CN110257553A - A kind of KASP molecule labelling method for identifying resistance gene of rice blast Pigm - Google Patents
A kind of KASP molecule labelling method for identifying resistance gene of rice blast Pigm Download PDFInfo
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Abstract
The present invention relates to a kind of KASP molecule labelling methods for identifying resistance gene of rice blast Pigm, belong to biotechnology engineering field.According to paddy plum No. 4, there are the nucleotide variations of G-C at the Pigm-R2 gene start codon upstream 515bp of the site Pigm with Xian, round-grained rice kind has been sequenced, KASP molecular labeling primer is designed, fluorescent quantitative PCR is carried out to the DNA of different rice materials and determines its Pigm genotype.Whether the method for the present invention can not only fast and accurately be identified containing Pigm gene in Rice Germplasm Resources or breeding population, and can realize the high throughput detection to more sample material simultaneously.Have the advantages that compared with common molecular labelling technique more quick, efficient.Therefore, which can be further improved the efficiency of selection to the disease-resistant homozygous genotype containing Pigm in breeding, accelerate the breeding process of anti-rice blast rice kind.
Description
One, technical field
The present invention relates to a kind of KASP molecule labelling methods for identifying resistance gene of rice blast Pigm, belong to biological skill
Art engineering field, identification, screening and the breed breeding of the dedicated blast resisting of trans-genetic hybrid rice containing Pigm germ plasm resource.
Two, background technique
Rice blast is by sac fungus (Magnaporthe oryzae (Hebert) Barr., Invisible element Pyricularia
Oryae Cav.) caused by a kind of fungal disease, the whole world every year because caused by rice blast production loss account for rice total output
10~30% (Wilson R A et al., Under pressure:investigating the biology of plant
infection by Magnaporthe oryzae,Nature Reviews Microbiology,2009,7(3):185-
195).In China, rice blast per there is different degrees of generation every year, year occurring area be about 4.0 × 106~6.0 × 106hm2,
Lose paddy 6.0 × 105~1.45 × 106T becomes an important factor for restricting Rice Production, also causes to grain security serious hidden
Suffer from (Lu Minghong etc., the rice blast repeating transmission analysis of causes in 2014 and treatment method research, Chinese plant protection guide, 2015,35 (6):
35-39).Currently, there are mainly two types of methods for rice blast prevention and treatment, first is that this not only will increase production cost using chemical pesticide, and
And it has a negative impact to agricultural product and Environmental security；Second is that excavating and cultivating disease-resistant variety using Resistance resource.It was verified that
It is most economical, safe and effective approach using varietal resistance.Therefore, rice blast resistance is that variety certification evaluates kind in the process
The key index of superiority and inferiority and the important goal of rice modification.However, Pyricularia oryzae has genetic diversity abundant and variation
Property, varietal resistance is easily lost.It is always the hot spot of breeder's concern that how breeding, which has wide spectrum, durable resistance rice varieties,.
Pigm is derived from wide spectrum, a Durable resistance gene in China Sichuan Local Indica Rice paddy plum No. 4.It is right
The 29 strong pathogenic strain that country variant and area are collected shows highly resistance and immune, it is anti-compose Pi2, P40 than homogenic cluster,
Wider (Wu Y Y et al., the Development and evaluation of Near-isogenic of Pi9, Piz, Pizt
lines with different blast resistance alleles at the Piz locus injaponica
rice from the lower region ofthe Yangtze River,China,Plant Dis.2017,101(7):
1283-1291).Correlative study shows that the site Pigm is the gene cluster comprising 13 NBS-LRR class disease-resistant genes, wherein
R6 is the disease-resistant gene (PigmR) in the site Pigm, and R8 is susceptible gene (PigmS).PigmR itself can form homologous dimerization
Body plays broad-spectrum disease resistance function, but mass of 1000 kernel is caused to reduce；And it can be competing with PigmR by the PigmS albumen of epigenetic regulation
It strives to form the broad spectrum resistance that heterodimer inhibits PigmR to mediate, slows down pathogenic evolution of the pathogen to PigmR, make
It generates durable resistance, while improving setting percentage, offset mass of 1000 kernel reduce to yield influence (DengYW et al.,
Epigenetic regulation of antagonistic receptors confers rice blast resistance
with yield balance,Science,2017,355(6328):962-965).Due to Pigm gene have wide spectrum, persistently
Resistance and the advantages of do not influence yield, thus utilized extensively in the disease-resistant improvement of long-grained nonglutinous rice especially indica Hybrid Rice, pass through paddy
Sterile line river paddy A, middle paddy A, Gu Feng A, wide anti-13A and its cross combination that plum No. 4 and its derived varieties select are in production
Show good resistance.
Traditional rice blast resistance breeding is to be selected under abundant onset condition according to the phenotype of plant, and this mode is not
Only time-consuming effort and easily not high by environmental disturbances accuracy.Utilize base difference existing for target gene, exploitation specificity
It is to improve the best approach of the anti-rice blast rice efficiency of selection of gene containing Pigm that molecular labeling, which carries out assisted Selection,.To improve Pigm
The efficiency of selection of gene, existing research person identifies its resistant genotype using different types of molecular labeling, and filters out one
A little new materials with target gene homozygosis.However, these labels also expose problems during actual selection, such as
S29742, S95477 are the labels of close linkage, and not only polymorphism is poor in different parents, but also accuracy is not high；And
Although M26205, M80375, M80410 and target gene isolate, the polymorphism between parent, as dominant marker
Disease-resistant homozygous and heterozygous genotypes (Deng Y W et al., Genetic characterization and can not effectively be distinguished
fine mapping ofthe blast resistance locus Pigm(t)tightly linked to Pi2and
Pi9in a broad-spectrum resistant Chinese variety,TheorAppl Genet,2006,113(4):
705-713；Deng Y W et al.,Epigenetic regulation ofantagonistic receptors
confers rice blast resistance with yield balance,Science,2017,355(6328):962-
965).2018, the once equal one section of distinguished sequence that No. 4 genomes of paddy plum are obtained by sequencing means of raw member, and develop insertion/
(Insertion/Deletion, InDel) label is lacked, realizes the specific detection to Pigm gene, but be related to polyacrylamide
The detection of electrophoresis, process is relatively cumbersome (once to give birth to member etc., the exploitation of Pigm specific selection markers and its in japonica rice panicle blast resistance
Utilization in breeding, rice in China science, 2018,32 (5): 453-461).
Competitive ApoE gene (KompetitiveAllele-Specific PCR, KASP) molecular labeling
It is built upon allele specific amplification (Amplification Refractory Mutation System, ARMS) and height
A kind of novel SNP classifying method on the fluorescence detection basis of sensitivity.Its principle is designed for allele SNP site
Two forward primers and a general reverse primer, every forward primer have specific sequence, can be from different fluorescent marker knots
It closes.With from the DNA of the forward primer of different fluorescence binding sequences and general reverse primer PCR amplification sample to be tested, equipotential
Variation can be able to reflection (He C L, et al.SNP genotyping:the KASP by different fluorescence signals
assay.Methods Mol Biol,2014,1145,75-86).The technology is since flux is high, stability is good and cheap
The advantages that and be widely used in mesh in the excavation, identification and breeding process of crop germplasm resource important character associated alleles
Mark screening (Rosas J E, et al., One-step, the codominant detection of of genotype
imidazolinone resistance mutations in weedy rice(Oryza sativa L.),Electronic
J Biotechnol,2014,17(2):95-101；Field space etc., the KASP marker development in the site anti-soybean cyst nematode Heterodera glycines SCN3-11
And utilization, Acta Agronomica Sinica, 2018,44 (11): 1600-1611).
Therefore, the identification and breed breeding efficiency to improve the rice blast of gene containing Pigm Resistant gerplasm resource, it is necessary to build
A kind of Genotype of new based on PCR technology is found to distinguish the different genotype of Pigm.
Three, summary of the invention
Technical problem: the present invention is directed to the time-consuming effort of traditional rice blast resistance breeding of gene containing Pigm and is easily done by environment
It is not high to disturb accuracy, and existing molecular labeling polymorphism in different parents is poor, accuracy is not high, detection process is relatively cumbersome etc.
Disadvantage.By largely comparing and versatility needs of later-stage utilization specific position exploitation label between different cultivars
It carries out verifying and finds the variant sites in the plum of gene valley containing Pigm No. 4 with versatility, design KASP molecular labeling primer is come fast
Speed, the genotype of precise Identification rice Pigm, to reach the identification of the rice blast Resistant gerplasm resource high-efficiency of gene containing Pigm and kind
Quickly select the purpose of selection.
Technical solution:
A kind of KASP molecule labelling method for identifying resistance gene of rice blast Pigm, it is characterised in that:
Pigm gene KASP primer sequence are as follows:
Forward primer Pigm-F-G sequence is 5 '-AAAAATGAAAATAAAAATGGTATGATGGTTTCG-3 '
Forward primer Pigm-F-C sequence is 5 '-AAAAATGAAAATAAAAATGGTATGATGGTTTCC-3 '
Reversed 5 '-AGGGATGAAACGGCTCGAAAACGAA-3 ' of universal primer Pigm-R sequence.
The KASP molecule labelling method of the detection resistance gene of rice blast Pigm, which is characterized in that synthesizing
When Pigm gene KASP molecular labeling primer, the 5 ' ends of the forward primer Pigm-F-G are plus the glimmering of Fluoresceincarboxylic acid (FAM)
Optical signal label；5 ' the ends of forward primer Pigm-F-C add the fluorescence signal mark of 5- chlordene fluorescein phosphoramidate (HEX)
Label.
The molecule labelling method, it is characterised in that:
(1) extraction of rice plant genomic DNA；
(2) same PCR reaction system is added in molecular labeling primer described in claim 1,2, and is arranged 2 with double steamings
Water replaces the blank control of sample template DNA, to Rice Germplasm Resources or breeding population plant on fluorescence quantitative PCR instrument
DNA is expanded；
(3) data analysis carries out on Step One Software v2.2.2 software.
10 μ L reaction systems include 0.1 μ L, 2 × KASP of sample template DNA (10ng/uL) 1.0 μ L, fluorescent primer mix anti-
Answer 5.0 μ L of mixed liquor, 3.9 μ L of distilled water.
Reaction condition includes 94 DEG C of activation 15min；94 DEG C of denaturation 20sec, 61~55 DEG C of annealing 60sec (each circulations
Reduce by 0.6 DEG C), 10 circulations；94 DEG C of denaturation 20sec, 55 DEG C of annealing 60sec, 26 recycle,
Differentiated according to the color of datagram and position, wherein the blue dot expression close to Y-axis is to carry G equipotential to become
The homozygote of the different resistant gene containing Pigm；It indicates to be carrying C allelic variation without Pigm resistance close to the red spots of X-axis
The homozygote of gene；X, green dot among Y-axis indicate be carry simultaneously G/C allelic variation the resistant gene containing Pigm it is miscellaneous
It is fit；Black box indicates it is the blank control that sample template DNA is replaced with distilled water.
The molecule labelling method can be applied in the anti-rice blast rice breeding of gene containing Pigm.
Beneficial effect
There are many different cultivars Pigm gene nucleotide variant sites, but to find the common variant sites between different cultivars
With regard to more difficult.By taking Xian, round-grained rice sequencing kind 93-11 and OryzasativaLcv.Nipponbare as an example, their Pigm-R2 gene orders corresponding with Gu Mei 4
Consistency on nucleotide is only 94~95% (gene order is about 8500bp), it means that Gu Mei 4 and OryzasativaLcv.Nipponbare,
The nucleotide diversity site of 93-11 is 425~510, and different interracial nucleotide diversities are increasingly complex, some nucleotide
Variation may be variant in some kind, and may then not have difference in another kind, therefore to find different cultivars
Between common function variant sites it is just more difficult.It needs largely to compare and the later-stage utilization specific position is developed
The versatility between different cultivars is marked to be verified.The ingredient for being also required to some fortune simultaneously can just be found.
The present invention is by introducing the blast resisting of gene containing Pigm place rice variety paddy plum No. 4, to paddy plum No. 4 and 4
Xian, the round-grained rice kind (basis that OryzasativaLcv.Nipponbare, 93-11, precious Shan 97, the bright site extensive 63) Pigm Pigm-R2 genome sequence compare is sequenced
On, specific nucle variant sites are found, discovery paddy plum No. 4 has the nucleotide site of covariation with above-mentioned 4 kinds
There are 23, wherein 12 are 1~3 nucleotides inserted/missing, is not suitable for carrying out the label design of four primer molecules, and it is remaining
11 sites then have and have different mononucleotide differences with paddy plum No. 4, design KASP molecule mark for wherein 10 sites
Note, fails to detect variant KASP fluorescence signal, thus it is speculated that there is also other homologous cores in different cultivars in these sites
Nucleotide sequence.
Fortunately, Pigm-R2 gene start codon upstream 515bp in the plum of gene valley containing Pigm No. 4 is had found
Locate and has been sequenced without a single base variation (G/C), the KASP molecule mark of successful design existing for Pigm trans-genetic hybrid rice kind
Pigm resistant gene variant sites different genotype can be identified by remembering, can not only be fast and accurately using the labeling method
It identifies the different genotype of Pigm in Rice Germplasm Resources or breeding population, and is able to achieve the high throughput to more sample to be tested
Detection, has the advantages that more efficiently compared with common molecular labelling technique.Therefore, it can be further improved in breeding to containing
The efficiency of selection of the disease-resistant homozygous genotype of Pigm accelerates the breeding process of anti-rice blast rice kind.
" a kind of KASP molecule labelling method for identifying resistance gene of rice blast Pigm " provided by the present invention, has
Following advantages:
(1) molecular labeling provided by the invention is according to the special of the variance site of No. 4 Pigm genes of paddy plum design
Property label, it can directly reflect the genotype and phenotype of plant, there is no due to label and it is intergenic heredity exchange and cause
The mistake of genotype identification；
(2) molecule labelling method provided by the invention, which is able to achieve, originates Pigm-R2 gene in the plum of gene valley containing Pigm No. 4
Single base variation (G/C) is detected at the 515bp of codon upstream.Since reported rice blast resistance variant sites are more,
It is difficult effectively to be distinguished by phenotype, the detection to above-mentioned site may be implemented by means of the present invention, without logical
Sequencing and a large amount of, complicated genetic tests are crossed to determine its variant sites；
(3) molecule labelling method provided by the invention can be educated effective for the auxiliary of the disease resisting rice kind of rice blast containing Pigm
Kind.Due to mainly sufficiently being sent out by natural occurrence or the method for artificial infection in traditional breeding method the identification of rice blast resistance
Carry out what observation obtained according to the phenotype that plant falls ill in the case where disease.This mode is not only by the shadow in vine growth and development stage
It rings, and time-consuming effort, not high vulnerable to environmental disturbances accuracy.Utilize a kind of identification resistance gene of rice blast Pigm's
KASP molecule labelling method can extract DNA after germination and be identified the homozygous genotype of Pigm gene, selected, be washed in a pan
The single plant for eliminating a large amount of non-targeted genotype, effectively improves the efficiency of selection of the anti-rice blast rice kind of gene containing Pigm, shortening is educated
Kind period and reduction breeding cost.
(4) compared with the InDel molecular labeling of existing identification Pigm genotype, the present invention uses KASP molecule labelling method
Pigm resistant gene variant sites different genotype is identified, its homozygous and heterozygous genotypes can not only be effectively distinguished, and
And can also not have to take a long time carry out electrophoresis detection, it is highly efficient, quick, it is suitable for a large amount of rice pest insects and breeding
Material is identified and is selected.
Four, Detailed description of the invention
Fig. 1 is used to detect the KASP molecular labeling layout strategy of Pigm genotype
(position of the digital representation base in No. 4 Pigm genome sequences of paddy plum；Shadow representation is in paddy plum No. 4, Japan
Fine, bright extensive 63, the base that precious Shan 97 and 93-11 have differences；Shade italic indicates special single nucleotide variations；Overstriking letter
Indicate Pigm gene KASP primer sequence)
Detection of Fig. 2 KASP molecular labeling to different rice varieties (strain) Pigm genotype
(black box represents the blank control (A) without template DNA；Close to the blue dot (B) of Y-axis and close to the red of X-axis
Color (C) dot, which respectively represents the homozygote-Gu Mei 4 for carrying the resistant gene containing Pigm of G allelic variation and carries C equipotential, to be become
Different homozygote-the IRBL9-W (Pi9) without Pigm resistant gene, IRBLz5-CA-1 (Pi2), IRBLz-Fu (Piz),
IRBLzt-T (Piz-t), Zhenhui 084,93-11, in extensive 8006, Cheng Hui 727, Restorer line Yihui1577, it is red extensive 589, first extensive 527, it is bright extensive
63, navigate No. 1, Jin Hui 2, wide extensive 998, Ning Xiang 1B, river perfume (or spice) 29B, an aromatic plant metioned in ancient books 93-11B, Zhenshan 97B, 03S, Bph68S, P88S, Peiai
64S, OryzasativaLcv.Nipponbare, more light, a product, zhenfu 10, Chu's round-grained rice 39, finish round-grained rice 44, precious agriculture 34, Zhejiang round-grained rice 97, good 58, southern round-grained rice 505, southern round-grained rice
9108, Huaidao 9, Henan round-grained rice 8, face rice 17, holy rice 20, peaceful round-grained rice 40, her round-grained rice 12, new rice No. 32, saliva rice 263, Shen Nong
265, distant round-grained rice 10, salt is rich 47, lucky round-grained rice 88 and imperial round-grained rice 29)
Fig. 3 KASP molecular labeling is to No. 4 F of Huaidao 9/Gu Mei2Detection (the black side of population segment single plant Pigm genotype
Frame represents the blank control (A) without DNA profiling；Blue dot (B) close to Y-axis, the red spots (C) and X, Y-axis close to X-axis
Intermediate green (D), which respectively represents, to be carried the homozygote of the resistant gene containing Pigm of G allelic variation, carries C allelic variation not
The homozygote of the resistant gene containing Pigm and carry simultaneously G/C allelic variation the resistant gene containing Pigm heterozygote and)
Fig. 4 parent's susceptible variety Huaidao 9, disease-resistant variety paddy plum No. 4 and its F1Single plant, F2Group's panicle blast artificial infection
The incidence of identification
(A: be free of the homozygotic rice varieties Huaidao 9 of Pigm resistant gene；B: the rice varieties of the resistant gene containing Pigm
Paddy plum No. 4；C: No. 4 F of Huaidao 9/Gu Mei of the heterozygote of resistant gene containing Pigm1Single plant；D: Huaidao 9/Gu Mei No. 4 F2Group
Plant part)
Fig. 5 using Pigm gene orderly improvement long-grained nonglutinous rice keep be an aromatic plant metioned in ancient books 93-11B rice blast resistance technology path
The holding of Fig. 6 long-grained nonglutinous rice is an aromatic plant metioned in ancient books 93-11B and the disease-resistant Improved lines of the rice blast of gene containing Pigm
(A: being an aromatic plant metioned in ancient books 93-11B single plant without the homozygotic long-grained nonglutinous rice holding of Pigm resistant gene；B: resistant gene containing Pigm is pure
The fit disease-resistant Improved lines single plant of rice blast)
Five, specific embodiment
A kind of " KASP molecule labelling method for identifying resistance gene of rice blast Pigm " its specific implementation step is as follows:
(1) test material (following material be public, Jiangsu Province agricultural germ plasm resource mid-term library can externally provide)
The material of homozygous genotype containing Pigm: Gu Mei 4, Sichuan Local Indica Rice.
Not anti-Pigm homozygous genotype material: general sense kind Lijiang xintuanheigu is 4 near isogenic lines of recurrent parent
IRBL9-W (Pi9), IRBLz5-CA-1 (Pi2), IRBLz-Fu (Piz), IRBLzt-T (Piz-t) (International Rice is drawn,
IRRI), Indica Rice Restorer Lines material Zhenhui 084 (Jiangsu Hilly Ground Zhenjiang Agriculture Science Research Institute), 93-11 (Lixiahe District
Regional Institute of agricultural sciences), in extensive 8006 (China Paddy Rice Inst-Zhejiang), (the sichuan agriculture academy of sciences crop of Cheng Hui 727
Research institute), Restorer line Yihui1577 (Sichuan Yibin City Academy of Agricultural Sciences), red extensive 589 (Guizhou Research Institute of Rice), first extensive 207 (Hunan
Hybrid rice research center), bright extensive 63 (Sanming, Fujian Province city Institute of agricultural sciences), boat No. 1 (Fujian Academy of Agricultural Sciences's rice and kernel
Research institute), Jin Hui 2 (Guangxi Yang Lijian breeding), wide extensive 998 (Inst. of Rice, Guangdong Academy of Agricultural Sciences)；Long-grained nonglutinous rice is kept
Based material rather perfume 1B (Cereal Crops Research Inst., Jiangsu Agricultural Science Academy), river perfume (or spice) 29B (sichuan agriculture academy of sciences crop investigations
Institute), an aromatic plant metioned in ancient books 93-11B (Anhui Quanyin Gaoke Seed Industry Co.ltd), Zhenshan 97B (Wenzhou, Zhejiang Province Institute of agricultural sciences)；
Long-grained nonglutinous rice two-line sterile line 03S (AnHui QuanYin Agriculture High Science Research Institute), Bph68S (Hubei Wuhan University school of life and health sciences),
P88S (Hunan Research Centre for Hybrid Rice), Peiai 64S (Hunan Research Centre for Hybrid Rice)；Conventional japonica rice kind includes Japan
Fine (Aichi, Japan agriculture examination hall), more light (Prefectura de Fukui, Japan agricultural experiment station), a product (South Korea), zhenfu 10 (South Korea), Chu
Round-grained rice 39 (Dongzhou Period in Chuxiong state Institute of agricultural sciences), finish round-grained rice 44 (Guizhou Bijie District Agricultural Science Institute), precious agriculture 34 (on
Sea market Baoshan District agricultural seed stock breeding station), Zhejiang round-grained rice 97 (Zhejiang Academy of Agricultural Science crop and nuclear technology research on utilization institute), good 58
(Jiaxing City, Zhejiang Province academy of agricultural science), southern round-grained rice 505 (Cereal Crops Research Inst., Jiangsu Agricultural Science Academy), southern round-grained rice 9108
(Cereal Crops Research Inst., Jiangsu Agricultural Science Academy), Huaidao 9 (Jiangsu Xuhuai Zone Huaiyin Agricultural Sciences Institute), Henan round-grained rice
No. 8 (Xinxiang City Agricultural Science Inst., Henan Prov.), face rice 17 (Shandong Province Yinan County rice research institute), holy 20 (Shandong Province of rice
Rice research institute, Academy of Agricultural Sciences), peaceful round-grained rice 40 (crops research institute, Ningxia Academy of Agri-Forestry Sciences), her No. 12 (Xinjiang Yili of China of round-grained rice
State Institute of agricultural sciences), new rice No. 32 (Xinjiang Yili of China state Institute of agricultural sciences), (the Tianjin rice research of saliva rice 263
Institute), Shennong-265 (ShenYang, Liaoning Province agriculture university), distant round-grained rice 10 (Liaoning Rice Research Institute), (Liaoning Province is saline and alkaline for salt rich 47
Ground utilizes research institute), lucky round-grained rice 88 (Rice Inst., Jiling Academy of Agricultural Science), 29 (Heilongjiang Institute of Agricultural Sciences's water of imperial round-grained rice
Rice research institute).
Other vegetable materials include with susceptible japonica rice variety Huaidao 9 for female parent, and disease-resistant rice variety paddy plum No. 4 are male parent
Hybridize the F of combo1Plant and 300 F2Single plant.
Contain ZB used by the artificial resistance inoculated identification of panicle blast3The bacterium solution of biological strain is planted by Jiangsu Province Agriculture Science Institute
Object Protective strategy externally provides, and spore concentration is about 5 × 104A/mL.The above plant and rice blast bacterial strain material are known
Public material, vegetable material Jiangsu Province agricultural germ plasm resource mid-term library freely can externally provide, and contain ZB3The bacterium solution of biological strain
It can externally be provided with compensation by Plant Protection Inst., Jiangsu Academy of Agriculture.Particular reference are as follows: in national rice data
The heart (http://www.ricedata.cn/variety/index.htm), Deng et al., Genetic
characterization and fine mapping of the blast resistance locus Pigm(t)
tightly linked to Pi2and Pi9in a broad-spectrum resistant Chinese variety,
Theoretical and Applied Genetics,2006,113(4):705-713；Chen Feng etc., South Korea introduce rice varieties
Identification and Utilization assessment, Shandong agricultural sciences, 2016,48 (11): 26-28；First east woods etc., rice is high-quality, high yield, Duo Kangxin
The breeding and popularization of kind " zhenfu 10 ", Yanbian University's agricultural journal, 2001,23 (2): 111-114；Fu Chongyun etc., Lijing
Newly roll into a ball black paddy near isogenic lines blast resisting analysis, Acta Agronomica Sinica, 2006,32 (6): 799-804；Lu Fan etc., Jiangsu Province's rice blast
The differentiation of Biological Strains of The Pest and correlation with rice varieties, the differentiation of Jiangsu Province's physiological races of rice blast fungus and and rice
The correlation of kind, Agricultural University Of Nanjing's journal, 1999,22 (4): 31-34)
(2) nucleic acid sequence analysis
No. 4 Pigm locus gene group sequence 178704bp of paddy plum, by 13 NBS-LRR class disease-resistant gene (Pigm-R1
~Pigm-R13) and largely turn element (1 Ty3/gypsy class retrotransposon, 13 long terminal repeats and 33
DNA transposons) composition.Since Pigm-R2 gene is widely present in wild rice and cultivated rice in the site Pigm, the guarantor with height
Keeping property, while in view of the coding sequence homology of Pigm-R2 and Pigm-R4, Pigm-R6, Pigm-R8, Pi9, Pi2 are higher,
Therefore selection Pigm-R2 gene 5 ' end upstream (10445~11580bp) sequence and intron sequences and sequencing japonica rice variety Japan
Fine genome sequence carries out sequence analysis, and in relevant range, there are 271 mononucleotides are more with OryzasativaLcv.Nipponbare for discovery paddy plum No. 4
State property makes a variation (https: //www.ncbi.nlm.nih.gov/).The sequence for selecting each 200bp of SNP site upstream and downstream, utilizes China
In middle agriculture university's genome database bright extensive 63, precious Shan 97 and 93-11 genome sequence compared again (http: //
Rice.hzau.edu.cn/), discovery paddy plum No. 4 then has on 11 mononucleotide sites and paddy plum 4 with above-mentioned 4 kinds
Number different difference fails to detect variant KASP fluorescence letter for the KASP molecular labeling of wherein 10 sites design
Number, thus it is speculated that there is also other homologous nucleotide sequences in different cultivars in these sites.Fortunately, one is had found to contain
It at Pigm-R2 gene start codon upstream 515bp and has been sequenced without Pigm trans-genetic hybrid rice kind in Pigm gene valley plum No. 4
An existing single base makes a variation (G/C), and the KASP label of successful design can be to Pigm resistant gene variant sites different genes
Type is identified.Further the cloned sequence containing Pi9, Pi2 rice varieties 75-1-127 and C101A51 corresponding site is compared
To (https: //www.ncbi.nlm.nih.gov/), finding the SNP site, there is also differences in other kinds.This shows
The single nucleotide variations may specifically be present in No. 4 Pigm locus gene group sequences of Gu Mei (Fig. 1).
(3) exploitation of molecular labeling
The full length sequence of Nipponbare Pigm gene is downloaded, selects specific single nucleotide variant sites upstream and downstream each
The sequence of 50bp marks 5 primer sequences, the Tm value of comprehensive analysis primer, hairpin structure, two using the design of oligo 7.57
Complementary case between aggressiveness, primer with etc. the software of many factors give a mark situation, finishing screen selects the highest primer of score value
Combination, and transfer to LGC company, Britain Beijing office (https: //www.lgcgroup.com/cn) synthesis respectively band FAM and
The KASP primer (Fig. 1) of HEX.
(4) Markers for Detection genotype and resistance inoculated identification phenotype are mutually authenticated
(1) DNA is extracted
30 days after rice transplanting, rice varieties (strain), F are collected1Hybrid, F2The fresh young leaflet tablet of group, by CTAB
Method extracts genomic DNA (Murray M G, et al., Rapid isolation of high molecular weight
plant DNA,NucleicAcids Res,1980,8(19):4321-4325).Specific steps are as follows: 2-3cm rice tender leaf is taken to fill
Enter in the centrifuge tube of 2mL, is clayed into power after liquid nitrogen frozen with grind away stick；65 DEG C of thermostat water baths are added into ground material
The DNA extracting solution of 700 μ L 2%CTAB containing mass concentration ratio of middle preheating 30min, after mixing, is put into 35 DEG C of thermostat water bath
During which middle 30min shakes up once every 10min；Take out centrifuge tube, in draught cupboard every pipe be added 24:1 (chloroform: isoamyl alcohol=
24:1) 700 μ L, mixes well, and is put into 1,2000rpm in centrifuge and is centrifuged 15min, it is made to be divided into three layers；Aspirate supernatant 400
In the centrifuge tube of μ L and another 1.5mL sterilizing, 400 μ L isopropanols of -20 DEG C of pre-coolings are added, slowly mix, in -20 DEG C of refrigerators
Free settling 30min.Then, 10000rpm is centrifuged 10min, abandons supernatant；70% ethyl alcohol, 400 μ L is added to wash 1 time, abandons supernatant,
It air-dries；The TE buffer of 100-200 μ L is added, room-temperature dissolution sets -20 DEG C of preservations at DNA solution 1-2d；
(2) molecular labeling amplification and Genotyping
Same PCR reaction system is added in Pigm gene KASP molecular labeling primer, and is arranged 2 and mould is replaced with distilled water
The blank control of version DNA, expands the DNA of Rice Germplasm Resources or breeding population plant on fluorescence quantitative PCR instrument；
10 μ L reaction systems include 1.0 μ L of DNA (10ng/uL), 0.1 μ L, 2 × KASP Master of fluorescent primer mix
Mix5.0 μ L, 3.9 μ L of distilled water.
Reaction condition includes 94 DEG C of activation 15min；94 DEG C of denaturation 20sec, 61~55 DEG C of annealing 60sec (each circulations
Reduce by 0.6 DEG C), 10 circulations；94 DEG C of denaturation 20sec, 55 DEG C of annealing 60sec, 26 recycle.
PCR result is analyzed using Step One Software v2.2.2 software.
(3) parent's Huaidao 9, Gu Mei 4 and its F1Single plant, F2Group's single plant panicle blast Resistance Identification and evaluation
Contain ZB used by the artificial resistance inoculated identification of rotten neck3The bacterium solution of biological strain, spore concentration is about 5 ×
104A/mL.5 days before rice plant booting cut, to No. 4 F of Huaidao 9/Gu Mei2The single plant of group carries out injection inoculation, often
Strain 3 spikes of rice of injection, every fringe are inoculated with 1mL bacterium solution.Inoculation selection, to avoid the evaporation of bacterium solution, influences after that afternoon 3:00
Effect of inoculation.The loss late of panicle blast incidence survey is the average value of 3 tassel loss lates, and severity Scaling is referring to NY/T 2646-
2014 carry out (The Ministry of Agriculture of the People's Republic of China, MOA's .NY/T 2646-2014 rice varieties test blast resistance identification and evaluation
The Beijing technical regulation: China Standards Press, 2014).Wherein, 0 grade (highly resistance, HR): disease-free；1 grade (anti-, R): sprig stalk hair
Disease, fringe average loss rate≤5%；3 grades (in resist, MR): main shaft or fringe neck morbidity, 5% < fringe average loss rate≤20%；5 grades
(middle sense, MS): main shaft or fringe neck morbidity, grain half is flat, 20% < fringe average loss rate≤50%；7 grades (sense, S): fringe neck hair
Disease, most of shrivelled kernel, between 50% < fringe average loss rate≤70%；9 grades (height sense, HS): fringe neck morbidity, fringe average loss rate >
70%, wherein 0,1,3 grade be denoted as it is disease-resistant, 5,7,9 grades be denoted as it is susceptible.
(4) interpretation of result
1. KASP molecular labeling is to different cultivars (strain) Pigm genotyping
48 parts of Xian, japonica rice variety (strain) are carried out on real-time fluorescence quantitative PCR instrument using KASP molecular labeling primer
It expands and carries out Genotyping, the results showed that the molecular labeling primer can clearly separate two kinds of genotype, wherein close
The blue dot of Y-axis is the homozygote for carrying the resistant gene containing Pigm of G allelic variation, and the red spots close to X-axis are to carry C
The homozygote without Pigm resistant gene of allelic variation, black box are blank control (Fig. 2).It is leaned on according to the judgement of point sample sequence
Nearly Y-axis blue dot is kind paddy plum No. 4 of the homozygous genotype containing Pigm, other 47 parts represented close to the red spots of X-axis
Material is the kind (strain) without Pigm homozygous genotype.
2. KASP molecular labeling is to No. 4 F of Huaidao 9/Gu Mei2Group's Pigm genotyping
It is verifying KASP molecular labeling to the detection effect of heterozygous genotypes, to No. 4 F of Huaidao 9/Gu Mei2Group 300
The DNA of single plant carries out genotyping, and KASP primer still can effectively be distinguished (Fig. 3) to three kinds of genotype.Wherein lean on
The blue dot of nearly Y-axis is the homozygote for carrying the resistant gene containing Pigm of G allelic variation, and the red spots close to X-axis are to take
The homozygote without Pigm resistant gene with C allelic variation, the green dot among X, Y-axis are to carry G/C allelic variation
The heterozygote of the resistant gene containing Pigm, black box are blank control.Homozygote, the base of resistance containing Pigm of the resistant gene containing Pigm
The heterozygote of cause and homozygotic plant number without Pigm resistant gene are respectively 62,166 and 72 plants (table 1).Statistical
Analysis result meets the segregation ratio for 1: 2: 1, meets the law of segregation (χ of 1 pair of gene2=4.08 < χ2 0.05,2, 0.10 < P < 0.25).
3. No. 4 F of Huaidao 9/Gu Mei2The relationship of group difference Pigm genotype single plant and panicle blast resistance
It is artificial using panicle blast for the relationship probed between the Pigm genotype and rice blast resistance that molecular markers for identification goes out
F of the method for inoculation to susceptible variety Huaidao 9, disease-resistant variety paddy plum No. 4 and hybridization1Plant and F2The single plant of group carries out
Resistance Identification.Huaidao 9, Gu Mei 4 and its F1Fringe loss late be respectively 82.65%, 6.82% and 7.65%, corresponding disease
Feelings grade is respectively high sense, resists, is anti-.By to F2300 single plants of group carry out the statistics of state of an illness grade, find close to Y-axis
Blue dot is the homozygote of the resistant gene containing Pigm for carrying G allelic variation and the green dot among X, Y-axis is carrying
It is flat that 228 single plants of heterozygote of the resistant gene containing Pigm of G/C allelic variation all show as highly resistance, anti-and middle water resistant, and leans on
The red spots of nearly X-axis are only 67 tables in 72 single plants of homozygote without Pigm resistant gene for carry C allelic variation
Be now middle sense, sense and high sense, and have 5 single plants show it is anti-and in resist (Fig. 4).
No. 4 F of 1 2017 years Huaidao 9/Gu Mei of table2Resistance of 3 kinds of Pigm genotype single plants to panicle blast in group
For exclude as be inoculated with it is improper caused by judge by accident, continue collect 5 single plants seed.Kind continues at strain within 2018
Inoculation, each 5 plants of strain inoculation, every plant of 3 fringes, and count state of an illness grade.As a result 5 single plants have 4 to show as middle sense, 1 table
It is now sense.This not only illustrates that the four primer molecules labeling method can fast and accurately identify Pigm different genotype, together
When Resistant segregation than also confirm that the resistance in Gu Mei 4 be by Pigm this to dominant gene control.
(6) molecule labelling method of the present invention is used for the breeding of the anti-rice blast rice of gene containing Pigm
Selecting the rice paddy of Pigm containing resistant gene plum No. 4 is donor, and sense rice blast long-grained nonglutinous rice high-combining ability holding is an aromatic plant metioned in ancient books 93-
11B is receptor, using the strategy of back cross breeding, the rice blast resistance of orderly improvement an aromatic plant metioned in ancient books 93-11B.In continuous backcross and selfing
In the process, tracing detection is carried out using KASP label of the invention, and combines the Systematic selection of economical character, until BC2F5In generation, obtains
With rice blast resistance, other economical characters and original parent an aromatic plant metioned in ancient books 93-11B almost the same improvement strain (Fig. 5, Fig. 6).This table
It is bright using molecule labelling method of the invention can the Pigm gene to rice breeding group select, realize blast resisting water
The efficient breeding of rice varieties.
Sequence table
<110>Jiangsu Province Agriculture Science Institute
<120>a kind of KASP molecule labelling method for identifying resistance gene of rice blast Pigm
<141> 2019-08-05
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aaaaatgaaa ataaaaatgg tatgatggtt tcg 33
<210> 2
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aaaaatgaaa ataaaaatgg tatgatggtt tcc 33
<210> 3
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
agggatgaaa cggctcgaaa acgaa 25
Claims (6)
1. a kind of KASP molecule labelling method for identifying resistance gene of rice blast Pigm, which is characterized in that
Pigm gene KASP primer sequence are as follows:
Forward primer Pigm-F-G sequence is 5 '-aaaaatgaaaataaaaatggtatgatggtttcg-3 '
Forward primer Pigm-F-C sequence is 5 '-aaaaatgaaaataaaaatggtatgatggtttcc-3 '
Reversed 5 '-agggatgaaacggctcgaaaacgaa-3 ' of universal primer Pigm-R sequence.
2. the KASP molecule labelling method of identification resistance gene of rice blast Pigm according to claim 1, feature
It is, when synthesizing Pigm gene KASP molecular labeling primer described in claim 1, the 5 ' of the forward primer Pigm-F-G
End adds the fluorescence signal label of Fluoresceincarboxylic acid FAM；5 ' the ends of forward primer Pigm-F-C add 5- chlordene fluorescein amino
The fluorescence signal label of phosphate HEX.
3. molecule labelling method according to claim 2, it is characterised in that:
(1) extraction of rice plant genomic DNA；
(2) same PCR reaction system is added in molecular labeling primer as claimed in claim 2, and is arranged 2 and is replaced with distilled water
The blank control of sample template DNA carries out the DNA of Rice Germplasm Resources or breeding population plant on fluorescence quantitative PCR instrument
Amplification；
(3) data analysis carries out on Step One Software v2.2.2 software.
4. molecule labelling method according to claim 3, it is characterised in that:
10 μ L reaction systems include that 0.1 μ L, 2 × KASP reaction of DNA1.0 μ L, fluorescent primer mix of sample template 10ng/uL is mixed
Close 5.0 μ L of liquid, 3.9 μ L of distilled water；
Reaction condition includes 94 DEG C of activation 15min；94 DEG C of denaturation 20sec, 61~55 DEG C of annealing 60sec, each circulation reduce
0.6 DEG C, 10 circulations；94 DEG C of denaturation 20sec, 55 DEG C of annealing 60sec, 26 recycle.
5. molecule labelling method according to claim 3 or 4, it is characterised in that:
Differentiated according to the color of Step One Software v2.2.2 software data analysis chart and position, wherein close to Y
The blue dot of axis indicates it is the homozygote for carrying the resistant gene containing Pigm of G allelic variation；Red spots close to X-axis indicate
It is the homozygote without Pigm resistant gene for carrying C allelic variation；X, the green dot expression among Y-axis is to carry G/ simultaneously
The heterozygote of the resistant gene containing Pigm of C allelic variation；Black box indicates it is the blank that sample template DNA is replaced with distilled water
Control.
6. application of the molecule labelling method described in one of claim 1-5 in the anti-rice blast rice breeding of gene containing Pigm.
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