CN107435068A - The exploitation and application of blast resisting Pi2 gene specific molecular labelings - Google Patents

The exploitation and application of blast resisting Pi2 gene specific molecular labelings Download PDF

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CN107435068A
CN107435068A CN201710610688.9A CN201710610688A CN107435068A CN 107435068 A CN107435068 A CN 107435068A CN 201710610688 A CN201710610688 A CN 201710610688A CN 107435068 A CN107435068 A CN 107435068A
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金名捺
丘式浚
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SHENZHEN XINGWANG BIOLOGICAL SEED INDUSTRY Co Ltd
Shenzhen Institute of Molecular Crop Design
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Abstract

The present invention relates to a kind of blast resistingPi2The development and application of gene specific molecular labeling, belongs to agricultural biological technical field, and in particular to a kind of rice blast resistant genePi2Specific molecular marker high-resolution melting curve analysis（High‑Resolution Melting Curve Analysis,HRM）Rice blast resistance genePi2Specific molecular marker primer, and utilize the primer detection rice blast resistance genePi2Molecule labelling method.The present invention carries out the diminution of amplified fragments and the optimization of detection architecture to original mark, greatly improves the gene in molecular marker assisted selection breeding, gene pyramiding breeding, and the efficiency utilized in transgenic breeding.

Description

The exploitation and application of blast resisting Pi2 gene specific molecular labelings
Technical field
The invention belongs to agricultural biological technical field, and in particular to a kind of rice blast resistance gene Pi2 special molecular mark The high-resolution melting curve analysis (High-Resolution Melting Curve Analysis, HRM) of note, and utilize The method of the primer detection rice blast resistance gene Pi2.
Background technology
Rice is one of most important cereal crops in the world, there are about the world population of half using rice as staple food.By son Rice blast caused by capsule bacterium (Magnapothe oryzae) is the extensive important disease for occurring to influence Rice Production in each rice region in the world One of evil, can all bring about great losses to Rice Production every year.From the point of view of environmental protection and agricultural sustainable development, seed selection and kind It is preventing and treating rice blast most safely and effectively method to plant disease-resistant variety.Traditional paddy disease-resistant breeding depend critically upon Resistance Identification and The selection of plant phenotype, limited by breeder's experience and germ onset condition, it is easy to cause the loss of disease-resistant gene, select It is often relatively low to select efficiency.Molecular marker assisted selection is by the genotype of the molecular labeling with gene close linkage come to purpose base Because being selected, because it is not influenceed by environmental factor, Select gene reliability is high.In recent years, rice anti-rice blast base The clone of cause achieves impressive progress, and the exploitation of the molecular labeling of rice blast resistance gene is for cultivating anti-rice blast rice kind It is significant.
Up to the present, at least 9 rice blast resistance bases in the Pi2/9 gene clusters at rice Short arm of chromosome 6 end Because (Pi2, Piz, Piz-t, Pi40, Pigm, Pi9, Pi26, Pi50, Pi2-2) oneself through being positioned, these genes are considered to There is in terms of rice blast resistance breeding important application value (Jiang et al., 2012), wherein Pi9, Pi2, Piz-t and Pigm oneself through by successful clone (Qu et al.2006, Zhou et al.2006, Deng et al.2016).Pi2 genes are derived from Colombia's rice variety 5173, be at present cloned one important dominant broad-spectrum resistance gene of rice blast (Liu et al., 2002), scholar's Liu equality (2003) is inoculated with using 75 rice blast bacterial strains in the different regions source being collected into, and finds Pi2 Anti- spectrum may be up to 85.53%.Pi2 parent C101A51 is carried to coming from 13 national 43 rice blast bacterial strains 36 performance resistances (Liu et al, 2002).
Pi2 encodes a NBS-LRR albuminoid, and containing 2 intrones, length is 3839bp and 116bp respectively, Pi2's CDNA total lengths are 3332bp, include 117bp 5'UTR and 116bp 3'UTR.Pi2 albumen includes 1032 amino acid, contains 1 Nucleotide binding site (NBS) and 3 full asphalt mixture areas (LRR).9 resistance bases be present in the genome area where Pi2 Because of the candidate gene of feature, their very high homologies in sequence, the sequence identity on nucleotide level reaches 95.6%；In ammonia On base sour water is flat, the coded product that Pi2 and Pi9 sequence identity are up to 96%, Pi2 and Piz-t only has in 3 LRRs regions The difference of 8 amino acid；And in this 8 mutating acid, only 1 be located at xxLxLxx motifs in (Zhou et al., 2006)。
High-resolution melting curve analysis (High Resolution Melting, HRM) is one to grow up in recent years Kind SNP (Single Nucleotide Polymorphism, SNP) and mutation research technology, are by Utah What university and Edward scientific ＆ technical corporation developed cooperatively.It is expanded by monitoring temperature-rise period double center chain DNA fluorescent dyes in real time with PCR Increase production the combination situation of thing, to judge whether SNP, and different SNP sites, whether be that heterozygote etc. can all influence to melt The peak shape of curve, therefore HRM analyses can effectively distinguish different SNP sites and different genotype.This detection method is not by prominent Become the limitation of base position and type, directly operation high-resolution melts after PCR terminates, you can completes to sample genotype Analysis.For the detection method because its is easy to operate, quick, cost is low, without designing probe, as a result accurately, and realizes really Stopped pipe operation and by universal concern.In plant breeding, HRM is easily understood because its operation is analyzed with later data, Have in mutation scanning, Genotyping, the germplasm identification based on specific gene and function labeling development etc. wide Application prospect (Luo Wenlong etc., 2011).
Research finds that Pi2 is multiple allele with the Piz-t at the site and Pi9, according to the complete of Pi2, Pi9 and Piz-t Genome sequence, the SNP site being had differences by sequence analysis 3 multiple alleles of searching, is developed as based on HRM skills The gene specific molecular labeling of art detection, and high-throughout assisted Selection system in foundation, can further improve Pi2 in water Breeding Efficiency in rice resistance breeding.In our previous studies 201410817042.4, detection Pi2 base is developed Because of specific molecular marker, the PCR primer length of labeled primer amplification is 495bp, but in the detection work of reality, we It was found that the testing result of the primer is not very stable.Studies have reported that the effect of HRM parting of the amplified production within 200bp When most preferably, more than 200bp, the probability that mispairing occurs for amplified production can increase (Liew et al., 2004), that is, with expansion Increase production the increase of thing fragment, the Stability and veracity of HRM detections can be reduced gradually.Therefore, in order to increase the HRM of Pi2 genes The Stability and veracity of detection, the diminution and detection of our the necessary progress amplified fragments on the basis of original mark The optimization of system.
The content of the invention
Rice blast resistance gene Pi2 and Pi9, Piz-t are all the multiple allele on Piz sites, they all by into Work(clone (https://www.ricedata.cn/gene/list/87.htm, national rice data center), its coding region sequence Can download and obtain from NCBI DNA sequence data storehouse Genbank, indexed number be respectively DQ352453, DQ285630, DQ352040.These sequences are analyzed by Multiple Sequence Alignment software DNAMAN, 6 blast resistant gene Pi2's of discovery Specific SNP site (such as Fig. 1), they are located at Pi2 the 787th, the 788th, the 789th and the 792nd bit codon respectively (GCA GGA ATC TCA GAT GGT, are shown in the sequence in red block in Fig. 1, and overstriking represents Pi2 gene specific SNP), these Difference site significantly can make a distinction resistant gene Pi2 and predisposing genes and multiple allele Pi9, Piz-t.
In order to develop more stable detection blast resistant gene Pi2 HRM primer, we are with 201410817042.4 In primer Pi2-HRM the genomic DNA (such as table 1) of 12 parts of rice materials to be measured is expanded, by these amplification produce After the sequencing result of thing is compared, 6 blast resistant gene Pi2 specific SNP site both sides conservative region again Primer is designed, the sequence in Fig. 2 red blocks is Pi2 specific sequence, and we develop 1 left primer altogether and 6 right sides are drawn Thing, primer sequence are as follows：
Pi2-HRM2-1F：5'-GCAGCCATTGAGAAGCTCTC-3'(SEQ ID NO:1)；
Pi2-HRM2-1R：5'-AGGGGAGGAGGAGATGAAAT-3'(SEQ ID NO:2)；
Pi2-HRM2-2R：5'-GCTGCTCAATCCAGTTAGGC-3'(SEQ ID NO:3)；
Pi2-HRM2-3R：5'-CCCCAAGTATCAGCATGGTT-3'(SEQ ID NO:4)；
Pi2-HRM2-4R：5'-AGCTTCTCCCCAAGGTAAGC-3'(SEQ ID NO:5)；
Pi2-HRM2-5R：5'-TGTTCTAAGATTTGGGAATGCTC-3'(SEQ ID NO:6)；
Pi2-HRM2-6R：5'-TCTTTTCCAACAGGGGTGAG-3'(SEQ ID NO:7)；
The genomic DNA of 12 parts of rice materials to be measured is carried out with 6 right primers pairings respectively by left primer amplification and HRM is detected, and finds only have Pi2-HRM2-1F and Pi2-HRM2-3R primer pairs to enter well to 12 parts of rice materials to be measured Row is distinguished, i.e., the sequencing result of HRM genotyping result and amplified production is completely the same (such as Fig. 3 and Fig. 4).
The present invention provides a kind of method for quickly and accurately being identified and being detected blast resistant gene Pi2 using molecular labeling, A pair are provided simultaneously and combines the molecular labeling primer that HRM is used to detect blast resistant gene Pi2, and the primer is according to Pi2 genes The specific SNP designs of sequence, can combine the molecule assisted selection that HRM is used for blast resistant gene Pi2, be divided with improving The efficiency of sub- marker assisted selection and the efficiency of the identification of disease-resistant variety and seed selection.
The present invention provides following solution：
Combine HRM and be used to identifying and detecting blast resistant gene Pi2 molecular labeling primers for a pair, thereon, downstream sequence It is divided into：
Pi2-HRM2-1F：5'-GCAGCCATTGAGAAGCTCTC-3'(SEQ ID NO:1)；
Pi2-HRM2-3R：5'-CCCCAAGTATCAGCATGGTT-3'(SEQ ID NO:4)；
Above-mentioned primer is designed according to the specific SNP of Pi2 gene orders, and its gene specific SNP is located at Pi2 respectively (GCA GGA ATC TCA GAT GGT, add for the 787th, the 788th, the 789th of gene coding region and the 792nd bit codon It is thick to represent Pi2 gene specific SNP).
The nucleotide sequence of the amplified production of above-mentioned primer is following (blast resistant gene Pi2 specificity SNP is highlighted)：
5'-gcagccattgagaagctctcttccctccaatctctccatgtggatgctgcAGGaAtctcagatGGt ggaacacttgagtgcctagattctatttcatctcctcctcccctactgaggacactcgtgttggatggaattcttga ggagatgcctaactggattgagcagctcactcacctgaagaagatctacttattgaggagcaaactaaaggaaggta aaaccatgctgatacttgggg-3'(SEQ ID NO:8)
A kind of combination HRM identifications and detection rice blast resistant gene Pi2 method：12 parts are expanded using above-mentioned primer to treat The genomic DNA (such as table 1) of rice material is surveyed, takes amplified production to carry out high-resolution with HRM instrument Light Scanner 96 Melting curve collection analysis, it is found that the amplified production of 12 rice varieties is divided into 6 types (see Fig. 3), wherein orange Curve is No. 8 rice materials " China accounts for " and No. 11 rice materials " BL122 ".Previous studies show that they all contain rice blast Resistant gene Pi2." China accounts for " (it is disclosed, apply for notification number：CNA004577E), be using quoted from Malay SCO2-S6 as Basic material, the restorer formed by test cross repeatedly and systematic breeding, positive control material " China accounts for " of the invention is Guangdong Shanxi Academy of Agricultural Sciences Zhu little Yuan researcher presents.Then, our amplified productions to this 12 parts of rice materials carry out sequencing discovery, They are also divided into 6 types in sequence, and (see Fig. 4) completely the same with the classification results of HRM partings, these results are said Bright, rice material DNA combinations HRM to be measured method is expanded with primer Pi2-HRM2 can carry out rice blast resistance gene Pi2's Identification and detection.If the high-resolution melting curve and positive control of detected materials --- rice anti-rice blast kind " China accounts for " Melting curve is completely the same (such as Fig. 3), then rice anti-rice blast material to be measured contains blast resistant gene Pi2；Otherwise, wait to measure and monitor the growth of standing timber Blast resistant gene Pi2 is not contained in material.
Meanwhile we to other 23 widely used sterile lines and keep material to carry out Pi2 identified for genes, find them The gene is not all contained, wherein orange curve is positive control material " China accounts for " (see Fig. 5).Therefore, molecular labeling can be passed through The means of assisted Selection, with Pi2-HRM2 primer combinations HRM method, Pi2 gene trackings are imported into these sterile lines and guarantor In holding and being, so as to improve the resistance using these sterile lines and Hybrid Rice Combination that maintainer is parent to rice blast, to be high-quality, more Anti- and high yield high-efficient breeding lays the foundation.
A kind of anti-rice blast rice material molecule mark auxiliary selection method：With the rice containing blast resistant gene Pi2 Material (donor parents of this patent are that China accounts for) is parent, with other rice material (acceptor parents of this patent without Pi2 genes Originally have five rich B, PA64S, Huang Huazhan, military fortune round-grained rice 7) hybridized, extraction respectively each hybridizes backcrossing BC1F2Separate offspring's Genomic DNA, performing PCR amplification is entered to them with primer Pi2-HRM2.The high-resolution of the amplified production of each colony melts bent Line has all been divided into three types well：The expression offspring consistent with the melting curve (orange curve) of positive control " China accounts for " Contain Pi2 genes in individual；(five rich B --- red curve, PA64S --- greens are followed successively by with the melting curve of negative control Curve, Huang Huazhan --- blue curve, military fortune round-grained rice 7 --- cyan curve) Pi2 is not contained in consistent expression offspring individuals Gene；Different rice materials individual containing heterozygous Pi2 in each colony, its high-resolution melting curve be grey (such as Fig. 6, Fig. 7, Fig. 8, Fig. 9).Five rich B, PA64S, Huang Huazhan, military fortune round-grained rice 7 represent four kinds of single times without Pi2 genes respectively Type, analysis is carried out by offspring's genotype separation to them and finds that they all meet 1:2:1, with reference to above to rice material In whether the qualification result containing Pi2, illustrate molecular labeling Pi2-HRM2 HRM detection can be used at present three systems keep System, two-line sterile line, indica conventional rice and conventional japonica rice Pi2 genes genetic improvement process.
The DNA that above-mentioned primer Pi2-HRM2 can be extracted preferentially to rice seedling carries out molecular markers for identification, after detection " homozygous/heterozygous " blast resistant gene Pi2 whether is carried in generation individual, so as to not contain blast resistant gene Pi2's Individual is rejected, and is analyzed with reference to the background scans of full-length genome, can obtain importing blast resistant gene Pi2 donors simultaneously faster The minimum individual of parent's cultural introduction, so as to improve the efficiency of rice material improvement.In view of HRM detections require relatively low to PCR, can Quickly to prepare pcr template by simple crushing " tube method ", the detection efficiency of molecular labeling is improved.Simple crushing " tube method " The STb gene preferred process for extracting rice material is as follows：2-4cm blades are taken, directly add extract solution (10mM Tris-HCl, 0.5mM EDTA, 0.15M KCl) grinding, after boiling water bath 10min, stratification takes supernatant (1 μ L) to can be used as DNA profiling and enter performing PCR expanding Increase.
Present invention also offers a kind of PCR kit of the rice Pi2 genetic tests based on HRM technologies, it includes primer Pi2-HRM2-1F：5'-GCAGCCATTGAGAAGCTCTC-3'(SEQ ID NO:And Pi2-HRM2-3R 1)：5'- CCCCAAGTATCAGCATGGTT-3'(SEQ ID NO:4)；And PCR reagent and fluorescent dye, for detecting in rice material Whether Pi2 genes are contained.
Present invention also offers a kind of application method of kit, it the step of it is as follows：Extract sample to be tested DNA；Prepare PCR reaction systems comprising sample to be tested DNA, primer pair, PCR kit and EvaGreen fluorescent dyes enter performing PCR amplification and HRM analysis scannings are carried out to PCR primer.
The present invention is had the following advantages relative to prior art and effect：
(1) molecular labeling provided by the invention almost occurs without false positive：Traditional PCR amplifications combine restriction enzyme Detection method interpretation is come with " the whetheing there is " of electrophoretic band, often false positive is higher.Before digestion, if PCR primer without Purifying then easily causes restriction enzyme enzymatic activity to reduce and can not cut so as to cause false positive；If PCR primer carries out pure Changing then easily causes the amount of recovery amplified fragments extremely low or even disappears, and influences the interpretation of digestion rear electrophoresis result and causes false sun Property.Molecular labeling provided by the invention great simplicity and sensitivity, PCR primer in detection directly carry out high-resolution melting Curve gathers, and reaches " zero loss ", its testing result almost occurs without false positive；
(2) molecular labeling provided by the invention has inexpensive, high-throughout feature in actual applications：At present, high score Resolution melting curve analysis method is to be only second to the middle high flux SNP detection method of DNA chip, can disposably be adopted in 10min The high-resolution of 96 PCR primer samples of collection melts, hence it is evident that more simple and efficient than traditional restriction enzyme digestion and electrophoresis detection method；And should The PCR system of molecular labeling only needs 10 μ l, is the half of regular-PCR system, therefore even if needs the saturation of 0.04 yuan/system glimmering Photoinitiator dye, its cost are also not above regular-PCR, in the production practices especially suitable for large-scale molecular assisted selection；
(3) molecular labeling provided by the invention can significantly decrease the human cost and time cost of pcr template preparation： In actual applications, requirement of the SNP detection method of traditional regular-PCR combination restriction enzyme to pcr template purity be very Height, generally require the extracting method to be wasted time and energy using CTAB etc..The molecular labeling of the present invention is less demanding to pcr template, leads to Cross the extract solution that simple crushing " tube method " obtains and can be used as pcr template, substantially increase the simplicity of Molecular Detection.
(4) what this method detected is the variation of Indel or SNP classes, and this kind of variation frequency in individual is very high, therefore can To be easier to find the molecular labeling for being suitable as tracking gene, the efficiency of molecular marker assisted selection breeding is greatly improved.
Therefore, combination HRM provided by the present invention is used for the molecular labeling primer for detecting blast resistant gene Pi2, Yi Jili Had broad application prospects with the molecule labelling method of the primer detection blast resistant gene Pi2 in production practices, profit Utilization ratio of the gene in molecular marker assisted selection breeding can be improved with this mark.
Brief description of the drawings
Fig. 1, Multiple Sequence Alignment software DNAMAN analyze resistant gene Pi2 and multiple allele Piz-t, Pi9 gene sequence Row uniformity (specific sequence that resistant gene Pi2 is represented in red block).
The gene that Fig. 2, Multiple Sequence Alignment software BioEdit analyze resistant gene Pi2 in 12 parts of rice materials to be detected is special The conservative (specific sequence that resistant gene Pi2 is represented in red block) of different in nature site both sides.
(red curve represents No. 1 " treasure to the high-resolution melting curve of Fig. 3, primer Pi2-HRM2 12 rice varieties of detection " the high-resolution melting curve for the Pi2 genes that normal S " contains, Grey curves represent No. 2 " the rich B " in day and No. 4 for Shan 97B " and No. 3 " the high-resolution melting curve for the Pi2 genes that the rich S " of Lay contains, blue curve represent No. 5 " five rich B ", No. 10 " Gu Mei tetra- " The high-resolution melting curve of the Pi2 genes contained with No. 12 " Huang Huazhan ", green curve represent No. 6 " PA64S " and No. 7 The high-resolution melting curve for the Pi2 genes that " P88S " contains, orange curve represent No. 8 " China accounts for " and No. 11 " BL122 " and contained Pi2 genes high-resolution melting curve, cyan curve represents the high-resolution for the Pi2 genes that No. 9 " forces fortune round-grained rice 7 " are contained Melting curve, figure top represent melting curve, and grid represents the detection case of counter sample Pi2 genes).
Fig. 4, Multiple Sequence Alignment software BioEdit analyze the gene order of 12 parts of rice material amplified productions to be detected.
Fig. 5, primer Pi2-HRM2 detect 23 rice sterile lines and keep the high-resolution melting curve of based material (orange Curve represents the high-resolution melting curve of the Pi2 genes of positive control material " China accounts for ", Grey curves and red curve equal generation The high-resolution melting curve of Pi2 genes in other 23 rice materials to be measured of table, figure top represent the melting curve of standardization, Grid represents the detection case of counter sample Pi2 genes).
Fig. 6, primer Pi2-HRM2 detection " five rich B " backcrossings " China accounts for " BC1F2The high-resolution melting curve of segregating population (orange curve represents positive control " China accounts for " and hybridization F2Melted for the individual high-resolution containing Pi2 genes in segregating population Solution curve, red curve represent negative control " five rich B " and hybridization F2For the individual height that Pi2 genes are not contained in segregating population Resolution ratio melting curve, Grey curves represent hybridization F2For the individual high-resolution containing heterozygous Pi2 genes in segregating population Rate melting curve, figure top represent the melting curve of standardization, and grid represents the detection case of counter sample Pi2 genes).
Fig. 7, primer Pi2-HRM2 detect " PA64S " backcrossing " China accounts for " BC1F2The high-resolution melting curve of segregating population (orange curve represents positive control " China accounts for " and hybridization F2Melted for the individual high-resolution containing Pi2 genes in segregating population Solution curve, green curve represent negative control " PA64S " and hybridization F2For the individual height that Pi2 genes are not contained in segregating population Resolution ratio melting curve, Grey curves represent hybridization F2For the individual high-resolution containing heterozygous Pi2 genes in segregating population Rate melting curve, figure top represent the melting curve of standardization, and grid represents the detection case of counter sample Pi2 genes).
Fig. 8, primer Pi2-HRM2 detect " Huang Huazhan " backcrossing " China accounts for " BC1F2The high-resolution melting curve of segregating population (orange curve represents positive control " China accounts for " and hybridization F2Melted for the individual high-resolution containing Pi2 genes in segregating population Solution curve, blue curve represent negative control " Huang Huazhan " and hybridization F2For not containing the individual of Pi2 genes in segregating population High-resolution melting curve, Grey curves represent hybridization F2For the individual high score containing heterozygous Pi2 genes in segregating population Resolution melting curve, figure top represent the melting curve of standardization, and grid represents the detection case of counter sample Pi2 genes).
Fig. 9, primer Pi2-HRM2 detection " force fortune round-grained rice 7 " backcrossing " China accounts for " BC1F2The high-resolution of segregating population melts bent (orange curve represents positive control " China accounts for " and hybridization F to line2For the individual high-resolution containing Pi2 genes in segregating population Melting curve, cyan curve represent negative control " force fortune round-grained rice 7 " and hybridization F2For that Pi2 genes are not contained in segregating population The high-resolution melting curve of body, Grey curves represent hybridization F2For containing the individual of heterozygous Pi2 genes in segregating population High-resolution melting curve, figure top represent the melting curve of standardization, and grid represents the detection feelings of counter sample Pi2 genes Condition).
Figure 10, primer Pi2-HRM detect " PA64S " backcrossing " China accounts for " BC1F2The high-resolution melting curve of segregating population (orange curve represents positive control " China accounts for " and hybridization F2Melted for the individual high-resolution containing Pi2 genes in segregating population Solution curve, green curve represent PA64S and hybridization F2Melted for the individual high-resolution that Pi2 genes are free of in segregating population bent Line).
Embodiment
Further detailed description is done to the present invention with reference to embodiment and accompanying drawing, but embodiments of the present invention are unlimited In this.Method therefor is the side described in conventional molecular biology, tissue culture technique and agronomy handbook unless otherwise instructed Method.Specific steps can be found in：《Molecular Cloning:A Laboratory Manual(3rd edition)》 (Sambrook, J., Russell, David W., 2001, Cold Spring Harbor),《Plant Propagation by Tissue Culture》(Edwin F.George,Michael A.Hall,Geert-Jan De Klerk,2008, Springer)。
Embodiment 1：The comparison of Pi2 allele coding region sequences and specific snp analysis
Rice blast resistance gene Pi2 and Pi9, Piz-t are all the multiple allele on Piz sites, they all by into Work(clone (https://www.ricedata.cn/gene/list/87.htm, national rice data center), its coding region sequence Can download and obtain from NCBI DNA sequence data storehouse Genbank, indexed number be respectively DQ352453, DQ285630, DQ352040.These sequences are analyzed by Multiple Sequence Alignment software DNAMAN, 6 blast resistant gene Pi2's of discovery Specific SNP site (such as Fig. 1), they are located at Pi2 the 787th, the 788th, the 789th and the 792nd bit codon respectively (GCA GGA ATC TCA GAT GGT, are shown in the sequence in red block in Fig. 1, and overstriking represents Pi2 gene specific SNP), these Difference site significantly can make a distinction Pi2 resistant genes and predisposing genes and multiple allele Pi9, Piz-t.
In our previous studies 201410817042.4, detection Pi2 gene specific molecule mark is developed Note, the PCR primer length of labeled primer amplification is 495bp, but in the detection work of reality, it has been found that the inspection of the primer It is not very stable to survey result, and cannot distinguish between that resistance is homozygous and heterozygous, as a result sees Figure 10.Studies have reported that amplified production The best results of HRM partings within 200bp, during more than 200bp, the probability that mispairing occurs for amplified production can increase (Liew Et al., 2004), that is, the increase with amplified production fragment, the Stability and veracity of HRM detections can be reduced gradually. Therefore, in order to increase the Stability and veracity of the HRM of Pi2 genes detections, we are necessary on the basis of original mark Carry out the diminution of amplified fragments and the optimization of detection architecture.
Embodiment 2：Pi2 specific Functions molecular labeling primer designs to be detected with HRM
(1) design of primers
According to the design principle of HRM primer, primer is separately designed in Pi2 specificity SNP upstream and downstream, we are altogether 1 left primer and 6 right primers are developed, primer sequence is as follows：
Pi2-HRM2-1F：5'-GCAGCCATTGAGAAGCTCTC-3'(SEQ ID NO:1)；
Pi2-HRM2-1R：5'-AGGGGAGGAGGAGATGAAAT-3'(SEQ ID NO:2)；
Pi2-HRM2-2R：5'-GCTGCTCAATCCAGTTAGGC-3'(SEQ ID NO:3)；
Pi2-HRM2-3R：5'-CCCCAAGTATCAGCATGGTT-3'(SEQ ID NO:4)；
Pi2-HRM2-4R：5'-AGCTTCTCCCCAAGGTAAGC-3'(SEQ ID NO:5)；
Pi2-HRM2-5R：5'-TGTTCTAAGATTTGGGAATGCTC-3'(SEQ ID NO:6)；
Pi2-HRM2-6R：5'-TCTTTTCCAACAGGGGTGAG-3'(SEQ ID NO:7)；
The genomic DNA (such as table 1) of 12 parts of rice materials to be measured, pcr amplification reaction system are expanded using above-mentioned 6 pairs of primers It is as follows：
10×PCR Buffer(Mg2+plus)：2μL
dNTPs(2.5mM each)：0.4μL
Pi2-HRMF(10μM)：0.4μL
Pi2-HRMR(10μM)：0.4μL
TakaRa TaqTM(5U/μL)：0.2μL
DNA profiling (50-100ng/ μ L)：1μL
ddH2O：Complement to 20 μ L.
PCR Thermal cycling conditions are as follows：94 DEG C 3 minutes；94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；72 DEG C 2 minutes.
Sequencing result is analyzed (see Fig. 3 and Fig. 4)：In the amplified production of 6 pairs of primers, only molecular labeling primer is to Pi2- HRM2-1F and Pi2-HRM2-3R amplified production sequencing result and HRM genotyping results is completely the same, and the upstream of the primer pair is drawn Thing is at -50bp to -31bp, and anti-sense primer is at 157bp to 176bp.
Table 1, the variety name for Pi2 Polymorphism Analysis
(2) HRM is detected
By Pi2 specific Function molecular labeling primer Pi2-HRM2 to the genomic DNAs of 12 parts of rice materials to be measured (such as Table 1) expanded, pcr amplification reaction system is as follows：
Pcr amplification reaction system is as follows：
10×PCR Buffer(Mg2+plus)：1μL
dNTPs(2.5mM each)：0.1μL
Pi2-HRMF(10μM)：0.1μL
Pi2-HRMR(10μM)：0.1μL
20×EvaGreen：0.1μL
TakaRa TaqTM(5U/μL)：0.1μL
DNA profiling (2-3ng/ μ L)：1μL
ddH2O:Complement to 10 μ L.
In order to prevent system from evaporating, need that the covering of 15 μ L-20 μ L mineral oil is added dropwise before PCR amplifications.
PCR Thermal cycling conditions are as follows：94 DEG C 3 minutes；94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 10 seconds, 40 circulation；72 DEG C 1 minute；95 DEG C 1 minute；25 DEG C are transferred to 4 DEG C of refrigerators after 1 minute and preserve.
After PCR terminates, product is fully transferred to 96 hole PCR plates of white background black surround.In order to facilitate the orifice plate of subsequent correction 96 Temperature difference between hole, each hole add 1 μ L temperature internal reference (Internal Temperature Controls) and add 15 μ L- After the covering of 20 μ L mineral oil, you can carry out the collection of high-resolution melting curve and analysis.
Described temperature internal reference base sequence is as follows：
InTemF：5 ＇-
ATCGTGATTTCTATAGTTATCTAAGTAGTTGGCATTAATAATTTCATTTT-3 ＇ (SEQ ID NO:9)；
InTemR：5 ＇-
AAAATGAAATTATTAATGCCAACTACTTAGATAACTATAGAAATCACGAT-3 ＇ (SEQ ID NO:10)；
After above-mentioned complementary oligonucleotide InTemF and InTemR synthesis, 800 μ LddH of 1OD amount2O melts, then Reaction system is as follows：
Saturation NaCl：1μL
InTemF：1μL
InTemR：1μL
ddH2O:Complement to 10 μ L.
After being denatured 3 minutes under the conditions of 95 DEG C, room temperature renaturation is naturally cooled to, you can as temperature internal reference.
HRM genotyping results are analyzed (see Fig. 3)：Amplified production is taken to carry out high-resolution with HRM instrument Light Scanner 96 Rate melting curve collection analysis, 6 types (see Fig. 3) can be divided into well by finding the amplified production of 12 rice varieties, its In orange curve be No. 8 rice materials " China accounts for " and No. 11 rice materials " BL122 ".Previous studies show that they all contain There is rice blast resistance gene Pi2." China accounts for " (it is disclosed, apply for notification number：CNA004577E), it is with quoted from Malay Material based on SCO2-S6, the restorer formed by test cross repeatedly and systematic breeding, positive control material of the invention " China Account for " presented for Guangdong Academy of Agricultural Sciences Zhu little Yuan researcher.The sequencing result of 12 parts of rice materials to be detected and HRM partings Classification results are completely the same (see Fig. 3 and Fig. 4), illustrate the side that rice material DNA combinations HRM to be measured is expanded with primer Pi2-HRM2 Method can carry out rice blast resistance gene Pi2 identification and detection.If the high-resolution melting curve and the positive of detected materials are right According to --- the melting curve of rice anti-rice blast kind " China accounts for " is completely the same (such as Fig. 3), then rice anti-rice blast material to be measured Contain blast resistant gene Pi2；Otherwise, blast resistant gene Pi2 is not contained in detected materials.Therefore, rice restorer " China Account for " can be as the donor kind of homozygous blast resisting Pi2 genes, and " China accounts for " filial generation material can be used as the anti-rice of heterozygous The positive control material of seasonal febrile diseases Pi2 genes.
Embodiment 3：Applied analysis of the Pi2 specific Function molecular labelings in assistant breeding
To producing upper widely used sterile line and keeping material to carry out Pi2 identified for genes, it is found that they all do not contain this Gene, wherein blue curve are positive control material " China accounts for " (see Fig. 5), with reference to Fig. 3 and Fig. 4 result, can pass through molecule The means of marker assisted selection, the method detected with Pi2-HRM2 primer combinations HRM, Pi2 gene trackings are imported into and not contained The high-quality conventional Rice of the gene, sterile line and keep in based material, so as to improve wait to improve high-quality conventional Rice and with these not Educate be with maintainer for parent Hybrid Rice Combination to the resistance of rice blast, established for high-quality, more anti-and high yield high-efficient breedings Basis.
With the rice material (donor parents of the invention are that China accounts for) containing blast resistant gene Pi2 for parent, with other Rice material (receptor parent of this patent has five rich B, PA64S, Huang Huazhan, military fortune round-grained rice 7) without Pi2 genes carries out miscellaneous Hand over, each hybridization is returned BC for extraction respectively1F2The genomic DNA of offspring is separated, performing PCR is entered to them with primer Pi2-HRM2 and expanded Increase.The high-resolution melting curve of the amplified production of each colony has been divided into three types well：With positive control " China Account for " the consistent expression offspring individuals of melting curve in contain Pi2 genes；The table consistent with the melting curve of negative control Show and Pi2 genes are not contained in offspring individuals；Different rice materials individual containing heterozygous Pi2, its high-resolution in each colony Rate melting curve is grey (such as Fig. 6, Fig. 7, Fig. 8, Fig. 9).And developed with our previous studies 201410817042.4 Detection Pi2 gene specific molecular labeling Pi2-HRM sample in Fig. 7 is detected, it is found that it can not be regional very well Point resistance is homozygous and heterozygous, as a result sees Figure 10.Different rice materials individual containing heterozygous Pi2 in each colony, its High-resolution melting curve is consistent.Four kinds of rice materials to be improved in this patent represent four kinds and are free of Pi2 genes respectively Haplotype, find that they all meet 1 by carrying out analysis to the separation of their offspring's genotype:2:1 segregation ratio, with reference to Above in rice material whether the qualification result containing Pi2, illustrate molecular labeling Pi2-HRM2 HRM detection can be used for mesh Preceding is the genetic improvement process of the Pi2 genes of maintainer, two-line sterile line, indica conventional rice and conventional japonica rice three.
SEQUENCE LISTING
<110>Shenzhen Crop Molecular Design Breeding Institute
Shenzhen Xingwang Biological Seed Industry Co., Ltd.
<120>The exploitation and application of blast resisting Pi2 gene specific molecular labelings
<130>
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 1
gcagccattg agaagctctc 20
<210> 2
<211> 20
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<213>It is artificial synthesized
<400> 2
aggggaggag gagatgaaat 20
<210> 3
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 3
gctgctcaat ccagttaggc 20
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<213>It is artificial synthesized
<400> 4
ccccaagtat cagcatggtt 20
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<211> 20
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<213>It is artificial synthesized
<400> 5
agcttctccc caaggtaagc 20
<210> 6
<211> 23
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<213>It is artificial synthesized
<400> 6
tgttctaaga tttgggaatg ctc 23
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<211> 20
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<400> 7
tcttttccaa caggggtgag 20
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<213> Oryza sativa
<400> 8
gcagccattg agaagctctc ttccctccaa tctctccatg tggatgctgc aggaatctca 60
gatggtggaa cacttgagtg cctagattct atttcatctc ctcctcccct actgaggaca 120
ctcgtgttgg atggaattct tgaggagatg cctaactgga ttgagcagct cactcacctg 180
aagaagatct acttattgag gagcaaacta aaggaaggta aaaccatgct gatacttggg 240
g 241
<210> 9
<211> 50
<212> DNA
<213>It is artificial synthesized
<400> 9
atcgtgattt ctatagttat ctaagtagtt ggcattaata atttcatttt 50
<210> 10
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<213>It is artificial synthesized
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aaaatgaaat tattaatgcc aactacttag ataactatag aaatcacgat 50

Claims (10)

  1. It is 1. a kind ofPi2The detection primer of gene, it is characterised in that the forward primer of the primer is 5'- GCAGCCATTGAGAAGCTCTC -3', reverse primer are 5'- CCCCAAGTATCAGCATGGTT -3'.
  2. A kind of 2. rice blast resistant genePi2HRM detection methods, it is characterised in that the HRM detection architectures include a pair Detection primer, the forward primer of the detection primer is 5'-GCAGCCATTGAGAAGCTCTC-3', and reverse primer is 5'- CCCCAAGTATCAGCATGGTT-3'。
  3. 3. HRM detection methods according to claim 2, it is characterised in that the described method comprises the following steps：
    a）Extract sample to be tested DNA；
    b）The PCR reaction systems comprising testing sample DNA, detection primer, PCR kit and EvaGreen fluorescent dyes are prepared to enter Performing PCR expands；With
    c）HRM analysis scannings are carried out to PCR primer.
  4. 4. HRM detection methods according to claim 3, wherein described extraction testing sample DNA method is：Sampling, Directly plus extract solution is ground, and after boiling water bath 10min, it is testing sample DNA that stratification, which takes supernatant, wherein described extract solution Include 10mM Tris-HCl, 0.5mM EDTA and 0.15M KCl.
  5. 5. HRM detection methods according to claim 3, wherein described PCR reaction systems include 1 μ L pcr templates, 0.5U rTaq archaeal dna polymerases, 1 × PCR buffer solutions, 250 μM of dNTP, 0.1 μM of forward and reverse primer and 0.1 μ L 20 × EvaGreen fluorescent dyes.
  6. 6. HRM detection methods according to claim 3, wherein described PCR amplification conditions are：94 DEG C 3 minutes；94℃30 Second, 58 DEG C 30 seconds, 72 DEG C 10 seconds, 40 circulation；72 DEG C 1 minute；And then 95 DEG C are denatured 1 minute；25 DEG C of renaturation 1 minute.
  7. A kind of 7. rice blast resistant gene based on HRM technologiesPi2Detection kit, it is characterised in that the detection reagent A pair of detection primers are included in box, the forward primer of the detection primer is 5'-GCAGCCATTGAGAAGCTCTC-3', reversely Primer is 5'-CCCCAAGTATCAGCATGGTT-3'.
  8. 8. detection kit according to claim 7, wherein described kit also includes PCR reagent and fluorescent dye.
  9. 9. the application method of claim 7-8 any described detection kit, it is characterised in that the application method includes Following steps：
    a）Extract sample to be tested DNA；
    b）The PCR reaction systems comprising sample to be tested DNA, primer pair, PCR kit and EvaGreen fluorescent dyes are prepared to carry out PCR is expanded；With
    c）HRM analysis scannings are carried out to PCR primer.
  10. 10. the described primer of claim 1, claim 2-6 any described HRM detection methods, claim 7-9 it Any described kit is in detection rice blast resistant genePi2In application.
CN201710610688.9A 2017-07-25 2017-07-25 The exploitation and application of blast resisting Pi2 gene specific molecular labelings Pending CN107435068A (en)

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