CN108300802A - The general molecular label of detection blast resisting multiple allele Pi-2/gm/zt a kind of and application - Google Patents
The general molecular label of detection blast resisting multiple allele Pi-2/gm/zt a kind of and application Download PDFInfo
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Abstract
The invention discloses the general molecular label of detection blast resisting multiple allele Pi 2/gm/zt a kind of and applications.The invention also discloses a kind of methods of detection said gene, include the following steps：Extract rice sample gene group DNA to be detected；PCR amplification is carried out using primer pair；It detects resistant gene donor parents and needs to improve material receptor parent with the presence or absence of polymorphism, if there is polymorphism, judge whether rice sample to be measured contains blast resistant gene Pi 2 or Pi gm or Pi zt genes according to pcr amplification product.The primer pair sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；The present invention can improve the molecule assisted Selection efficiency of anti-rice blast rice new varieties.
Description
Technical field
The invention belongs to rice molecular heredity and thremmatology field, specifically, it is multiple etc. to be related to a kind of detection blast resisting
The general molecular label of position gene Pi-2/gm/zt and application.
Background technology
Rice blast is a kind of global rice disease caused by fungi (Magnaporthe oryzae), is China's water
One of three major diseases in rice production have the characteristics that occurrence scope is wide, epidemic rate is fast, destructive big, pathogen easily makes a variation,
It effectively causes the rice underproduction and reduces rice quality.It was verified that the selection and breeding of resistant rice cultivars are control rice blast with plantation
Most effective measure.However, China has a vast territory, South And North Rice Regions natural climate condition difference is big, and cropping system is also not quite similar,
Physiological races of rice blast fungus type is rich and varied, and there are significant Resistant Differences in different rice regions for identical resistant gene, then
Add dominant race easily to change, disease-resistant variety is caused gradually to lose its resistance in several Nian Houhui of plantation.Therefore, reasonably
Broad-spectrum resistance gene or the multiple disease-resistant genes of polymerization are excavated and utilized, are to obtain lasting, broad-spectrum disease resistance kind important channel.
Traditional rice resistance breeding depends on the identification of resistant phenotype, does not require nothing more than rich experiences and sharp sight that breeder has
Power is examined, and qualification result is also highly prone to the influence of environment and human factor and causes error, Breeding Efficiency is low.With point
The generation and development of sub- labelling technique, it is convenient, not affected by environment the advantages that make its application value and foreground increasingly by
Concern.By developing the molecular labeling with target gene close linkage, especially its specific Function is developed in gene internal
Marker carries out molecular marker assisted selection, not only greatly accelerates breeding paces, but also can reduce breeding cost, improves
Efficiency of selection, therefore be to cultivate that blast resisting is most economical has using the kind that molecular breeding technology selection and breeding contain blast resistant gene
The method of effect.
In recent years, with the rapid development of Protocols in Molecular Biology, rice blast resistance gene is positioned, is cloned successively.Mesh
Preceding a blast resistant gene is accurately positioned at least more than 90, wherein 26 successful clones.These gene distributions exist
Except on the 3rd extrachromosomal each article of chromosome of rice, plurality of gene cluster is distributed on the 6th and 11 chromosomes of rice.
The sites Pi-2 near the 6th chromosome centromere are rice blast resistance gene close quarters, have up to the present cloned 5
Multiple allele is Pi-gm, Pi-2, Pi-zt, Pi-9 and Pi-50 respectively.At the grain of o.11 chromosome long arm proximal end
The sites Pi-1 are also rice blast resistance gene close quarters, have up to the present cloned 6 multiple alleles, be respectively Pi-1,
Pi-k, Pi-kh, Pi-kp, Pi-ke and Pi-km.Research has shown that single NBS- in Pi-gm, Pi-2, Pi-zt, Pi-9 and Pi-50
LRR genes have had the anti-pest effect of wide spectrum, wherein the difference of only 8 amino acid residues in the regions LRR of Pi2 and Pi-zt just generates
Different product and resistance.The rice blast resistance of different rice region Pi-gm, Pi-2, Pi-zt, Pi-9 and Pi-50 exist significant
Difference.There is good utility value as Pi-zt is not good enough in Hubei resistance, but in Jiangxi.The Pi- of rice varieties " Gu Mei 4 "
Gm genes are one of the genes that anti-spectrum is wide and resistance is lasting, to being shown in more than the 30 strong pathogenic strain of various regions and nations
Highly resistance is immune, but feels 1 bacterial strain of enshi, therefore rationally using multiple allele to different rice blast biological strains
Resistant Difference imported into the different multiple alleles in same site in Parent, by hybridizing combo, further increases hybrid paddy rice
Rice blast resistance, to extend the plantation service life of kind.
Invention content
In view of this, the present invention is directed to above-mentioned problem, a kind of detection blast resisting multiple allele Pi-2/ is provided
The general molecular of gm/zt marks and application, by detecting point with blast resisting multiple allele Pi-2/gm/zt close linkages
Son label, it may be determined that whether parental rice or filial generation contain blast resistant gene Pi-2 or Pi-gm or Pi-zt, to
Improve the molecule assisted Selection efficiency of anti-rice blast rice new varieties.
In order to solve the above-mentioned technical problem, the invention discloses a kind of detection blast resisting multiple allele Pi-2/gm/zt
General molecular label primer pair, which is：
F:5 '-GCAGGGTAACTCCTATTT-3 ', nucleotide sequence is as shown in SEQ IDNO.1；
R:5 '-AATCAGACCTTACATCACAG-3 ', nucleotide sequence is as shown in SEQ ID NO.2.
The invention also discloses a kind of methods of detection blast resisting multiple allele Pi-2/gm/zt, including following step
Suddenly：
1) rice sample gene group DNA to be detected is extracted；
2) PCR amplification is carried out using primer pair described in claim 1；
3) resistant gene donor parents are detected and need to improve material receptor parent with the presence or absence of polymorphism, if there is polymorphic
Property, then judged using following conditions：
If there is individual 480bp or occurs 480bp and 269bp simultaneously in pcr amplification product, rice to be detected
Sample contains blast resistant gene Pi-2 or Pi-gm or Pi-zt gene；Otherwise, then rice sample to be detected is free of blast resisting
Gene Pi-2 or Pi-gm or Pi-zt gene.
Optionally, the PCR reaction systems of the PCR amplification in step 2 are：10xPCR reaction buffers 2ul, 5pM primers F and
R each 1ul, 10mM dNTP0.5ul, DNA sample 2ul, Taq DNA polymerase 0.2ul add ddH2O complements to 20ul.
Optionally, the PCR reaction conditions of the PCR amplification in step 2 are：95 DEG C of pre-degenerations 5 minutes, 95 DEG C are denaturalized 30 seconds,
55 DEG C are annealed 30 seconds, and 72 DEG C extend 1 minute, and totally 35 cycles, are placed at 72 DEG C of heat preservations 10 minutes, 10 DEG C.
The invention also discloses a kind of application of above-mentioned method in Molecular Breeding for Blast Resistance.
The invention also discloses a kind of kits containing above-mentioned primer pair.
The invention also discloses a kind of application of above-mentioned kit in Molecular Breeding for Blast Resistance.
Compared with prior art, the present invention can be obtained including following technique effect：
1) present invention using these three allele during being directed respectively into different kinds, the molecule of required development
When marker-assisted breeding, the detection of previous label needs to do three times using corresponding three primers for its donor gene respectively
PCR detect, could detect each single plant whether the information containing resistant gene, and use side of the present invention need to only use a primer
As soon as do time PCR detection, can obtain each single plant whether the information containing resistant gene, reduce PCR numbers of repetition, reduce
Labor intensity improves work efficiency.
2) polymorphism is good between three resistant gene donor materials of the invention and other background materials, and anti-sense material PCR expands
Base sequence difference has that there are about 220 bases between the DNA fragmentation of increasing, it is only necessary to the electrophoresis 30 in 2.0% Ago-Gel
Minute, it can clearly analyze whether each single plant contains resistant gene, shorten the Molecular Detection time in laboratory, improve
Detection efficiency.
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technique effect.
Description of the drawings
Attached drawing described herein is used to provide further understanding of the present invention, and constitutes the part of the present invention, this hair
Bright illustrative embodiments and their description are not constituted improper limitations of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is the electrophoretogram of 26 materials of InDel-2gmzt primer detections of the present invention；The segment of top is in each band
Amplified fragments possessed by target gene, wherein 1, Gu Mei No. 4 (pi-gm), 2, IRBB5 (pi-2), 3, Gumei2 (pi-
Gm), 4, IRBL11 (pi-zt), 5, paddy B, 6, good fortune her B, 7, China account for (pi-2), 8, middle group 14 (pi-2), 9, in extensive 8006,
10,9311,11, another name for Sichuan Province extensive 527,12, Nipponbare, 13, spring river 06,14, Jiahe 212,15, Zhenshan 97B, 16, high mountain 4B, 17,
JR7599,18, JR3385,19, IR24,20, the rich A (pi-1, pi-2) of peace, 21,02428,22, R1128,23, the extensive 934 (pi- in Jiangxi
Gm), 24, Jiangxi extensive 986 (pi-gm), 25, bright extensive 86 (pi-zt), 26, early anti-B (pi-2)；
Fig. 2 is 48 of InDel-2gmzt primer detections R9113/ of the present invention //No. 4 BC2F2 of R9113//R9113/ paddy plum
The electrophoretogram of single plant；The segment of top is amplified fragments possessed by target gene in each band, wherein 1 is Gu Mei 4,
2-47 is 46 BC2F2 single plants, and 48 be R9113；
Fig. 3 is that early aobvious B//morning shows 48 blast resisting strains that B/ pacifies rich BBC1F4 to InDel-2gmzt primer detections of the present invention
The electrophoretogram of system；The segment of top is amplified fragments possessed by target gene in each band, wherein 1 is peace rich B, 2-47
It is early aobvious B for the rice blast resistance single plant of 46 BC1F4,48；
Fig. 4 is InDel-2gmzt primer detections R350/ ///R350/ of the present invention //R350//R350/BL11 (Pi-zt)
The electrophoretogram of 48 strains of BC3F2；The segment of top is amplified fragments possessed by target gene in each band, wherein 1
It is BL11,2 be R350, and 3-48 is the single plant of 46 BC3F2；
Fig. 5 be organized in InDel-2gmzt primer detections of the present invention 14/R9113//R9113BC1F2 strains, JR7599/ ///
The electricity of JR7599/ //No. 4 BC2F2 strains of JR7599//JR7599/ paddy plum and R463/ //R463//R463/BL11BC2F2 strains
Swimming figure；The segment of top is amplified fragments possessed by target gene in each band,
Wherein, 1-1 to 6-7 is 47 single plants of the BC1F2 in middle group of 14/R9113//R9113BC1F2 strain, during 6-8 is
Middle group 14 (Pi-2) in 14/ //R9113//R9113BC1F2 strains of group；12-8 and 18-8 be JR7599/ ///JR7599/ //
Paddy plum No. 4 in No. 4 BC2F2 strains of JR7599//JR7599/ paddy plum, 7-1 to 18-7 be JR7599/ ///JR7599/ //
94 single plants of BC2F2 in No. 4 BC2F2 strains of JR7599//JR7599/ paddy plum；24-8 is R463/ //R463//R463/
BL11 (Pi-zt) in BL11BC2F2 strains, 19-1 to 24-7 are R463/ //R463//R463/BL11BC2F2 strains
47 single plants of BC2F2.
Specific implementation mode
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.
Embodiment 1 detects the foundation of the molecule labelling method of rice anti-rice blast base multiple allele
1) base that Nipponbare is used near each 100kb of gene Pi-2 upstream and downstream, in NCB works website (http://
Www.ncbi.nlm.nih.gov/) sequence of Nipponbare is compared with the sequence of rice variety 9311 with blast program,
Obtain 15 sites be inserted into or lacked with base, 15 pairs of primers devised with Primer5, extraction with Pi-2, Pi-9,
The donor material and Nipponbare of Pi-gm, Pi-zt resistant gene and 9311 DNA carry out PCR amplification, 4% with 15 pairs of primers
Ago-Gel in be separated by electrophoresis, after being dyed with GelRed, under gel imager ultraviolet lamp scanning take pictures and analyze.15 pairs
Primer amplification as a result, it has been found that, primer I nDel-9 (label is named as InDel-2gmzt) is with Pi-2, Pi-gm, Pi-zt resistance
There is extraordinary polymorphism between the donor material and Nipponbare and 9311 of gene.
InDel-2gmzt primer information：
F:5 '-GCAGGGTAACTCCTATTT-3 ', nucleotide sequence is as shown in SEQ IDNO.1；
R:5 '-AATCAGACCTTACATCACAG-3 ', nucleotide sequence is as shown in SEQID NO.2；
55 DEG C of annealing temperature.
2) a small amount of blade [number of 26 parent materials is taken：1, Gu Mei No. 4 (pi-gm), 2, IRBB5 (pi-2), 3, Gu Mei
No. 2 (pi-gm), 4, IRBL11 (pi-zt), 5, paddy B, 6, good fortune her B, 7, China account for (pi-2), 8, middle group 14 (pi-2), 9, in it is extensive
8006,10,9311,11, another name for Sichuan Province extensive 527,12, Nipponbare, 13, spring river 06,14, Jiahe 212,15, Zhenshan 97B, 16, high mountain 4B, 17,
JR7599,18, JR3385,19, IR24,20, the rich A (pi-1pi-2) of peace, 21,02428,22, R1128,23, the extensive 934 (pi- in Jiangxi
Gm), 24, Jiangxi extensive 986 (pi-gm), 25, bright extensive 86 (pi-zt), 26, early anti-B (pi-2), DNA is extracted, DNA is extracted according to 2%
Method extraction after the small modifications of CTAB, carries out PCR amplification, 2.0% with InDel-9 primers (being named as InDel-2gmzt)
Ago-Gel in be separated by electrophoresis, after being dyed with GelRed, under gel imager ultraviolet lamp scanning take pictures and analyze, have
The material of Pi-2, Pi-gm, Pi-zt resistant gene has extraordinary polymorphism with other materials without resistant gene, but
Also purpose band can be amplified by two materials with japonica rice blood source occur.Close linkage InDel-2gmzt polymorphic rates are high,
There are polymorphic between 84.3% receptor parent and donor parents Pi-2/gm/zt；The PCR product difference of InDel-2gmzt compared with
Greatly, the marker genetype of material can be quickly detected.By with Markers for Detection material to be checked, it can be determined that whether it carries
Disease-resistant gene Pi-2 or Pi-gm or Pi-zt, sequence difference effectiveness results are shown in Fig. 1, as shown in Figure 1, resistant gene donor material with
Polymorphism between other many parents has significant difference, is easy to distinguish.
3) DNA extraction according to 2%CTAB it is simple modification after method extract (MURRAYM G,
THOMPSON W F.Rapid isolation of high molecular weight
PlantDNA.Nuceleic Acids Research, 1980,8 (19):4321-4325, according to molecular marker assisted selection
It needs, eliminates and dry half an hours with the DNA 50 degree of temperature of baking oven for increasing extraction with 70% alcohol eluted dna step).
PCR reaction systems are calculated as with 20ul：10xPCR reaction buffers 2ul, 5pM primers F and R each 1ul, 10mM dNTP0.5ul, DNA
Sample 2ul, Taq DNA polymerase 0.2ul, adds ddH2O complements to 20ul.
PCR reaction conditions are：95 DEG C of pre-degenerations 5 minutes, 95 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 1 point
Clock, totally 35 cycles, are placed at 72 DEG C of heat preservations 10 minutes, 10 DEG C.
Wherein, the nucleotide sequence of primers F is as shown in SEQ ID NO.1；The nucleotide sequence of primer R such as SEQ ID
Shown in NO.2；Each 1OD of front and back primer of synthesis is directly diluted to the use of 5pM concentration.
4) PCR electrophoresis detections interpretation of result：
The donor material of three blast resistant genes Pi-2, Pi-gm and Pi-zt, pcr amplification product 480bp, 84.3%
Receptor parent and donor parents Pi-2/gm/zt between there are polymorphic, but 02428 and R1128 this 2 materials can also amplify
With with the above-mentioned similarly the same segment of resistant gene donor parents, therefore, these three resistance bases are done using the molecular labeling
The molecule assisted Selection of cause first has to detection resistant gene donor parents and needs to improve material receptor parent with the presence or absence of polymorphic
Property.If there is polymorphism, the label can be utilized, in subsequent molecular marker assisted selection, independent 480bp or simultaneously
When there is 480bp and 269bp, then rice sample to be detected contains blast resistant gene Pi-2 or Pi-gm or Pi-zt gene；It is no
Then, then rice sample to be detected is free of blast resistant gene Pi-2 or Pi-gm or Pi-zt gene.
Embodiment 2
Take 46 single-strain blade InDel-2gmzt primers of the BC2F2 of R9113/ //R9113//R9113/ paddy plum No. 4
It is detected, experimentation program as above.1 is Gu Mei 4, and 2-47 is 46 BC2F2 single plants, and 48 be R9113；As shown in Fig. 2,
21 single plants of wherein number 2,5,6,7,8,9,11,12,15,19,20,22,24,26,29,33,34,36,37,43,47 are taken
11 single plants of the heterozygous with gene Pi-gm, number 10,14,16,21,27,28,30,31,32,41,44 carry gene
Pi-gm's is homozygous.
Embodiment 3
The morning selected with Soybean seed lipoxygenase shows the BC1F4 strains that B//morning aobvious B/ pacifies rich B, is planted in Jinggang Mountain rice
Seasonal febrile diseases spontaneous induction identifies garden, and the single-strain blade in 46 strains of blast resisting is taken to be detected with InDe1587 primers, real
The process of testing is same as above.1 is the rich B of peace, and 2-47 is the rice blast resistance single plant of 46 BC1F4, and 48 be early aobvious B.As shown in figure 3, wherein
5 single plants of number 16,17,20,25,30 carry the heterozygous of gene Pi-2, number 2,3,7,8,10,11,12,13,15,
18、19、21、22、23、24、26、27、28、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47
35 single plants carry the homozygous of gene Pi-2,6 single plants of number 4,5,6,9,14,29 do not carry gene Pi-2 and
It is because this 6 strains carry the result of gene Pi-1 with rice blast resistance.
Embodiment 4
Take the BC3F2 of Soybean seed lipoxygenase selection R350/ ///R350/ //R350//R350/BL11 (Pi-zt)
Seed, be planted in Jinggang Mountain natural rice blast and induce identification garden, take the single-strain blade in 46 strains of blast resisting, extract
DNA carries out PCR detections, experimentation program as above with InDel-2gmzt primers.1 is BL11, and 2 be R350, and 3-48 is 46
BC3F2 single plants.As shown in figure 4, wherein number 4,5,7,8,12,13,14,16,18,19,21,22,24,26,27,28,29,
30,32,34,35,36,37,38,39,40,43,44,45,46,47,48 32 single plants carry the heterozygous of gene Pi-zt,
9 single plants of number 3,6,10,11,15,23,25,31,42 carry the homozygous of gene Pi-zt.Number 9,17,20,33,
41 5 single plants do not carry gene Pi-zt and have rice blast resistance, this possible 5 strains also carry other resistance bases
Cause.
Embodiment 5
Choose the strain that three differences of plantation answer backcrossing of the resistance allele through the resistant gene of molecular marker assisted selection
System：Middle group 14 (Pi-2)/R9113//R9113BC1F2, it is middle group therefrom to take 47 plants of blades, number 1-1 to 6-7, wherein 6-8
14；JR7599/ ///JR7599/ //No. 4 BC2F2 of JR7599//JR7599/ paddy plum, therefrom takes 94 plants of blades, number be 7-1 extremely
18-8, wherein 12-8 and 18-8 are Gu Mei 4；R463/ //R463//R463/BL11 (Pi-zt) BC2F2 therefrom takes 47 plants of leaves
Piece, number 19-1 to 24-7, wherein 24-8 are BL11, extract DNA respectively, with InDel-2gmzt primers simultaneously in two pieces of PCR plates
Middle progress PCR detections, DNA sample contains two different resistant genotypes, experimentation program as above in every piece of PCR plate.
As shown in figure 5, total 12 single plants of 1-2,1-6,1-7,2-2,2-4,2-5,3-6,3-8,4-5,4-8,5-5 and 6-7 carry base
Because of homozygous, 7-3,7-6,8-4,8-8,9-4,9-7,9-8,10-4,10-7,11-1,11-6,11-7,12-2,12- of Pi-2
3,13-6,13-7,13-8,14-1,14-5,15-2,15-4,15-5,16-3,17-5,17-7,18-1,18-3 and 18-7 are total
28 single plants carry the homozygous of gene Pi-gm.19-1、19-4、19-6、20-2、20-3、20-6、20-7、21-2、22-7、
24-4 and 24-5 amounts to 11 single plants and carries the homozygous of gene Pi-zt.
Carry out rice molecular marker assisted selection, the work in laboratory is an important link, especially does PCR, PCR production
Object electrophoresis and interpretation of result are the work taken time and effort very much, it usually needs in the short time with determining target strain so as to field
Hybridization or backcrossing are completed in time.The primer is directed to when carrying out molecular improvements using these three multiple resistance alleles of difference can be with
It is general, the trouble for frequently changing primer is eliminated when being PCR, reduces amiss probability, and the polymorphism of primer is good, is only needed
Experimental period is shortened in 2% Ago-Gel with regard to separating after 30 minutes by electrophoresis.A usual staff one
What it can complete 800-1200 sample does the tasks such as PCR, electrophoresis and interpretation of result, improves conventional efficient.Although this draws
Object is the label with target resistant gene close linkage, but imported into 8 differences answering resistance allele using three differences
In the kind of the heredity back of the body, the strain of homozygous resistant gene is contained by the primer molecule assisted Selection, 2015-2017 is in Jiangxi
Jing Gangshan natural rice blast induces garden and identifies rice blast resistance, the resistance strain spontaneous induction rice blast through molecular marker assisted selection
The Resistance Identification of disease is with obvious effects, and concrete outcome is shown in Table 1.Although three differences are answered resistance allele and are imported in different heredity
In the kind of background, but the efficiency of molecular marker assisted selection is all higher than 90% or more, and respective material reaches 100%, shows
Good resistance improvement selection effect.
Table 1 is the strain and wheel with homozygous resistant gene through InDel-2gmzt molecular marker assisted selections of the present invention
Return the resistance result that parent's Jing Gangshan natural rice blast induces identification
Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not
It is confined to form disclosed herein, is not to be taken as excluding other embodiments, and can be used for various other combinations, modification
And environment, and can be carried out by the above teachings or related fields of technology or knowledge in the scope of the invention is set forth herein
Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then should all be weighed appended by invention
In the protection domain that profit requires.
Sequence table
<110>Inst. of Rice, Jiangx Prov. Academy of Agricultural Sciences
<120>The general molecular label of detection blast resisting multiple allele Pi-2/gm/zt a kind of and application
<130> 2018
<141> 2018-04-27
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 1
gcagggtaac tcctattt 18
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 2
aatcagacct tacatcacag 20
Claims (7)
1. a kind of primer pair of the general molecular label of detection blast resisting multiple allele Pi-2/gm/zt, which is characterized in that
The primer pair is：
F:5 '-GCAGGGTAACTCCTATTT-3 ', nucleotide sequence is as shown in SEQ ID NO.1；
R:5 '-AATCAGACCTTACATCACAG-3 ', nucleotide sequence is as shown in SEQ ID NO.2.
2. a kind of method of detection blast resisting multiple allele Pi-2/gm/zt, which is characterized in that include the following steps：
1) rice sample gene group DNA to be detected is extracted；
2) PCR amplification is carried out using primer pair described in claim 1；
3) it detection resistant gene donor parents and needs to improve material receptor parent and whether there is polymorphism, if there is polymorphism,
Then judged using following conditions：
If there is individual 480bp or occurs 480bp and 269bp simultaneously in pcr amplification product, rice sample to be detected
Contain blast resistant gene Pi-2 or Pi-gm or Pi-zt gene；Otherwise, then rice sample to be detected is free of blast resistant gene
Pi-2 or Pi-gm or Pi-zt genes.
3. the method for detection blast resisting multiple allele Pi-2/gm/zt according to claim 2, which is characterized in that step
The PCR reaction systems of PCR amplification in rapid 2 are：10xPCR reaction buffers 2ul, 5pM primers F and R each 1ul, 10mM
DNTP0.5ul, DNA sample 2ul, Taq DNA polymerase 0.2ul add ddH2O complements to 20ul.
4. the method for detection blast resisting multiple allele Pi-2/gm/zt according to claim 2, which is characterized in that step
The PCR reaction conditions of PCR amplification in rapid 2 are：95 DEG C of pre-degenerations 5 minutes, 95 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, 72 DEG C
Extend 1 minute, totally 35 cycles, are placed at 72 DEG C of heat preservations 10 minutes, 10 DEG C.
5. application of the method described in claim 2-4 any claims in Molecular Breeding for Blast Resistance.
6. the kit containing primer pair described in claim 1.
7. application of the kit described in claim 6 in Molecular Breeding for Blast Resistance.
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