the specific sequence difference between the target gene and the allele and developing the functional specific molecular marker, so that the selection reliability is high, and the breeding pace can be greatly accelerated.
Since the Pik locus located on the long arm end of chromosome 11 contains more than 5 alleles Pik-m, pik, pik-p, pik-h and Pik-s, etc., which have different resistance profiles and race specificities, this locus has been a popular study of rice blast resistance, and among them, pik-m, pik, pik-p, pik-h and Pik-s have been successfully identified and cloned, and related functional specific molecular markers for the above alleles have been gradually developed and used.
The rice blast resistance gene Pik-ZH is a new main effective disease resistance gene which is identified by the applicant from rice variety ZH11 (Zhonghua 11) and is allelic to Pik, the CDs total length is 3438bp by using a map cloning method, the detailed nucleic acid sequence is shown as SEQ ID NO.4, and the further analysis of the rice blast resistance gene Pik-ZH finds that the Pik-ZH sequence has specificity difference with partial sequences of Pik, pik-s, pik-h and Pik-p cloned at the current site, and the Pik-ZH gene can show stronger disease resistance reaction to Chinese strains D and KJ 201. Therefore, in order to more accurately and effectively apply the broad-spectrum resistance gene Pik-zh of rice blast to the rice resistance breeding work, development of a Pik-zh specific molecular marker which truly reflects the target gene, is convenient and easy to use is needed.
Disclosure of Invention
The invention provides a functional specificity molecular marker and application of a rice blast resistance gene Pik-zh, creatively discovers that the functional specificity molecular marker can truly reflect the rice blast broad-spectrum resistance gene Pik-zh, and simultaneously, the preparation of the Pik-zh specificity molecular marker is convenient and easy to use and has higher accuracy and stability, so that the rice blast broad-spectrum resistance gene Pik-zh can be better applied to rice resistance breeding.
The invention aims at realizing the following technical scheme:
one of the purposes of the invention is to provide a functional specific molecular marker of a rice blast resistance gene Pik-zh, wherein the functional specific molecular marker of the rice blast resistance gene Pik-zh is Pik-zh-InDel, and a molecular marker which is in a specific band type with the rice blast resistance gene Pik-zh is amplified from genome DNA of a rice variety carrying the rice blast resistance gene Pik-zh through a primer pair SEQ ID NO.1 and SEQ ID NO. 2; wherein, the nucleotide sequences of the primer pair SEQ ID NO.1 and SEQ ID NO.2 are shown as follows:
SEQ ID NO.1：5’-ACGAGTGGGATCGATACTG-3’；
SEQ ID NO.2：5’-GGTAGGACACCTGCCATCA-3’；
the rice variety carrying the rice blast resistance gene Pik-zh is flower 11 in the rice disease-resistant variety.
Further, the rice blast resistance gene Pik-zh comprises a gene fragment SEQ ID NO.3 which partially codes for NBS-LRR type proteins.
The invention also aims at providing a preparation method of the functional specificity molecular marker of the rice blast resistance gene Pik-zh, which specifically comprises the following steps:
(1) Sequence comparison is carried out on the Pik-zh locus of the rice blast resistance gene, and insertion/deletion InDel loci which are specific to Pik-zh and can be different from other rice blast allele resistance genes of the locus are screened;
(2) Designing a gene specific primer at the InDel locus by utilizing the InDel locus information obtained in the step (1);
(3) And (3) taking total DNA of a rice blast resistant variety carrying a rice blast resistant gene Pik-zh as a template, and performing PCR amplification to obtain a PCR product, namely a rice blast resistant gene Pik-zh gene specific molecular marker Pik-zh-InDel.
One of the purposes of the invention is also to apply the functional specific molecular marker of the rice blast resistance gene Pik-zh to the identification of the rice blast resistance gene of rice varieties.
The invention also provides a kit for identifying rice blast resistance of rice varieties, which comprises a primer pair with a nucleic acid sequence shown as SEQ ID NO.1 and SEQ ID NO. 2.
The invention has the beneficial effects that:
1. the invention is a functional specific molecular marker developed for a new major disease-resistant gene Pik-zh with Pik allele, and the functional specific molecular marker of Pik-zh has the advantages of low cost, high efficiency, high accuracy and the like in practical application; the Pik-zh functional specificity molecular marker is Pik-zh-InDel, the Pik-zh can be successfully distinguished from other non-Pik-zh genes at the site by using an electrophoresis detection method, and the detection efficiency is high; meanwhile, the Pik-zh specific molecular marker Pik-zh-InDel provided by the invention is a dominant marker, exists in the Pik-zh gene, and has a screening capability theoretical value of 100 percent on Pik-zh
2. The Pik-zh functional specificity molecular marker provided by the invention is Pik-zh-InDel only needs PCR combined with agarose gel electrophoresis or non-denaturing polyacrylamide gel electrophoresis, has low cost, high accuracy and high flux, and is particularly suitable for agricultural production practice and germplasm resource screening of Pik-zh under any genetic background, transgenic breeding, gene polymerization and resistance breeding based on MAS technology, and the breeding efficiency and accuracy are greatly improved.
Drawings
FIG. 1 shows the amplification using the primers F1 and R1 shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, with the total DNA of 6 selected representative varieties as templates.
Detailed Description
The present invention will be described in further detail with reference to the following examples and drawings, which are provided for the purpose of illustration only and are not to be construed as limiting the invention in any way.
Example 1
The preparation method of the functional specific molecular marker of the rice blast resistance gene Pik-zh specifically comprises the following steps:
(1) Analysis of the Pik-zh insertion/deletion (InDel) site:
downloading genome sequences of different alleles of Pik locus and corresponding loci of different rice varieties from a public database: pik-m (GenBank: GU 811860.1), pi1 (GenBank: HQ 606329.1), pik (GenBank: AB 616658.1), pik-h (GenBank: HQ 662330.1), pik-s (GenBank: HQ 662329.1), pik-p (GenBank: HM 035360.1), shuhui 498 (GenBank: CP 018167.1), japanese sunny (Locus: LOC-Os 11g 42010), and then sequence alignment is performed on the rice blast resistance gene Pik-zh site in a rice database, the genome sequences of the whole corresponding regions are not found in the rice donor varieties Japanese sunny, shuhui 498 and Pik alleles are sequenced, wherein the deleted and differentiated base parts in the alignment result are the identified InDel sites, and the alignment result is as follows: pik-zh:
Shuhui498：
Pik-m：
Pi1：
Pik：
Pik-h：
Pik-s：
Pik-p：
deletion of the corresponding sequence
Wherein, the boxed single base part of the Pic-zh gene with the difference detected after the comparison, the blank is the single base part of the Pic-zh gene with the deletion detected after the comparison, and further analysis shows that a section of sequence in the Pic-zh gene is extremely specific, and has 9bp difference compared with the sequences corresponding to Pic-m, pi1, pic-h, pic-s, pic-p and Shuhui 498, and the partial sequence corresponding to Nippon is completely deleted compared with the sequences corresponding to Nippon.
(2) Designing a gene specific primer at the InDel locus by using the InDel locus information obtained in the step (1):
according to the design principle of the InDel marker, designing a primer at the InDel difference site, wherein the base sequence of the primer pair is as follows: SEQ ID NO.1 (F1): 5'-ACGAGTGGGATCGATACTG-3';
SEQ ID NO.2(R1)：5’-GGTAGGACACCTGCCATCA-3’；
(3) PCR amplification was performed using total DNA of the rice blast resistant variety carrying the rice blast resistant gene Pik-zh as a template:
in this example, 6 representative rice varieties were selected, in order: flower 11, shuhui 498, CO39, nippon Tetepu, lijiang New-Paddy, and Tetepu;
the primers are used for carrying out PCR amplification by taking the total DNA of the 6 rice varieties as templates, and the PCR product obtained after the conventional PCR amplification is the specific molecular marker Pik-zh-InDel of the rice blast resistance gene Pik-zh gene;
wherein, the PCR amplification reaction system is as follows:
2x Reaction Mix:16μL；
primer SEQ ID NO.1 (F1) (10. Mu.m): 1 μl;
primer SEQ ID NO.1 (F1) (10. Mu.m): 1 μl;
DNA template (20-50 ng/. Mu.L) 2. Mu.L;
ddH 2 o: make up to 25 μl;
the PCR temperature cycle conditions were as follows: 94 ℃ for 5 minutes; 94 ℃ for 30 seconds; 55 ℃ for 30 seconds; 30 seconds at 72 ℃;35 cycles; 7 minutes at 72 ℃; preserving at 10 ℃;
after the PCR reaction is finished, 10-20ul of PCR sample products are taken to carry out electrophoresis detection on 3% agarose, the electrophoresis condition is 110V,40 minutes, after the electrophoresis is finished, a gel imaging system is utilized to carry out photographing and observe the electrophoresis result, and the fragment with the expected target size is positive, and the fragment without the target size or the fragment with the target size is negative.
Example 2
The application of the functional specificity molecular marker of the rice blast disease resistance gene Pik-zh in identifying rice blast resistance genes of rice varieties comprises the steps of firstly carrying out PCR amplification on genomes of 6 rice varieties to be detected by using a primer F1 shown in SEQ ID NO.1 and a primer R1 shown in SEQ ID NO. 2; secondly, detecting by agarose electrophoresis, if a nucleotide fragment with the molecular weight of 108bp is detected in the PCR product, the rice variety to be detected carries a rice blast resistance gene Pik-zh; if no nucleotide fragment with the molecular weight of 108bp is detected, the rice variety to be detected does not carry the rice blast resistance gene Pik-zh.
Referring to FIG. 1, amplification was performed using the primers F1 shown in SEQ ID NO.1 and R1 shown in SEQ ID NO.2 provided in example 1, with the total DNA of 6 selected representative varieties as a template; referring to FIG. 1, lane 1 is a DNA ladder, lane 2 is a fragment obtained by PCR using a flower 11 genome in a Pik-zh donor rice variety as a template, and the fragment is a specific band of a rice blast resistance gene Pik-zh, namely lane 2 is a Pik-zh-InDel gene specific molecular marker Pik-zh, and the fragment size is 108bp; the DNA templates of lanes 3-7 were, in order, shuhui 498, CO39, nippon Hei and tertepu; as shown in figure 1, the electrophoresis detection strip of PCR amplified products of the rice variety carrying the Pik-zh gene is shown as 108bp, the Zhonghua 11 and the tertepu contain the Pik-zh gene, the functional genes of the Pik-zh gene are not contained, the 108bp strip is not detected by electrophoresis, and the Shuhui 498, CO39, nippon and Lijiang new-cluster black grains do not contain the Pik-zh gene.
As shown in figure 1, the test results are identical with the design analysis, and the functional specificity molecular marker of the resistance gene Pik-zh can identify the rice blast resistance gene Pik-zh, is suitable for germplasm resource screening of Pik-zh under agricultural production practice and any genetic background, and greatly improves breeding efficiency and accuracy through transgenic breeding, gene polymerization and resistance breeding based on MAS technology.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.
Sequence listing
<110> institute of Rice at the national academy of agricultural sciences of Fujian province
<120> functional specific molecular markers of rice blast resistance gene Pik-zh and application thereof
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ggtaggacac ctgccatca 19
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gggagcagtg aaaacattgc gggcagcagt gggacgagtg ggatcgatac tgagcctgca 60
caggcacagc atgataatct ccctgctgtt cgagatgact acaagggaaa agggattctt 120
cttgatggca ggtgtcctac ctgcggccga gcgactaaaa ttgaagagga aac 173
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atggaggcgg ctgccatggc cgtaaccgca gccacggggg ccttggcgcc cgtgctagtg 60
aagctggccg ctttgctgga cgacggggag tgcaatcttc tggaggggag ccggagcgac 120
gcagagttca tcagatccga gctggaggcc gttcattctc tcctcacccc aaatatcttg 180
gggaggatgg gggatgacga tgcggcgtgc aaggatggct tgattgcgga ggtccgggag 240
ctgtcctacg acctggatga tgccgtcgac gacttcttgg agctcaattt cgagcagcga 300
agaagcgcaa gccctttcgg tgagctcaag gcaagagttg aggagcatgt ctccaatcgc 360
ttctctgact ggaagctacc ggcggcgagc cttccgccgt cgtcggtaca ccgccgagct 420
ggcttgccgc caccagatgc agagctggtg gggatggaca aacgtatgga agagctcacc 480
aaattgctgg aacaagggag caatgatgct tcacgatggc gcaagcgaaa accgcatttc 540
ccgctcagaa aaacagggct aaaggtacgg ttggatctcg attcttcaca aatatagctt 600
cctccgatag cagtgccaac gaatttgttt acttctctct tgattcttta atttggaagt 660
actgtataac aaacatggag ggatcgtcat ttaacttaat tttttttgtt gcagcaaaaa 720
atcgtgatca aggttgccat ggagggcaac aattgccgtt caaaagcaat ggctttagtt 780
gcgagcactg gaggagtgga ctcggttgcg ctcgcaggtg atctaagaga caagatagag 840
gtggtcggtt atggcattga ccccatcaag ctgatctccg cgctccggaa gaaggtgggc 900
catgcggagt tgctgcaggt cagccaagca aaaaaagatg tgaaggagac gacgccgatg 960
cttgcgccgg tgaaatccat atgtgaattt cacaaggtca aaacagtttg catccttgga 1020
ttgccaggtg gaggcaaaac aacggttgcc agagaattat atgacgcctt gggaacgcac 1080
ttcccatgcc gggttttcgt gtcagtctct ccaagttcta gtcccagtcc caatctcaca 1140
aagactcttg cagacatttt cgctcaagca caactaggag taactgatac acttagcaca 1200
ccatatggtg ggagtgggac cgggagagct cttcaacaac atctcatcga caacatatca 1260
gctttcctcc tcaacaaaaa gtaagcaata acttatatct cttatctagc ttgtccttaa 1320
tccggcttcc caaattaaag tgaacaggct atatgtccat ataatgttgg gttttttttt 1380
tctgtcctcg atgaggtata tatcaagata tgccaaactt tttttaatta gcgttacttt 1440
attctaccta gtaaatagat acataaatgg ggcaggcccc tcctttgtga caggccaatg 1500
gttttatgat gtggaaacaa ctcccttatc actgaagctc ttgctattac tcgtgatggt 1560
actgatcttg caaaggcttg cagtatttgt aaatccacag ggagtggtca tctttttttt 1620
ttaagataat gaatagaaat ctggcctcta tatagaaagc caaaggttac acaagtgcat 1680
acactccaaa tctcaaagct gagaaacaca aaaaacaaac gaccaaaaag aaaagacgat 1740
aagattaagt ttcaagcttt tcctccttgc ttcaaaaaac agggaaatgc agagctagcc 1800
cgctcttccc ttgccatgcc ctctcactgc cattgcaact accacaaaag ctcccctgtt 1860
tcgccgtgag cgaagggagg ggatggaact agtctgccgc tttgttgtcg ccaagcactg 1920
gtttcatgct gcccaaacaa cctatgcccc gctagcgata acgagtctgt tgccgccatg 1980
ctgttgtcgc tggagagcac cacttgtcgc tgctcagcta tgtgttaagt aagctactaa 2040
caatctctcc attctcttgt agactgctta taaaagattg tacttcaact tttgattaat 2100
gaaatagagt tcctgcctta caacaacaag aacgagagag agagagagag ccaatatcac 2160
gagttagcac tgtgaagact tcctgactgt tatgtcttca agaacagtat aaaagcattt 2220
gccactataa aaatatgata gccctcaaga tcattaaaaa tatgtcatgt tcttttacct 2280
tcataacaac cagtatcaca aatatgaaaa catttaaagg aaaatcagta tttgccattt 2340
atgtaaagaa aaactagact agaactctgg gcttttattt tagtgcacac tgtatacatg 2400
tgtgttcttt gacgtatata cttgatatgt tttcaaaaac ttttgggggc atatctgtaa 2460
cccatgcccc atgcatccaa agtagtagtg agacaacttt cgtatcaatg gacatgtggc 2520
attaaattta atcccaatat ttgttctctc gaagtcctga acatataatt ggacaaaatt 2580
atataattta attaggccaa ttgagccata tatacacaca ctataagaaa cttctacaaa 2640
acaaacattc gctcctttta ggttttgacc agtctataca tgaaccagcc aacggtgtgc 2700
tggtctgact ataaatgttt ttttagagag taaaggcacc ttcaagtacc tctctcgagg 2760
acagtaaaaa cacccatgct ttaaggttat gaacaacaaa gcatagaaca accacaagaa 2820
aaaaaaacaa ctagaacaag ttctggaact agttagggat tcgggaagcc attgcttgct 2880
ggcttgggac aagctatgcc tgccattgga ctatggtgga gactgggcat aaaggatctc 2940
aatctcttgg agattggcct atgtcctagg tggccatggc tgactgcaag gagatatatt 3000
tcttgcaacg gaaattttca ttgcaaaagc tgaaaatcgt tgcgatatgt acctattgtg 3060
acataattga atccattggg agccgtagca ataaatcccg tggtaatagg ctattgcaat 3120
gattttccga tggttgcata aaaaggtgct attacaatgg ttttgcattg ctattgcaac 3180
aacttgatat gtcataggta tttattgcaa cagtttgtac catgctgcaa taagatttcg 3240
caacacagac ctattcatta aatagcctat ttccacacat tcatttttgt ggcaataatt 3300
cagtgcgaca agcatgttct atagctataa cctgatgcaa cgggttagac ttgttgcagt 3360
aacttattag cacatttaat tttttactaa atttatattg attcaactat ttcaacatca 3420
ttcaatacag taaatactgg aaaatgcaag caatctccaa aaattcacca ataagagaaa 3480
cgaattaaat catatacaca attatttcac accgtacagt gcacttaagt gtaggaaata 3540
taatgctata tacatttcca atcctcaata caggttctgt aaataatgaa aagaaacgtc 3600
aatttgattt cttgaccggt ccctctgaga cacacagtgc tttcctcagc aacatcgttg 3660
ccgatctatg taacttgtaa actccttgtg cttaataaat tctggccagg gttacttcag 3720
cctgcatggc gaaaaaaaag aacttgttag ggagatcgca aacaaatgtt agctgcccca 3780
tttcttctct actgaccata aaagctctag gatcaggtca tgaaaacaat gccgtcagac 3840
cggaaatact caagattcag aacacgactc ctaggaactg caagtttaaa tttcaacaaa 3900
agcaaaatac aaccagatga caaactaata tatatcagct gactgttcat ctcaagtgga 3960
ctcacatctg tgaaattgtt agttctatgc atatatgcta atgtaatact aatatgctat 4020
tgaggcttat tactcttctc gacttttata ggtatctcat tgtaatcgat gacatttggc 4080
attgggaaga atgggaagtc atcagaaagt ccattcccaa gaatgatctg ggtggtagaa 4140
taatcatgac tactcgtctt aattcaatag ctgagaagtg ccacactgat gacaatgatg 4200
tttttgtcta cgaagttggg gatctagata ataatgatgc tttgtcgttg tcttggggga 4260
tagcaacaaa gtctggggca ggcaacagga tcggaactgg agaggataat ccatgctatg 4320
atattgtgaa catgtgttat ggtatgcctt tagcacttat ttggctgtcg tcagcattgg 4380
ttggagagat agaagaatta ggtggtgctg aagtgaaaaa atgtagggat ttgagacaca 4440
tagaggatgg tattttggac atcccatcct tacaaccatt ggcggagagt ttatgccttg 4500
gttataacca tcttcctctt tatctgagga ctttgttgtt gtactgtagt gcataccatt 4560
ggtctaacag aatcgaaagg ggtcgtctgg tcaggaggtg gattgcggaa ggatttgtgt 4620
cggaagagaa agaagcagaa ggttactttg gcgagcttat tgacagagga tggattacgc 4680
agcacggaga caacaacagt tataattact atgagatcca ccccgtgatg ctggccttcc 4740
tgagatgcaa gtccaaggag tacaattttt taacatgctt gggtctggga tctgatacta 4800
gtactagtgc atcctcccca aggttgattc gccggctgtc tcttcagggg gggtatccag 4860
tggactgctt gtcaagcatg agtatggatg tgtcacacac ttgcagcctt gtcgtccttg 4920
gtgacgtggc gcgatccaag ggaatcccct tctatatgtt taagcgcttg cgagtgttgg 4980
accttgaaga taataaggat atacaggatt ctcatctgca gggcatatgt gaacagttaa 5040
gcctcagagt gaggtacctt ggtctcaagg gaacgcggat ccgaaagctc cctcaggaga 5100
tgaggaagct gaagcatttg gagattttgt atgtggggag cactcggatc agtgaacttc 5160
cgcaagagat tggagagctg aagcatctgc ggattctgga cgtgagaaac acggacatca 5220
ctgagctccc actgcagata cgggagctgc agcatctgca cactctggac gtgaggaaca 5280
cgccaatcag tgagctcccg ccgcaggttg gcaagctgca gaatctcaag attatgtgcg 5340
tgaggagcac tggggttagg gagctcccaa aggagattgg ggagctgaat catctacaga 5400
ctctggacgt gagaaacacg agggtgagag agctgccatg gcaagctggc cagatctccc 5460
aatcgttgcg cgtgcttgcc ggtgacagtg gcgatggcgt gcggttgccc gaaggcgtct 5520
gcgaagctct gatcaacggt attccagggg ctacgcgtgc aaaatgcagg gaggttctgt 5580
ccatcgcgat catcgatcgt ttcggacctc cccttgttgg gatattcaaa gttcccggca 5640
gtcatatgcg tatcccgaag atgatcaaag accacttccg cgttctttct tgcctagaca 5700
tcaggctctg ccacaagctt gaggatgatg accaaaagtt cctcgccgag atgcccaacc 5760
tgcagacgct cgtgctgagg ttcgaggccc taccaagaca acccataacc atcaacggca 5820
caggcttcca gatgctggag agcttccgtg tcgacagccg ggtgccaagg atagccttcc 5880
atgaagacgc catgcccaac ctcaagcttc tcgagttcaa gttctacgcc ggcccagcaa 5940
gcaacgatcc catcggcatc accaacctga agagcctcca aaaggtggtc tttcggtgct 6000
cgccatggta caagagcgac gcccctggca tcagcgccac cattgacgtc gtgaagaaag 6060
aagccgagga gcatcccaac cggccgatca ccctcctcat caatgctggg tataaggaga 6120
tatcaattga gtcacatggg agcagtgaaa acattgcggg cagcagtggg acgagtggga 6180
tcgatactga gcctgcacag gcacagcatg ataatctccc tgctgttcga gatgactaca 6240
agggaaaagg gattcttctt gatggcaggt gtcctacctg cggccgagcg actaaaattg 6300
aagaggaaac ccaagatcga gtagcagata ttgaaattca aacagaaact actagctag 6359

Claims (3)

1. The functional specificity molecular marker of the rice blast resistance gene Pik-zh is characterized in that: the functional specific molecular marker of the rice blast resistance gene Pik-zh is Pik-zh-InDel, and the molecular marker which is in a specific band type with the rice blast resistance gene Pik-zh is amplified from genome DNA of rice varieties carrying the rice blast resistance gene Pik-zh through primer pairs SEQ ID NO.1 and SEQ ID NO. 2; the nucleotide sequence of the genome DNA is SEQ ID NO.4; wherein, the nucleotide sequences of the primer pair SEQ ID NO.1 and SEQ ID NO.2 are shown as follows:
SEQ ID NO.1：5’-ACGAGTGGGATCGATACTG-3’；
SEQ ID NO.2：5’-GGTAGGACACCTGCCATCA-3’；
the rice variety carrying the rice blast resistance gene Pik-zh is flower 11 in the rice disease-resistant variety.
2. The functional specific molecular marker of rice blast resistance gene Pik-zh as claimed in claim 1, wherein: the rice blast resistance gene Pik-zh comprises a gene fragment SEQ ID NO.3 for partially encoding NBS-LRR protein.
3. Use of a functionally specific molecular marker of the rice blast resistance gene Pik-zh according to any one of claims 1 to 2 for the identification of rice blast resistance genes of rice varieties.
CN202111045221.7A 2021-09-07 2021-09-07 Functional specificity molecular marker of rice blast resistance gene Pik-zh and application Active CN113789404B (en)

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CN107937600A (en) * 2018-01-19 2018-04-20 福建省农业科学院生物技术研究所 A kind of rice blast resistance gene seat Pik functional genes molecular labeling and its application
CN111978387A (en) * 2020-08-26 2020-11-24 武汉大学 Rice blast resistance gene Pikg, encoding protein and application thereof
CN113106104A (en) * 2021-04-20 2021-07-13 福建农林大学 Rice blast resistance related gene OsNAC29 and application thereof

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CN105950747A (en) * 2016-06-02 2016-09-21 福建省农业科学院生物技术研究所 Rice blast resistance gene Pi1 functional specificity molecular marker and application thereof
CN107937600A (en) * 2018-01-19 2018-04-20 福建省农业科学院生物技术研究所 A kind of rice blast resistance gene seat Pik functional genes molecular labeling and its application
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