CN104789655A - Molecular marker and application of rice blast-resistant gene Pik - Google Patents
Molecular marker and application of rice blast-resistant gene Pik Download PDFInfo
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Abstract
The invention belongs to the technical field of plant biology, and particularly discloses a molecular marker and application of a rice blast-resistant gene Pik. The molecular marker comprises a pair of outer primers Pik-O-F and Pik-O-R as well as a pair of inner primers Pik-T-F and Pik-C-R, and primer sequences are shown as SEQ ID NO: 1 to 4. When the molecular marker is used for identifying the genotype of the rice blast-resistant gene Pik, sequencing is not required, and only through simple PCR, genotyping can be performed on the rice blast-resistant gene to distinguish rice blast-resistant rice varieties from rice blast-susceptible varieties, so that molecular marker assistant selection of rice blast is achieved; the molecular marker can improve the detection efficiency of the Pik gene, is applicable to the molecular marker assistant selection of a rice improvement segregation population, can improve the breeding efficiency, and meets the requirement for large-scale molecular breeding.
Description
Technical field
The invention belongs to plant biotechnology field, be specifically related to a kind of resistance gene of rice blast
pikmolecule marker and application.
Background technology
Paddy rice is the important farm crop of China, and the stability of its output is directly connected to national economy.Rice blast is the destructive disease of paddy rice, causes great effect to the output of paddy rice, and the paddy rice of high resistant to rice blast is the common-denominator target that new rice variety is cultivated always.At present, excavate wide spectrum rice blast resistance gene or main effect QTL, by pyramiding breeding, cultivating the disease-resistant rice varieties of broad spectrum durable is the major measure controlling rice blast.Thus, the detection of rice blast resistance gene and the screening of rice blast resistance rice germplasm just seem particularly important.Traditional rice blast resistance detects, and is that the mode then inoculating Pyricularia oryzae by artificial leaf-cutting is identified, the method detected result less stable, cycle longer, technical qualification require high, cannot meet the qualification requirement at the low generation large group of breeding.The most effective Resistance Identification method should be directly select rice blast resistance gene.
Research shows, rice blast resistance gene
pikbe positioned at 11 chromosome long arm, have stronger, stable resistance to many Chinese rice blast microspecies.
piklocus by two continuous print NBS-LRR genoids (
pik-1with
pik-2) composition, two gene pairss
pikthe rice blast resistance of mediation is all essential.
pikthe resistance multiple allelomorphos of seat having been located or clone comprises
pik(donor Kusabue),
pikh(donor Tetep),
pik-H4(donor H4),
pikp(donor K60),
pikm(donor Tsuyuake),
piks(donor Norin),
pi1(donor LAC23) etc.Although
pikanti-spectrum difference to some extent between the different resistance multiple allelomorphoss at seat, but resistance is all better.
pikcorresponding Japan is fine (containing susceptible allelotrope
pik) genomic two genes are respectively LOC_Os11g46200(
pikm5-NP) and LOC_Os11g46210(
pikm6-NP); And
pik-2relatively not conservative between isoallele, special variation SNP can be excavated, for developing mark.Contriver has cloned the resistance multiple allelomorphos of paddy rice
pik-H4, will
pik-H4carry out Multiple sequence alignments with LOC_Os11g46210, find resistance multiple allelomorphos and Japan fine
pikthere is SNP variation at the 2173rd bit base place, wherein resistant gene is T, and susceptible gene is C.
Tetra-primer ARMS-PCR PCR(ARMS-PCR) special equipotential amplification can be carried out for SNP site.For known variant sites, design 1 pair of outer primer and 1 pair of specificity inner primer respectively in both sides, Auele Specific Primer 3' terminal bases must drop on the position of catastrophe point, thus optionally increase saltant type and wild-type genes of individuals.In same once amplification, wild-type is different with the expanding fragment length that mutated genes produces, the presence or absence of the particular bands be separated by product fragment differentiates individual genotype, a kind of codominance typing method, have easy and simple to handle, somatotype fast, the advantage such as low cost.
Summary of the invention
Technical problem to be solved by this invention overcomes above-mentioned technological deficiency, provides a kind of resistance gene of rice blast
pikmolecule marker.
Second object of the present invention is to provide above-mentioned molecule marker at qualification resistance gene of rice blast
pikgenotype or/and can paddy rice application in resisting rice blast bacteria.
3rd object of the present invention is to provide the application of above-mentioned molecule marker in rice breeding.
4th object of the present invention is to provide and utilizes above-mentioned molecular markers for identification resistance gene of rice blast
pikgenotypic method.
Can the 5th object of the present invention be to provide and utilize above-mentioned Markers for Detection paddy rice resisting rice blast bacteria or simultaneously detect resistance gene of rice blast
pikgenotype and paddy rice can the method for resisting rice blast bacteria.
The object of the invention is to be achieved by the following technical programs:
A molecule marker for resistance gene of rice blast, described molecule marker is made up of a pair outer primer Pik-O-F, Pik-O-R and a pair inner primer Pik-T-F, Pik-C-R, and primer sequence is as shown in SEQ ID NO:1 ~ 4.
Molecule marker of the present invention be for
pikthere is T/C SNP variation design and obtain in bit base place, gene coding region the 2173rd; 3 ' end the 3rd base of described inner primer Pik-T-F, Pik-C-R introduces base mismatch.
Present invention also offers above-mentioned molecule marker at qualification resistance gene of rice blast
pikcan genotype and/or paddy rice application in resisting rice blast bacteria.
Present invention also offers the application of above-mentioned molecule marker in rice breeding.
Present invention also offers and utilize above-mentioned molecular markers for identification resistance gene of rice blast
pikgenotypic method, particularly, comprises the following steps:
S1 testing sample DNA extraction;
S2 take sample DNA as template, builds PCR reaction system, utilizes described molecule marker to carry out pcr amplification;
S3 analyzes the product after pcr amplification, carry out result judgement, described result is judged to be: if there is 184bp and 260bp band, then show testing sample
pikfor disease-resistant gene type (T/T is homozygous); If there is 131bp and 260bp band, then show testing sample
pikfor susceptible genotype (C/C is homozygous); If there is 131bp, 184bp and 260bp band, then show testing sample
pikfor heterozygous (C/T heterozygous).
Present invention also offers and utilize above-mentioned Markers for Detection paddy rice can resisting rice blast bacteria or simultaneously detect resistance gene of rice blast
pikcan genotype and paddy rice the method for resisting rice blast bacteria, particularly, comprises the following steps:
S1 testing sample DNA extraction;
S2 take sample DNA as template, builds PCR reaction system, utilizes above-mentioned molecule marker to carry out pcr amplification;
S3 analyzes the product after pcr amplification, carry out result judgement, described result is judged to be: if there is 184bp and 260bp band, then show testing sample
pikfor disease-resistant gene type, testing sample is resisting rice blast bacteria kind; If there is 131bp and 260bp band, then show testing sample
pikfor susceptible genotype, testing sample is sense Pyricularia oryzae kind; If there is 131bp, 184bp and 260bp band, then show testing sample
pikfor heterozygous, testing sample is sense Pyricularia oryzae kind.
Preferably, described PCR reaction system is: DNA profiling 1.0 uL, Pik-T-F 0.4uL, Pik-C-R 0.4uL, Pik-O-F 0.4uL, Pik-O-R 0.4uL, 2 × Power Taq PCR Master Mix 7.5uL, all the other are by two steaming aqua sterilisa (ddH
2o) 15uL is complemented to.
Preferably, described PCR reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of 30sec, 55 DEG C of 30sec, and 72 DEG C of 30sec run 35 circulations altogether, and last 72 DEG C extend 10min.
Compared with prior art, the present invention has following beneficial effect:
The present invention is based on ARMS-PCR principle, designed and developed that a kind of amplified fragments is moderate, the molecule marker of the resistance gene of rice blast of high specificity, utilize this Marker Identification resistance gene of rice blast
pikgenotype, do not need, through order-checking, gene type to be carried out to resistance gene of rice blast only by simple PCR, distinguish resisting rice blast bacteria rice varieties and susceptible variety, achieve the molecular marker assisted selection to rice blast, molecule marker of the present invention can improve
pikthe detection efficiency of gene, is suitable for the molecular marker assisted selection of rice modification segregating population, improves breeding efficiency, meets the demand of large-scale molecular breeding.
Accompanying drawing explanation
Fig. 1 is functional indicia Pik-C/T design of primers schematic diagram; Wherein allelic variation site is respectively with red background mark, and primer base mismatch identifies with underscore.
Fig. 2 is the capillary electrophoresis detection and genotyping of functional indicia Pik-C/T amplified production; Wherein, 1: China accounts for; 2: Yue Feng newly accounts for; 3:H4 polymerization system " H436 "; 4:H4; 5: navigate extensive 1173; 6: China accounts for/H4 polymerization system, 7 ~ 8: Yue Feng newly accounts for/and H4 is polymerized system.
Fig. 3 is that functional label Pik-C/T combines full-automatic capillary electrophoresis detected result; Wherein M:DNA ladder; 1 ~ 2,4,6,9,11:C/C is homozygous; 3,5,7-8,10,12:C/T heterozygous.
Embodiment
Further illustrate content of the present invention below in conjunction with Figure of description and specific embodiment, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the simple modification do the inventive method, step or condition or replacement, all belong to scope of the present invention; If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
embodiment 1 paddy rice
pikthe design of primers of gene function mark Pik-C/T and amplified fragments analysis
1, design of primers
With the resistance multiple allelomorphos of having cloned
pik-H4carry out Multiple sequence alignments with LOC_Os11g46210, find resistance multiple allelomorphos and Japan fine
pikthere is SNP variation at the 2173rd bit base place, wherein resistant gene is T, and susceptible gene is C.The functional label Pik-C/T in difference site is contained in design.Described Pik-C/T is made up of 4 primers Pik-T-F, Pik-C-R, Pik-O-F and Pik-O-R, primer sequence (5 '-3 ') as follows:
Pik-T-F：TCTCCGTGAGGTGCATCTCAAAGTT
CGT
Pik-C-R：AACTTGGTTATTGCTTCTGCCCCA
GCG
Pik-O-F：GCAATGCCAGCACTCGAAATCATTGAAA
Pik-O-R：TGATAGAGGGGCAGCATCAGATGATTGG
(base with underscore in primer sequence is the base mismatch introduced, and the base adding frame is the variation base that will detect)
2, amplified fragments analysis
Disease-resistant allelotrope (T/T is homozygous), susceptible allelotrope (C/C is homozygous) and heterozygous (C/T heterozygous), the band that Pik-O-F and Pik-O-R can be utilized to amplify size be about 260 bp, this band may be used for pcr amplification whether monitoring.In addition, disease-resistant allelotrope (T/T is homozygous) can also utilize Pik-T-F and Pik-O-R to amplify the band of 184 bp; Susceptible allelotrope (C/C is homozygous) can utilize Pik-O-F and Pik-C-R to amplify the band of 131 bp; The band that heterozygous (C/T heterozygous) can amplify 184 bp can amplify again the band of 131 bp.
embodiment 2 paddy rice
pikgene function mark Pik-C/T identifies the disease-resistant gene type of 7 paddy rice
1, the extraction of oryza sativa genomic dna
With 7 rice varieties for material: China accounts for, Yue Feng newly accounts for, H4 polymerization system " H436 ", H4, boat is extensive 1173, China accounts for/H4 polymerization system, Yue Feng newly accounts for/and H4 is polymerized system.Choose paddy rice individual plant tender leaf, adopt CTAB method to extract oryza sativa genomic dna, concrete steps are as follows: (1) is got proper amount of fresh blade and is placed on and adds liquid nitrogen in 2 mL centrifuge tubes and smash to pieces, adds 1 mL CTAB extract, shake up in centrifuge tube; (2) be placed in water-bath or the thermostat container of 65 DEG C, shake gently once every 10min, take out after 30 ~ 45min; (3) after cooling 2 min, add chloroform-isoamyl alcohol (24:1) to full packages, acutely shake up and down, make both mix; (4) centrifuge tube puts into whizzer 15, takes out after centrifugal 10 min of 000 r/min; (5) careful Aspirate supernatant is in new sterile centrifugation tube, then adds the Virahol of 600 μ L precoolings, shakes gently up and down, Virahol is fully mixed with water layer; Place 20 min, DNA is fully precipitated for (6)-20 DEG C; (7) 10,000 r/min moment is centrifugal, outwells liquid immediately, then is stood upside down by centrifuge tube on the paper handkerchief spread out; (8) upright centrifuge tube after 1min, adds ethanol and the 80 μ L 3M NaAc solution of 720 μ L 70%, shakes gently, flick tip with finger, DNA is precipitated and swims in liquid; (9) at least place 30min, impurity is fully dissolved; (10) 10,000 r/min moment is centrifugal, outwells liquid immediately, adds the ethanol of 800 μ L 70% by DNA rinsing 20 ~ 30min again; Centrifugal 3 ~ the 5min of (11) 15,000 r/min, outwells liquid immediately, is stood upside down by centrifuge tube on the paper handkerchief spread out; (12) upright centrifuge tube after several minutes, ventilating kitchen inner drying DNA; (13) add 100 μ L 1 × TE Buffer, DNA is fully dissolved; (14)-20 DEG C of preservations, for subsequent use are placed in.
2, rice blast conventional identification method
Reference literature (Sun great Yuan, Zhou Danhua, Xiao Wuming, etc. utilize MAS technology cultivate high resistant to rice blast hybrid rice restorer line boat extensive 1173 [J]. North China agronomy report, 2014,29 (6): 121-125.) experimental technique.(1) by after the vernalization of rice strain dry seeds, bunch planting (30cm in seedling dish in order
3× 20cm
3× 8cm
3), often 28 caves broadcast by dish, and each strain is broadcast once (8 ~ 10 seed/caves).Rice seedling takes drought to educate cultivation, and grow to leaf one heart stage employing ammonium sulfate and apply fertilizer, often 0.5 g executed by dish, applies fertilizer altogether 3 times before connecing bacterium, when seedling grows to 3.5 ~ 4 leaves, adopts GD00-193 to inoculate; (2) artificial infection idenfication: the single spore separation and the Spore cultivation that first carry out Pyricularia oryzae.Wash lower spore suspension with sterile distilled water, connecing bacterial concentration is 5 × 10
4individual every milliliter.Adopt high-pressure fog bacterination process.30 ~ 40 ml spore suspensions are connect in each seedling-cultivation plate.To meet after bacterium water-retaining cultivation 24 h in 25 DEG C of darkrooms, then move to and cover screened negative room, envrionment temperature remains on 22 ~ 30 DEG C, and regular spraying and moisturizing makes relative humidity remain on more than 90%, promotes morbidity.Within after connecing bacterium 7 days, carry out state of an illness qualification.Disease scale is undertaken by 0 ~ 9 grade standard that the whole nation is unified, and be decided to be disease-resistant by sick level 0 ~ 3 grade, 4 ~ 9 grades are decided to be susceptible.Qualification result shows, H4 polymerization system " H436 ", H4, the extensive 1173 high resistant to rice blast bacterium GD00-193 of boat; China accounts for, Yue Feng newly accounts for high sense Pyricularia oryzae GD00-193.
3, the genotype of functional indicia Pik-C/T amplification qualification 7 rice varieties disease-resistant genes
Functional label Pik-C/T is utilized to carry out pcr amplification to the DNA of 7 rice varieties, reaction system is 15 μ L, comprise: DNA profiling 1.0 uL, Pik-T-F 0.4uL, Pik-C-R 0.4uL, Pik-O-F 0.4uL, Pik-O-R 0.4uL, 2 × Power Taq PCR Master Mix 7.5uL, all the other are by two steaming aqua sterilisa (ddH
2o) supply.
PCR reaction conditions: 94 DEG C of denaturation 5min, 94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 30sec run 35 circulations altogether, and last 72 DEG C extend 10min.
4, the capillary electrophoresis of amplified production detects and genotype judgement
Amplified production is carried out diluting and is transferred to the Fragment Analyzer Automated CE System(U.S., AATI) detect, operate according to the process specifications of test kit DNF-910-K2000, and use PROSize 2.0 data analysis software to carry out data analysis, genotype is judged:
Amplified production is 260 bp and 184 bp, two kinds of fragments, and show tags idiotype is that T/T is homozygous, as in Fig. 2,3rd ~ 5 are detected sample (H4 polymerization system " H436 ", H4, boat extensive 1173); Amplified production is 260 bp and 131 bp, two kinds of fragments, and show tags idiotype is that C/C is homozygous, as sample (China accounts for, Yue Feng newly account for) is detected in 1st ~ 2 in Fig. 2; Amplified production is 260 bp, 184 bp and 131 bp, tri-kinds of fragments; Show tags idiotype is C/T heterozygous, as sample (China accounts for/H4 polymerization system, Yue Feng newly accounts for/H4 be polymerized system) is detected in 6th ~ 7 in Fig. 2.
Use the result of traditional method qualification consistent with the result of using function Marker Identification of the present invention, demonstrate accuracy, the reliability of functional indicia Pik-C/T of the present invention at qualification rice blast resistance gene.
comparative example 1 utilizes functional indicia Pik-C/T to detect 7 rice varieties
pikgenotype
Experimental technique with embodiment 2, uniquely unlike, 2 inner primers of molecule marker used are as follows:
SEQ ID NO:5:5 '-TCTCCGTGAGGTGCATCTCAAAGTT
tgC-3 ' (C equipotential)
SEQ ID NO:6:5 '-AACTTGGTTATTGCTTCTGCCCCA
gcA-3 ' (T equipotential)
Utilize the molecule marker in this comparative example to identify 7 rice varieties described in embodiment 2, result shows: this primer sets can be effectively not right
pikeffective somatotype is carried out in this site, may be because the Tm difference of primer self causes amplification less than polymorphic fragment, cannot differentiate genotype.
embodiment 3 application function mark Pik-C/T identifies paddy rice individual plant
1, test material: with H4 polymerization system " H436 " for donor parents, Yue Feng newly accounts for as recurrent parent, backcrosses, the BC of generation
1f
2it is test material that backcross improvement is separated individual plant.
2, the extraction of oryza sativa genomic dna: with embodiment 2.
3, functional indicia Pik-C/T increases: with embodiment 2.
4, the capillary electrophoresis of amplified production detects and genotype judgement
The capillary electrophoresis detection method of amplified production, with described in embodiment 2, judges genotype:
Amplified production is 260 bp and 131 bp, two kinds of fragments, and show tags idiotype is C/C homozygous (in Fig. 3,1st ~ 2,4,6,9,11 are detected sample); Amplified production is 260 bp, 184bp and 131 bp, tri-kinds of fragments, and show tags idiotype is C/T heterozygous (the 3rd, 5,7 ~ 8,10,12 in Fig. 3 is detected sample).
SEQUENCE LISTING
<110> Agricultural University Of South China
The molecule marker of a <120> resistance gene of rice blast Pik and application
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> DNA
<213> Pik-T-F
<400> 1
tctccgtgag gtgcatctca aagttcgt 28
<210> 2
<211> 27
<212> DNA
<213> Pik-C-R
<400> 2
aacttggtta ttgcttctgc cccagcg 27
<210> 3
<211> 28
<212> DNA
<213> Pik-O-F
<400> 3
gcaatgccag cactcgaaat cattgaaa 28
<210> 4
<211> 28
<212> DNA
<213> Pik-O-R
<400> 4
tgatagaggg gcagcatcag atgattgg 28
<210> 5
<211> 28
<212> DNA
Inner primer forward sequence in <213> comparative example 1
<400> 5
tctccgtgag gtgcatctca aagtttgc 28
<210> 6
<211> 27
<212> DNA
Inner primer reverse sequence in <213> comparative example 1
<400> 6
aacttggtta ttgcttctgc cccagca 27
Claims (9)
1. a resistance gene of rice blast
pikmolecule marker, it is characterized in that, described molecule marker is made up of a pair outer primer Pik-O-F, Pik-O-R and a pair inner primer Pik-T-F, Pik-C-R, and primer sequence is as shown in SEQ ID NO:1 ~ 4.
2. molecule marker described in claim 1 is at qualification resistance gene of rice blast
pikcan genotype and/or paddy rice application in resisting rice blast bacteria.
3. the application of molecule marker described in claim 1 in rice breeding.
4. utilize molecular markers for identification resistance gene of rice blast described in claim 1
pikgenotypic method.
5. utilize Markers for Detection paddy rice described in claim 1 can resisting rice blast bacteria or simultaneously detect resistance gene of rice blast
pikcan genotype and paddy rice the method for resisting rice blast bacteria.
6. method according to claim 4, is characterized in that, comprises the following steps:
S1 testing sample DNA extraction;
S2 take sample DNA as template, builds PCR reaction system, utilizes molecule marker described in claim 1 to carry out pcr amplification;
S3 analyzes the product after pcr amplification, carry out result judgement, described result is judged to be: if there is 184bp and 260bp band, then show testing sample
pikfor disease-resistant gene type; If there is 131bp and 260bp band, then show testing sample
pikfor susceptible genotype; If there is 131bp, 184bp and 260bp band, then show testing sample
pikfor heterozygous.
7. method according to claim 5, is characterized in that, comprises the following steps:
S1 testing sample DNA extraction;
S2 take sample DNA as template, builds PCR reaction system, utilizes molecule marker described in claim 1 to carry out pcr amplification;
S3 analyzes the product after pcr amplification, carry out result judgement, described result is judged to be: if there is 184bp and 260bp band, then show testing sample
pikfor disease-resistant gene type, testing sample is resisting rice blast bacteria kind; If there is 131bp and 260bp band, then show testing sample
pikfor susceptible genotype, testing sample is sense Pyricularia oryzae kind; If there is 131bp, 184bp and 260bp band, then show testing sample
pikfor heterozygous, testing sample is sense Pyricularia oryzae kind.
8. the method according to claim 6 or 7, it is characterized in that, described PCR reaction system is: DNA profiling 1.0 uL, Pik-T-F 0.4uL, Pik-C-R 0.4uL, Pik-O-F 0.4uL, Pik-O-R 0.4uL, 2 × Power Taq PCR Master Mix 7.5uL, all the other complement to 15uL by two steaming aqua sterilisa.
9. the method according to claim 6 or 7, is characterized in that, described PCR reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of 30sec, 55 DEG C of 30sec, and 72 DEG C of 30sec run 35 circulations altogether, and last 72 DEG C extend 10min.
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| CN109207631A (en) * | 2018-11-15 | 2019-01-15 | 上海市农业生物基因中心 | A kind of Gene For Resistance To Rice Bacterial Blight xa5 specific molecular marker and its application |
| CN113999934A (en) * | 2021-12-14 | 2022-02-01 | 湖北省农业科学院粮食作物研究所 | Rice blast resistance Pik locus allele identification molecular marker and application thereof |
| CN113999934B (en) * | 2021-12-14 | 2022-08-05 | 湖北省农业科学院粮食作物研究所 | Rice blast resistance Pik locus allele identification molecular marker and application thereof |
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