CN106148335B - The molecular labeling of No. 4 blast resistant gene Pigm of paddy plum and its application - Google Patents
The molecular labeling of No. 4 blast resistant gene Pigm of paddy plum and its application Download PDFInfo
- Publication number
- CN106148335B CN106148335B CN201610852523.8A CN201610852523A CN106148335B CN 106148335 B CN106148335 B CN 106148335B CN 201610852523 A CN201610852523 A CN 201610852523A CN 106148335 B CN106148335 B CN 106148335B
- Authority
- CN
- China
- Prior art keywords
- pigm
- rice
- molecular labeling
- extensive
- molecular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the molecular labeling of No. 4 blast resistant gene Pigm of paddy plum a kind of and its applications.The molecular labeling includes No. 4 specific molecular marker Pigm-4 of paddy plum, the molecular labeling Pigm-4 is to include the DNA fragmentation having differences at No. 6 chromosome 10436119bp~10436192bp of rice OryzasativaLcv.Nipponbare genome, and No. 4 this section of sequence deletions of paddy plum, the upstream and downstream primer of Pigm-4 primer pair is respectively as shown in SEQ ID NO:2 and SEQ ID NO:3.Pigm upstream region of gene molecular labeling primer is to the upstream and downstream primer of Pigm-2 respectively as shown in SEQ ID NO:4 and SEQ ID NO:5.The invention also discloses application of the molecular labeling in the breeding of molecular marker assisted selection anti-rice blast rice and the blast resisting infection ability for passing through the method raising rice of molecular marker assisted selection.Molecular labeling specificity of the invention is good, chain even closer with target area.
Description
Technical field
The invention belongs to Rice molecular breeding technical fields, more particularly it relates to a kind of No. 4 blast resistings of paddy plum
The molecular labeling of gene Pigm and its application.
Background technique
Rice blast is one of three big Major Diseases of Rice Production in global range, and cultivating disease-resistant variety is prevention and treatment rice blast
Endanger most economical effective means.Traditional paddy disease-resistant breeding is to carry out Phenotypic Selection to plant by Resistance Identification, is expended
Time is long, and the limitation vulnerable to environmental condition, qualification result are easy to cause error, and efficiency of selection is lower.It is auxiliary by molecular labeling
It helps selection disease-resistant gene not influenced by above-mentioned unfavorable factor then, is one of the effective way for cultivating disease-resistant variety.
Paddy plum No. 4 are a region kinds, lasting rice blast resistance, disease-resistant major gene resistance Pigm with wide spectrum
(t) (hereinafter abbreviated as Pigm) is positioned between the 6th chromosome molecular labeling C5483 and C0428 (paper in the range of about 70kb
Source: Theor Appl Genet, 2006,113:705-713).But the molecular labeling announced in document is mostly CAPS mark
Note, complicated for operation and cost is higher, is not suitable for molecular marker assisted selection, remaining SSR and InDel label are mainly used for heredity
Positioning, it is lower that there are polymorphic rates in breeding, the lesser disadvantage of difference.Pan Cunhong etc. develops (patent application: CN
103497996A) for detecting the molecular labeling InDel587 of No. 4 blast resistant gene Pigm of paddy plum, but there are still about 15%
Receptor parent (especially indica type parent's ratio is higher) and paddy plum No. 4 without polymorphic, marking path target interval is farther out
(65.2kb) two defects.
In addition, existing research has proven to Gu Mei 4 disease-resistant main effect Pigm and is located on the seat Piz.It is had found on the seat Piz
The multiple alleles such as Pi2, Pi9, Pi-zt, Piz, Pi40, Pigm, but there is some difference for respective gene order and anti-spectrum,
It realizes and the utilization of Pigm is just needed to distinguish Pigm from other different multiple alleles, to realize Pigm is special
Among ground transformation to receptor kind.
Summary of the invention
Goal of the invention: for label existing in the prior art specificity it is insufficient and with target gene is chain untight asks
Topic, an object of the present invention are to provide the molecular labeling of No. 4 blast resistant gene Pigm of Gu Mei, the second object of the present invention is to
The primer for detecting the molecular labeling is provided, the third object of the present invention is to provide the application of the molecular labeling.
Technical solution: the molecular labeling of No. 4 blast resistant gene Pigm of paddy plum of the present invention, including molecular labeling
Pigm-4, the molecular labeling Pigm-4 be comprising be located at No. 6 chromosome 10436119bp of rice genome~
The DNA fragmentation (being located among AP005659) at 10436192bp, No. 4 this section of base deletions of Gu Mei.The present invention is in description gene
It is on the basis of rice OryzasativaLcv.Nipponbare genome when group base positions.
The fragment length of the molecular labeling Pigm-4 is 150~500bp.The fragment length is conducive to using electrophoresis
Method carries out recognition detection, and electrophoresis method is simple and easy, at low cost.Certainly, the fragment length of above-mentioned molecular labeling Pigm-4
Be not it is necessary, when being such as sequenced using other detection methods, then do not limited by above-mentioned length.
Further, the molecular labeling Pigm-4 is the DNA fragmentation comprising the sequence as shown in SEQ ID NO.1.Into
One step, the sequence of the molecular labeling Pigm-4 is as shown in SEQ ID NO.1.
Further, the molecular labeling further includes the molecular labeling being used cooperatively with minute mark note Pigm-4
Pigm-2, the molecular labeling Pigm-2 be comprising be located at No. 6 chromosome 10366681bp of rice genome~
DNA fragmentation at 10366821bp.Further, the molecular labeling Pigm-2 is to dye positioned at rice genome the 6th
DNA fragmentation at body 10366681bp~10366821bp.
Pigm-4 can distinguish the anti-sense difference of Pigm gene, because itself and target gene are there is also certain genetic distance,
The screening accuracy of single marking reaches 99.01% (rather than 100%), and two labels of Pigm-4 and Pigm-2 are used cooperatively
Shi Ze has completely included the whole fragment where Pigm, and the accuracy of screening reaches 100%.
The present invention also provides a kind of amplimer, primer is Pigm-4 primer pair or/and Pigm-2 primer pair, wherein
The upstream and downstream primer of Pigm-4 primer pair respectively as shown in SEQ ID NO:2 and SEQ ID NO:3, Pigm-2 primer pair it is upper,
Downstream primer is respectively as shown in SEQ ID NO:4 and SEQ ID NO:5.
The present invention also provides the molecular labeling answering in molecular marker assisted selection anti-rice blast rice breeding
With.
The application includes: that blast resistant gene Pigm is transferred to receptor parent, utilizes the Marker-assisted selection
The rice material of the Pigm containing disease-resistant gene.
When wherein, using the Marker-assisted selection rice material, using Pigm-4 primer pair or/and Pigm-2 primer
To expanding, wherein the upstream and downstream primer of Pigm-4 primer pair respectively as shown in SEQ ID NO:2 and SEQ ID NO:3,
The upstream and downstream primer of Pigm-2 primer pair is respectively as shown in SEQ ID NO:4 and SEQ ID NO:5.
The present invention also provides the molecular labelings to improve the anti-of rice in the method by molecular marker assisted selection
Application in rice blast infection ability.The described application includes: that blast resistant gene Pigm is transferred to receptor parent, using described
Marker-assisted selection Pigm containing disease-resistant gene rice material.When wherein, using the Marker-assisted selection rice material,
Above-mentioned Pigm-4 primer pair or/and Pigm-2 primer pair can be used to be expanded.
Compared with prior art, the invention has the benefit that
The present invention passes through to No. 4 genome respective section segmentation sequencings of blast resistant gene Pigm (t) donor kind paddy plum
Mode, it was found that close to No. 4 specific DNA sequences of paddy plum of Pigm (t) gene, and according to No. 4 distinguished sequences of paddy plum with it is known
The difference design of parent demonstrates specific molecular marker Pigm-4.
The present invention screens the specific molecular marker and No. 4 wide spectrum blast resistant gene Pigm close linkages of Gu Mei obtained,
Pigm-4 is only 14kb at a distance from target zone.
The present invention screening obtain the molecular labeling based on No. 4 genome specifics of paddy plum, can 100% distinguish paddy plum No. 4 (or
Transduceed using it as donor the target strain of distinguished sequence) with other kinds.
Molecular labeling Pigm-4 of the present invention can distinguish the anti-sense difference of Pigm gene, and the screening accuracy of single marking reaches
To 99.01%, and the accuracy for cooperating the candidate molecules in upstream region of gene area to mark Pigm-2 that can make screening reaches 100%.
The Pigm gene that the present invention is transduceed has wide spectrum blast resisting characteristic, will by molecular marker assisted selection
Pigm (t) imports receptor rice varieties, can be obviously improved the blast resisting resistance of rice varieties.
Detailed description of the invention
Fig. 1 is the partial sequence comparison result in SEQ ID NO:1 sequence and related known parent's respective section；Wherein product
Kind code 1 are as follows: Gu Mei 4 (GM4 contains Pigm), 2 are as follows: 9311 (long-grained nonglutinous rice controls), 3 are as follows: IR65482 (contains Pi40), and 4 are as follows:
Fukunishiki (contains Piz), and 5 are as follows: Nipponbare (japonica rice control, OryzasativaLcv.Nipponbare), 6 are as follows: C101LA51 (contains Pi2), and 7 are as follows:
75-1-127 (contains Pi9), and 8 are as follows: Toride 1 (contains Pi-zt).
Fig. 2 is that Pigm coherent detection marks Pigm-2, Pigm-4 and Indel587 to clone AP005659 target zone in BAC
Position view (AP005659 overall length 138870bp).
Fig. 3 is the electrophoretogram that specific marker Pigm-4 detects each parent material；Wherein No. 1 swimming lane be Marker (model:
I DNA ladder of 100bp-, be purchased from Shanghai JaRa Biotechnology Co., Ltd), 2-19 swimming lane be respectively as follows: Gu Mei 4,
2674 pure lines of IR65482, C101LA51,75-1-127, Toride 1, Fukunishiki, Gu Mei No. 4 numbers/force fortune, 9311, town it is extensive
82, town is extensive 832, Cheng Hui 177, Cheng Hui 727, elegant water 519, Wu Yun 2674, military fortune round-grained rice 27, town rice 88, town rice No. 18, town glutinous 19
Number；Swimming lane number is followed successively by 1 to 19 from left to right.
Fig. 4 is the electrophoretogram for marking military 2674//force of the fortune fortune No. 4 filial generation part single plants of 2674/ paddy plum of Pigm-4 detection
(BC1F2)；Wherein 1 and No. 5 swimming lane is Marker, and No. 2 swimming lanes are disease-resistant control paddy plum No. 4, and No. 3 swimming lanes are the military fortune of susceptible control
2674, remaining swimming lane is single plant in group；Swimming lane number is followed successively by 1 to 33 from left to right.
Specific embodiment
Combined with specific embodiments below, the present invention is furture elucidated, it should be understood that these embodiments are merely to illustrate the present invention
Rather than limit the scope of the invention, after the present invention has been read, those skilled in the art are to various equivalences of the invention
The modification of form falls within the application range as defined in the appended claims.
The acquisition and verifying of No. 4 specific gene group sequence mark Pigm-4 of 1 paddy plum of embodiment
1 test material
This research rice material used includes the genomic DNA of the donor kind of Pi9, Pi2, Piz, Pi-zt, Pi40:
The donor paddy plum No. 4 of 75-1-127, C101A51, Toride 1, Fukunishiki, IR65482 and Pigm；Receptor kind round-grained rice
Rice new lines force fortune 2674, long-grained nonglutinous rice receptor kind town be extensive 82, conventional japonica rice kind and typical hybrid paddy rice keep system, restorer
250 parts (9311, OryzasativaLcv.Nipponbare, it is middle spend 11, elegant water 519, imperial round-grained rice 41, even round-grained rice 4, even round-grained rice 6, even round-grained rice 7, even round-grained rice 11, even round-grained rice
No. 9, even round-grained rice 10, Xu rice No. 3, magnificent round-grained rice 3, magnificent round-grained rice 4, magnificent round-grained rice 5, magnificent round-grained rice 6, magnificent round-grained rice 7, Huaihe River rice No. 5, Huaihe River rice No. 6,
Huaihe River rice No. 7, Huaihe River rice No. 8, Huaidao 9, Huaihe River rice No. 10, Huaihe River rice No. 11, Huaihe River is No. 12 glutinous, Huaihe River rice No. 13, the excellent round-grained rice in Huaihe River 2, salt rice 6
Number, salt rice No. 8, salt rice No. 9, salt rice No. 11, salt round-grained rice 9, salt round-grained rice 10, salt round-grained rice 11, raise and educate round-grained rice 2, raise agriculture rice No. 1, is military
Mound round-grained rice 1 raises spoke round-grained rice 7, raises spoke round-grained rice 8, raise round-grained rice 4038, raise round-grained rice 4227, peaceful round-grained rice 1, peaceful round-grained rice 2, peaceful round-grained rice 3, peaceful round-grained rice 4
Number, peaceful round-grained rice 5, southern round-grained rice 49, southern round-grained rice 38, southern round-grained rice 40, southern round-grained rice 41, southern round-grained rice 42, southern round-grained rice 43, southern round-grained rice 44, southern round-grained rice 45, southern round-grained rice 46, south
Round-grained rice 47, southern round-grained rice 5055, Su Huxiang round-grained rice, logical round-grained rice 981, military round-grained rice 4, military round-grained rice 13, military round-grained rice 15, Wu-Yu-Geng 3, military fragrant round-grained rice 1,
Military fragrant round-grained rice 9, force perfume round-grained rice 14, force educates round-grained rice 18, force educates round-grained rice 20, force fortune round-grained rice 7, force transports round-grained rice 11, force transports round-grained rice 19, military
Transport round-grained rice 21, military fortune round-grained rice 23, military fortune round-grained rice 24, military fortune round-grained rice 27, military fortune round-grained rice 29, normal agriculture round-grained rice 3, normal agriculture round-grained rice 4, Chang Nong
Round-grained rice 5, normal agriculture round-grained rice 6, normal agriculture round-grained rice 7, Soviet Union's round-grained rice 8, Su Xiang round-grained rice 2, good 33, good 991, town rice 88, town rice 99, town rice No. 7,
Town rice No. 8, town rice No. 10, town rice No. 11, town rice No. 12, town rice No. 13, town rice No. 14, town rice No. 15, town rice No. 16, town rice 17
Number, town rice No. 18, town be No. 19 glutinous, Anhui is extensive 057, another name for Sichuan Province is extensive 498, An Xuan 6, Restorer line Yihui1577, in extensive 8006, R9308, Yanhui 559,
Lu is extensive 17, polyphyly 1, Gui Hui 582, Wen Hui 242, boat 1, extensive 9516, the five mountains silk seedling in Zhejiang, bright extensive 63, R6547, bright extensive 86, continuous
Extensive 577, interior perfume 5B, preferably extensive No. 10, grace is extensive 58, Mianhui725, hope extensive 006, wide extensive 218, Hua Hui 451, Guangdong product silk seedling 2, sub- extensive
420, survey that 253, Nan Hui 11, Yuejingsimiao, Zhejiang are extensive 501, spoke is extensive 728, interior perfume is No. 1 extensive, Hunan is extensive 263, Cheng Hui 177, R7954, far
Extensive No. 2, Cheng Hui 727, Fujian it is extensive 3301, it is bright extensive 100, in extensive 218, wide extensive R15, middle 9B, Pinghui 9901, prosperous extensive T114, in it is extensive
2286, first extensive No. 1, Gan is No. 3 extensive, Gan is extensive 319, Cheng Hui 448, wide extensive 998, Zhejiang are extensive 7954, Wen Hui 689, Cheng Hui 209, another name for Sichuan Province are extensive
538, praise extensive 99, Cheng Hui 3203, day extensive 198, happy extensive 188, Cheng Hui 19, good fortune extensive 673, Cheng Hui 761, Cheng Hui 425, the extensive 3T in Lu, at
Extensive 9348, preferably extensive 4245, refined extensive 2115, river is extensive 907, continuous extensive 663, anti-65, Yang Hui 136, Yang Hui 507, R6547, Yang Hui 818,
Anti- 98, China accounts for, Zhenhui 084, wide extensive 128, Yun Hui 310, town are extensive 41, Cheng Hui 210, another name for Sichuan Province are extensive 527, town is extensive 62, town is extensive 46, river is extensive
047, good fortune is extensive 7185, Yun Hui 68, Yun Hui 23, brocade are extensive 907, Yun Hui 26, Yun Hui 808, Yun Hui 40, prosperous extensive 121, interior extensive 182, bright extensive
62, interior extensive 3105, it is dual anti-it is bright account for, be early extensive 66, middle group 14, Cheng Hui 157, Su Hui 728, Su Hui 118, town be extensive 10003, D3745, peaceful
It is extensive it is wide anti-account for, town is extensive 100, continuous extensive 146, Hunan R702, third 4114, Lu is extensive 602, continuous extensive 3724, pearl is glutinous, R9368, Liu Fengxiang
It accounts for, China Resources 2, R476, R900, R6326, another name for Sichuan Province are extensive 498, continuous extensive 523, continuous extensive 662, Cheng Hui 178, wide extensive 116, wide extensive 290, wide
Extensive 710, wide extensive 138, Shan is extensive 721, Guanghui 122, wide extensive 169, wide extensive 428, bright extensive 70, bright extensive 2088, bright extensive 1259, purple extensive
100, drought it is No. 10 extensive, expensive extensive 2190, expensive extensive 5832, expensive extensive 168, red extensive 98, first extensive 207, it is early extensive 89, early extensive 90, bright extensive 69, bright
Extensive 70, Jin Hui 161, river be wide extensive 909, town is extensive 832, town Xian 1B, Y58S, Guangzhan 63S)；Wherein Pi9, Pi2, Piz, Pi-zt,
The donor kind DNA of Pi40 is provided by Inst. of Agricultural Science, Lixiahe Prefecture, Jiangsu Prov., other seed rice materials are collected by our unit.
Donor kind DNA is extracted to obtain by rice leaf using SDS method.
2 test methods
2.1 sequencings and marker development
According to (Yiwen Deng, Xudong Zhu, Ying Shen, the Zuhua He.Genetic such as Deng
characterization and Fine mapping of the blast resistance locus Pigm(t)
tightly linked to Pi2and Pi9in a broad-spectrum resistant Chinese
Variety.Theor Appl Genet, 2006,113:705-713.) positioning result, in http: //
The DNA sequence dna that rice varieties OryzasativaLcv.Nipponbare respective section is obtained on the website www.ncbi.nlm.nih.gov/ (is located at OryzasativaLcv.Nipponbare gene
No. 6 chromosome 10360000-10440000bp of group), using the relatively uniform distribution of 5 software of software Primer (every the left side 8kb
It is right) design amplification of 10 pairs of fragment length expected products in 500-1200bp and mark.Respectively with paddy plum No. 4 and OryzasativaLcv.Nipponbare,
9311 be template amplification, product sequencing compares, and develops specific molecular marker further according to comparison result.
2.2 detecting step
Step 1: DNA extracts (SDS method):
1, the blade of 2cm long is shredded in the centrifuge tube for setting 2ml, in addition steel ball, is then placed in the vacuum flask of dress liquid nitrogen
It is quick-frozen, it quickly fishes for and is placed on 48 punch dies, cover lid and be placed on sample grinding machine and shake 30s, remove centrifuge tube, pour out steel ball.
2, in the 2ml centrifuge tube of the blade ground containing liquid nitrogen, SDS (0.1M Tris-Hcl, PH 8.0 is added；
0.025M EDTA,PH 8.0；29.25g/L Nacl；12g/L SDS) 600 μ l, are placed in 65 DEG C of water-baths, 30min.
3,150 μ l KAc (PH 4.8) are added, place -20 DEG C of refrigerators, 30min.
4, be added and SDS isometric chloroform: isoamyl alcohol (volume ratio 24:1) solution places oscillator
It sufficiently shakes up, 20min.
5, it is centrifuged, 12000rpm, 4min, 200 μ l supernatants of transfer are set in 1.5ml centrifuge tube.
6, the dehydrated alcohol of 2 times of -20 DEG C of volume pre-coolings is added in supernatant, is placed in -20 DEG C of refrigerators, 20min.
7, it is centrifuged, 12000rpm, 4min, abandons supernatant, air-dry, 200 μ l ddH are added2O dissolution, this is DNA mother liquor.
8, mother liquor is diluted 10 times is DNA working solution.
9, take 1.5 μ l for pcr amplification reaction.
Step 2: PCR amplification:
Pcr amplification reaction carries out in PCR amplification instrument.
1, reaction system is as follows:
Total volume: 10 μ l.Under 1.5 μ l, 2mM SEQ ID NO:1 upstream primer of DNA, 0.5 μ l, 2mM SEQ ID NO:2
Swim 0.5 μ l, 10 × Taq Buffer (GENERAY, JaRa company) of primer, 1.2 μ l, 1mM dNTP0.3 μ l, 1000U Taq DNA
0.1 μ l of polymerase (GENERAY), adds ddH2O complements to 10 μ l.
2, the amplification program of PCR is as follows:
Step 3: PCR product detection and sequencing:
It is detected with 8% polyacrylamide gel electrophoresis, the 2 μ l of PCR product expanded is taken, with DNA Marker
(I DNA ladder of 100bp-) is compareed as molecular weight, electrophoresis 1 hour under 240V constant pressure.Silver staining display DNA band, passes through
It compares DNA Marker and finds target stripe, for the product that needs are sequenced, the remaining amplified production containing purpose band is sent
To Nanjing Genscript Biotechnology Co., Ltd.'s sequence verification；Detection for target gene then judges according to clip size anti-
Feel genotype.
3 test results
According to sequencing result we found in the 4th pair of product paddy plum No. 4 have with long-grained nonglutinous rice 9311 and japonica rice OryzasativaLcv.Nipponbare it is equal
There is significant DNA sequence dna difference, shows compared with japonica rice OryzasativaLcv.Nipponbare, 74 bases (bp) of Gu Mei 4 missings, deletion sites position
At No. 6 chromosome 10436119bp~10436192bp of OryzasativaLcv.Nipponbare rice genome, and 45bp is lacked compared with 9311
(Fig. 1).According to this difference, we devise specific marker Pigm-4, and primer sequence is SEQ ID NO:2 and SEQ ID
Shown in NO:3, material to be tested is analyzed with Pigm-4, discovery, which only has paddy plum No. 4 in 250 parts of materials in total, can amplify missing
The specific band of 74bp, so that the Pigm-4 label (Fig. 3) special for No. 4 genomes of paddy plum is demonstrated, and with
InDel587 is compared, and Pigm-4 is only 14kb at a distance from target zone, chain closer (Fig. 2).
Table 1 is used to detect the PCR label of screening Pigm
It is designed to obtain (the position marker upstream Pigm-2 of Pigm using universal method according to known Xian round-grained rice sequence difference later
Setting in No. 6 chromosome 10366681bp~10366821bp of OryzasativaLcv.Nipponbare rice genome), Pigm-2 expands donor paddy plum 4
Number with receptor parent force 2674, town it is extensive 82, town rice No. 18, find there is polymorphism between donor and 3 parts of acceptor materials, can be with
For further molecular marker assisted selection.
Embodiment 2 is cultivated using genome-specific markers Pigm-4 and marker upstream Pigm-2 transduction Pigm (t) gene
Wide spectrum rice blast resistant rice new varieties
The test material and test method of the present embodiment are substantially with embodiment 1, and donor kind used is paddy plum No. 4, receptor
Kind be military fortune 2674 (new lines having participated in the examination of Estimation of Paddy In Jiangsu Province area but being eliminated because rice blast is not up to standard,
Claim force 2674, those skilled in the art can be obtained by conventional route, in addition, receptor kind be not limited in it is military 2674), town it is extensive
82, it detects and is labeled as both wings label Pigm-2 and Pigm-4, the method for rice blast inoculated identification and judge is referring to Deng, Wu etc.
Document (Yiwen Deng, Xudong Zhu, Ying Shen, Zuhua He.Genetic characterization and
Fine mapping of the blast resistance locus Pigm(t)tightly linked to Pi2and
Pi9in a broad-spectrum resistant Chinese variety.Theor Appl Genet,2006,113:
705–713.
Yunyu Wu,Ling Yu,Cunhong Pan,Zhengyuan Dai,Yuhong Li,Ning Xiao,
Xiaoxiang Zhang,Hongjuan Ji,Niansheng Huang,Buhong Zhao,Changhai Zhou,
Guangqing Liu,Xiaojing Liu,Xuebiao Pan,Chengzhi Liang,Aihong Li.Development
of near-isogenic lines with different alleles of Piz locus and analysis of
their breeding effect under Yangdao 6background.Mol Breeding,2016,36:1-12
(online).)。
Amount to 652 single plants to BC1F2 derived from force fortune 2674//military fortune 2674/ paddy plum No. 4 to detect, find
Pigm-2 label carries the individual difference of Pigm/Pigm (i.e. AA type), Pigm/pigm (i.e. Aa type), pigm/pigm (i.e. aa type)
It is 165:330:157 plants, and Pigm-4 is marked with Pigm/Pigm (i.e. AA type), Pigm/pigm (i.e. Aa type), pigm/pigm
The individual of (i.e. aa type) is 162:330:160 plants respectively, the ratio of the 1:2:1 of equal coincidence statistics meaning, but has 6 exchanges
Single plant (i.e. the inconsistent single plant of two marker genetypes), we carry out inoculated identification to 102 single plants randomly selected later,
It was found that there is 73 individuals to show as blast resisting in 75 individuals in total of AA and Aa, 2 individuals show as it is susceptible, and this 2
Susceptible single plant is exactly that (one is that Pigm-2 is Aa type, and Pigm-4 is aa type by 2 in 6 exchange single plant；Another be for
Pigm-2 is aa type, and Pigm-4 is Aa type), and 27 individuals that two labels are aa type show as susceptible (Fig. 4).
Amount to 24 single plants to BC1F1 derived from the extensive 82/ paddy plum in the extensive 82//town in town No. 4 using above-mentioned two label to examine
Survey, Pigm/pigm (i.e. Aa type), pigm/pigm (i.e. aa type) individual be 13:11 plant respectively, do not find exchange single plant, accord with
The ratio of the 1:1 of statistical significance is closed, we carry out all equal tables of individual of inoculated identification to 13 heterozygotes (i.e. Aa type) single plant
It is now disease-resistant.
By embodiment 2 it can be seen that Pigm can be obviously improved the blast resisting characteristic of rice varieties, Pigm-4 can be with
The anti-sense difference of Pigm gene is distinguished, the screening accuracy of single marking reaches 99.01%, and Pigm-4 is marked with Pigm-2 two
The accuracy that note is screened when being used cooperatively reaches 100%.
Claims (6)
1. a kind of molecular labeling of No. 4 blast resistant gene Pigm of paddy plum, which is characterized in that it is molecular labeling Pigm-4, it is described
Molecular labeling Pigm-4 be include be located at No. 6 chromosome 10436119bp ~ 10436192bp of rice genome at DNA piece
Section, No. 4 this section of base deletions of Gu Mei, the sequence of the molecular labeling Pigm-4 is as shown in SEQ ID NO.1.
2. molecular labeling according to claim 1, which is characterized in that further include making with molecular labeling Pigm-4 cooperation
Molecular labeling Pigm-2, the molecular labeling Pigm-2 are comprising being located at No. 6 chromosome of rice genome
DNA fragmentation at the bp of 10366681 bp ~ 10366821.
3. described in any item molecular labelings are in molecular marker assisted selection anti-rice blast rice breeding according to claim 1 ~ 2
Application.
4. application according to claim 3 characterized by comprising blast resistant gene Pigm is transferred to receptor parent,
Utilize the rice material of Marker-assisted selection Pigm containing disease-resistant gene.
5. application according to claim 4, which is characterized in that when using the Marker-assisted selection rice material, adopt
It is expanded with Pigm-4 primer pair and Pigm-2 primer pair, wherein the upstream and downstream primer of Pigm-4 primer pair is respectively such as SEQ
Shown in ID NO:2 and SEQ ID NO:3, the upstream and downstream primer of Pigm-2 primer pair is respectively such as SEQ ID NO:4 and SEQ ID
Shown in NO:5.
6. described in any item molecular labelings are improving rice by the method for molecular marker assisted selection according to claim 1 ~ 2
Blast resisting infection ability in application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610852523.8A CN106148335B (en) | 2016-09-26 | 2016-09-26 | The molecular labeling of No. 4 blast resistant gene Pigm of paddy plum and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610852523.8A CN106148335B (en) | 2016-09-26 | 2016-09-26 | The molecular labeling of No. 4 blast resistant gene Pigm of paddy plum and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106148335A CN106148335A (en) | 2016-11-23 |
| CN106148335B true CN106148335B (en) | 2019-05-31 |
Family
ID=57341462
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610852523.8A Active CN106148335B (en) | 2016-09-26 | 2016-09-26 | The molecular labeling of No. 4 blast resistant gene Pigm of paddy plum and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106148335B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106929585B (en) * | 2016-12-29 | 2019-09-10 | 深圳兴旺生物种业有限公司 | The detection method and its application of blast resistant gene Pigm |
| CN106755471B (en) * | 2017-01-08 | 2017-12-08 | 王依然 | Rice blast resistant gene Pilk (t) linked marker and its primer special |
| CN107034308A (en) * | 2017-06-27 | 2017-08-11 | 上海市农业生物基因中心 | The molecular labeling of Rice Resistance seasonal febrile diseases gene Pigm a kind of and its application |
| CN107164547B (en) * | 2017-07-19 | 2020-05-26 | 安徽丰大种业股份有限公司 | Molecular marker closely linked with rice blast resistance gene, primer and application thereof |
| CN109929838B (en) * | 2017-12-15 | 2023-01-03 | 中国种子集团有限公司 | Rice genome recombinant nucleic acid fragment RecCR012612 and detection method thereof |
| CN109929837B (en) * | 2017-12-15 | 2022-11-22 | 中国种子集团有限公司 | Rice genome recombinant nucleic acid fragment RecCR012613 and detection method thereof |
| CN107988419A (en) * | 2018-01-19 | 2018-05-04 | 福建省农业科学院生物技术研究所 | A kind of rice blast resistance gene Pigm specific Functions molecular labeling and its application |
| CN109628627B (en) * | 2018-12-11 | 2022-03-18 | 华智生物技术有限公司 | Development and application of SNP (single nucleotide polymorphism) marker of broad-spectrum rice blast resistance gene Pigm of rice |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103497996A (en) * | 2013-09-22 | 2014-01-08 | 江苏里下河地区农业科学研究所 | Molecular marker InDe1587 for detecting rice blast resistant gene Pigm(t) of rice #4 |
-
2016
- 2016-09-26 CN CN201610852523.8A patent/CN106148335B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103497996A (en) * | 2013-09-22 | 2014-01-08 | 江苏里下河地区农业科学研究所 | Molecular marker InDe1587 for detecting rice blast resistant gene Pigm(t) of rice #4 |
Non-Patent Citations (1)
| Title |
|---|
| 水稻广谱抗瘟基因Pigm紧密连锁分子标记及其育种应用;梁毅等;《杂交水稻》;20130715;第28卷(第4期);摘要及第63页左栏最后一段、第65页最后一段、第66页第一段、第67页左栏最后一段、第68页左栏第1-3段图1 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106148335A (en) | 2016-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106148335B (en) | The molecular labeling of No. 4 blast resistant gene Pigm of paddy plum and its application | |
| CN103305510B (en) | Rice blast resistance gene Pi9 gene specificity molecular marker Pi9SNP as well as preparation and application thereof | |
| CN104073487B (en) | The molecule marker of rice blast resistant gene Pi2 and application thereof | |
| CN103320437B (en) | Gene-specific molecular marker Pi2SNP of rice blast-resistant gene Pi2 as well as preparation method and application thereof | |
| CN105950747A (en) | Rice blast resistance gene Pi1 functional specificity molecular marker and application thereof | |
| CN103820476A (en) | Gene relevant to wheat thousand seed weight, functional marker and application thereof | |
| CN108588272A (en) | A kind of molecular labeling and its application with Plant Height in Wheat and spike length character main effect QTL compact linkage | |
| CN103866026A (en) | Rice cold-resistant major gene identification method and special primer thereof | |
| CN104450932B (en) | Rice blast resistance gene Pi2 gene specific molecular labelings Pi2SSR and its preparation method and application | |
| CN102899400B (en) | Molecule marking method for rice anti-rice blast gene Pigm | |
| CN103789306A (en) | SNP (Single Nucleotide Polymorphism) molecular marker of rice blast resistance gene Pia, and detection method and application of SNP molecular marker | |
| CN109628627A (en) | The SNP marker development and application of broad-spectrum rice-blast resistant gene of paddy rice Pigm | |
| CN108504773B (en) | Molecular marker of major QTL (quantitative trait loci) site for grain weight and silique length of brassica napus and application of molecular marker | |
| CN109402291A (en) | A kind of identification muskmelon female flower inserted part InDel molecular marker and primer thereof and application | |
| CN104561348B (en) | The detection Oryza sativa L. height grain allelic specific PCR molecular markers of weight | |
| CN107201395B (en) | Molecular marker of major gene Bph30 for resisting brown planthopper of rice and application thereof | |
| CN105296472B (en) | The molecular labeling and its screening technique in Sand Pear ' faint scent ' pericarp brown character gene site entirely | |
| CN109735648A (en) | A kind of method and its dedicated kit for screening different mass of 1000 kernel wheats | |
| CN108504772A (en) | The molecular labeling of rice premature gene and application | |
| CN108456740A (en) | One Rice Resistance To Rice Blast site ' Pi-jx ' and its Indel labeled primers and Breeding Application | |
| CN109251996B (en) | dCAPS marker for detecting low temperature resistant gene COLD1 genotype of rice and application | |
| CN106701749A (en) | Rice blast resistance gene Piz-t functional specific molecular marker and detection method and application thereof | |
| CN110468229A (en) | Rice wide spectrum high resistance to hoja blanca gene Xa45's (t) isolates molecular labeling Hxjy-1 | |
| CN115786564A (en) | Rice Pi-ta and Ptr dominant functional molecular marker and application thereof | |
| CN108179221A (en) | Detect the specific molecular marker of high mass of 1000 kernel allele on rice mass of 1000 kernel QTL qTGW10.2a |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |