CN106148335A - The molecular marker of No. 4 blast resistant gene Pigm of paddy prunus mume (sieb.) sieb.et zucc. and application thereof - Google Patents
The molecular marker of No. 4 blast resistant gene Pigm of paddy prunus mume (sieb.) sieb.et zucc. and application thereof Download PDFInfo
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Abstract
The invention discloses molecular marker and the application thereof of a kind of No. 4 blast resistant gene Pigm of paddy prunus mume (sieb.) sieb.et zucc..Described molecular marker includes No. 4 specific molecular marker Pigm 4 of Gu Mei, described molecular marker Pigm 4 is to include to be positioned at the DNA fragmentation that there are differences at Oryza sativa L. Japan No. 6 chromosome 10436119bp～10436192bp of fine genome, and No. 4 this section of sequence deletions of paddy prunus mume (sieb.) sieb.et zucc., 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 marker primer 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 described molecular marker in molecular marker assisted selection anti-rice blast rice breeding and by the application in the blast resisting infection ability of the method raising Oryza sativa L. of molecular marker assisted selection.The molecular marker specificity of the present invention is good, chain tightr with target area.
Description
Technical field
The invention belongs to Rice molecular breeding technical field, more particularly it relates to an No. 4 blast resistings of paddy prunus mume (sieb.) sieb.et zucc.
The molecular marker of gene Pigm and application thereof.
Background technology
Rice blast is one of three big Major Diseases of Rice Production in global range, and cultivating disease-resistant variety is preventing and treating rice blast
Endanger most economical effective means.Traditional paddy disease-resistant breeding is, by Resistance Identification, plant is carried out Phenotypic Selection, expends
Time is long, and is easily limited by environmental condition, and qualification result easily causes error, and efficiency of selection is relatively low.Auxiliary by molecular marker
Help selection disease-resistant gene not affected by above-mentioned unfavorable factor, be one of effective way cultivating disease-resistant variety.
Paddy prunus mume (sieb.) sieb.et zucc. No. 4 is an endemicity kind, has wide spectrum and lasting rice blast resistance, its disease-resistant major gene resistance Pigm
T () (hereinafter abbreviated as Pigm) is positioned between the 6th chromosome molecular marker C5483 and C0428 (paper in the range of about 70kb
Source: Theor Appl Genet, 2006,113:705-713).But the molecular marker announced in document is mostly CAPS mark
Note, operation is complicated and cost is higher, is not suitable for molecular marker assisted selection, and remaining SSR and InDel labelling is mainly used for heredity
Location, breeding exists polymorphic rate relatively low, the shortcoming that difference is less.Pan Cunhong etc. develop (patent application: CN
103497996A) for detecting the molecular marker InDel587 of No. 4 blast resistant gene Pigm of paddy prunus mume (sieb.) sieb.et zucc., but still suffer from about 15%
Receptor parent (the most indica type parent's ratio is higher) with paddy prunus mume (sieb.) sieb.et zucc. No. 4 without polymorphic, marking path target interval is farther out
(65.2kb) two defects.
It is positioned on Piz seat it addition, existing research has proven to the disease-resistant main effect Pigm of Gu Mei 4.Piz has been found that on seat
The multiple alleless such as Pi2, Pi9, Pi-zt, Piz, Pi40, Pigm, but there is some difference for respective gene order and anti-spectrum,
Realize being accomplished by the utilization of Pigm making a distinction different from other for Pigm multiple alleless, thus realize Pigm is special
Ground transformation is among receptor kind.
Summary of the invention
Goal of the invention: not enough for labelling specificity present in prior art and chain with target gene untight ask
Topic, an object of the present invention is to provide the molecular marker of No. 4 blast resistant gene Pigm of Gu Mei, and the two of the purpose of the present invention are
Thering is provided the primer for detecting described molecular marker, the three of the purpose of the present invention are to provide the application of described molecular marker.
Technical scheme: the molecular marker of No. 4 blast resistant gene Pigm of paddy prunus mume (sieb.) sieb.et zucc. of the present invention, including molecular marker
Pigm-4, described molecular marker Pigm-4 for comprise be positioned at No. 6 chromosome 10436119bp of rice genome～
The DNA fragmentation (being positioned among AP005659) at 10436192bp, No. 4 this section of base deletions of Gu Mei.The present invention is describing gene
During group base positions, it is on the basis of the fine genome of Oryza sativa L. Japan.
The fragment length of described molecular marker Pigm-4 is 150～500bp.This fragment length facilitates views with electrophoresis
Method is identified detection, and electrophoresis method is simple, low cost.Certainly, the fragment length of above-mentioned molecular marker Pigm-4
It is not necessary, when using other detection methods such as order-checking etc., is not limited by above-mentioned length.
Further, described molecular marker Pigm-4 is to comprise the DNA fragmentation of sequence as shown in SEQ ID NO.1.Enter
One step, the sequence of described molecular marker Pigm-4 is as shown in SEQ ID NO.1.
Further, described molecular marker, also include with described minute mark note Pigm-4 with the use of molecular marker
Pigm-2, described molecular marker Pigm-2 for comprise be positioned at No. 6 chromosome 10366681bp of rice genome～
DNA fragmentation at 10366821bp.Further, described molecular marker Pigm-2 is for being positioned at rice genome the 6th dyeing
DNA fragmentation at body 10366681bp～10366821bp.
Pigm-4 can distinguish the anti-sense difference of Pigm gene, because itself and target gene there is also certain genetic distance,
The screening accuracy of single marking reaches 99.01% (rather than 100%), and two labellings of Pigm-4 Yu Pigm-2 with the use of
Shi Ze has completely included the whole fragment at Pigm place, and the accuracy of screening reaches 100%.
Present invention also offers a kind of amplimer, primer is Pigm-4 primer to 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 upper,
Downstream primer is respectively as shown in SEQ ID NO:4 and SEQ ID NO:5.
Present invention also offers described molecular marker answering in molecular marker assisted selection anti-rice blast rice breeding
With.
Described application includes: blast resistant gene Pigm is proceeded to receptor parent, the Marker-assisted selection described in utilization
Rice material containing disease-resistant gene Pigm.
Wherein, during Marker-assisted selection rice material described in utilization, use Pigm-4 primer to or/and Pigm-2 primer
To expanding, wherein the upstream and downstream primer of Pigm-4 primer pair is 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.
Present invention also offers described molecular marker and improve the anti-of Oryza sativa L. by the method for molecular marker assisted selection
Application in rice blast infection ability.Described application includes: blast resistant gene Pigm proceeds to receptor parent, utilizes described
Marker-assisted selection containing the rice material of disease-resistant gene Pigm.Wherein, during Marker-assisted selection rice material described in utilization,
Above-mentioned Pigm-4 primer can be used or/and Pigm-2 primer is to expanding.
Compared with prior art, the invention have the benefit that
The present invention is by checking order to No. 4 genome respective section segmentations of blast resistant gene Pigm (t) donor kind paddy prunus mume (sieb.) sieb.et zucc.
Mode, it was found that No. 4 specific DNA sequences of paddy prunus mume (sieb.) sieb.et zucc. of next-door neighbour's Pigm (t) gene, and according to No. 4 distinguished sequences of paddy prunus mume (sieb.) sieb.et zucc. with known
The difference of parent designs, demonstrates specific molecular marker Pigm-4.
The present invention screens specific molecular marker and No. 4 wide spectrum blast resistant gene Pigm close linkages of paddy prunus mume (sieb.) sieb.et zucc. of acquisition,
Pigm-4 is only 14kb with the distance of target zone.
The present invention screening obtain molecular marker based on No. 4 genome specifics of paddy prunus mume (sieb.) sieb.et zucc., can 100% distinguish paddy prunus mume (sieb.) sieb.et zucc. No. 4 (or
Transduceed using it as donor the target strain of distinguished sequence) with other kind.
Molecular marker 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 coordinate the candidate molecules labelling Pigm-2 in upstream region of gene district that the accuracy of screening can be made to reach 100%.
The Pigm gene that the present invention is transduceed has wide spectrum blast resisting characteristic, by molecular marker assisted selection, and will
Pigm (t) imports receptor rice varieties, can be obviously improved the blast resisting resistance of rice varieties.
Accompanying drawing explanation
Fig. 1 is SEQ ID NO:1 sequence and the partial sequence comparison result in related known parent's respective section；Wherein product
Planting code 1 is: Gu Mei 4 (GM4, containing Pigm), and 2 are: 9311 (long-grained nonglutinous rice comparisons), and 3 are: IR65482 (containing Pi40), and 4 are:
Fukunishiki (containing Piz), 5 are: Nipponbare (japonica rice compares, and Japan is fine), and 6 are: C101LA51 (containing Pi2), and 7 are:
75-1-127 (containing Pi9), 8 are: Toride 1 (containing Pi-zt).
Fig. 2 is that Pigm coherent detection labelling Pigm-2, Pigm-4 and Indel587 clone AP005659 target zone at BAC
Position view (AP005659 total length 138870bp).
Fig. 3 is the electrophoretogram that specific marker Pigm-4 detects each parent material；Wherein No. 1 swimming lane be Marker (model:
100bp-I DNA ladder, purchased from JaRa bio tech ltd, Shanghai), 2-19 swimming lane be respectively as follows: Gu Mei 4,
IR65482, C101LA51,75-1-127, Toride 1, Fukunishiki, Gu Mei No. 4 numbers/force fortune 2674 pure lines, 9311, town extensive
82, town is extensive 832, it is extensive 177 to become, it is extensive 727 to become, elegant water 519, military fortune 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 of labelling Pigm-4 detection force fortune 2674//military fortune No. 4 filial generation part individual plants of 2674/ paddy prunus mume (sieb.) sieb.et zucc.
(BC1F2)；Wherein 1 and No. 5 swimming lane is Marker, and No. 2 swimming lanes are disease-resistant comparison paddy prunus mume (sieb.) sieb.et zucc. No. 4, and No. 3 swimming lanes are susceptible comparison force fortune
2674, remaining swimming lane is individual plant in colony；Swimming lane number is followed successively by 1 to 33 from left to right.
Detailed description of the invention
Below in conjunction with specific embodiment, it is further elucidated with the present invention, it should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention, after having read the present invention, the those skilled in the art's various equivalences to the present invention
The amendment of form all falls within the application claims limited range.
The acquisition of No. 4 specific gene group sequence mark Pigm-4 of embodiment 1 paddy prunus mume (sieb.) sieb.et zucc. and checking
1 test material
The rice material of this institute includes the genomic DNA of the donor kind of Pi9, Pi2, Piz, Pi-zt, Pi40:
The donor paddy prunus mume (sieb.) sieb.et zucc. 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 are extensive 82, conventional japonica rice kind and typical hybrid paddy rice keep system, restorer
250 parts (9311, Japan fine, middle spend 11, elegant water 519, dragon 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, slowly rice No. 3, China's round-grained rice 3, China's round-grained rice 4, China's round-grained rice 5, China's round-grained rice 6, China's 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, glutinous No. 12 of Huaihe River, 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, military
Mound round-grained rice 1, raise spoke round-grained rice 7, raise 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, south round-grained rice 49, south round-grained rice 38, south round-grained rice 40, south round-grained rice 41, south round-grained rice 42, south round-grained rice 43, south round-grained rice 44, south round-grained rice 45, south round-grained rice 46, south
Round-grained rice 47, south round-grained rice 5055, Soviet Union's Shanghai perfume (or spice) 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, military fragrant round-grained rice 14, Wuyujing 18, Wuyujing 20, military fortune round-grained rice 7, military fortune round-grained rice 11, military fortune round-grained rice 19, military
Fortune 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, glutinous No. 19 of town, Anhui is extensive 057, another name for Sichuan Province is extensive 498, peace select No. 6, Restorer line Yihui1577, in extensive 8006, R9308, Yanhui 559,
Lu is extensive 17, polyphyly 1, osmanthus are extensive 582, literary composition is extensive 242, boat 1, extensive 9516, the five mountain silk Seedlings in Zhejiang, bright extensive 63, R6547, bright extensive 86, continuous
Fragrant 5B extensive 577, interior, the most extensive No. 10, grace is extensive 58, Mianhui725, prestige are extensive 006, the most extensive 218, China is extensive 451, Guangdong product silk Seedling 2, sub-extensive
420, survey 253, south is extensive 11, Yuejingsimiao, Zhejiang are extensive 501, spoke is extensive 728, extensive No. 1 of interior perfume (or spice), Hunan are extensive 263, it is extensive 177 to become, R7954, remote
Extensive No. 2, become extensive 727, Fujian extensive 3301, bright extensive 100, in extensive 218, wide extensive R15, middle 9B, Pinghui 9901, prosperous extensive T114, in extensive
2286, first extensive No. 1, extensive No. 3 of Gan, Gan are extensive 319, it is extensive 448 to become, the most extensive 998, Zhejiang is extensive 7954, temperature is extensive 689, it is extensive 209 to become, another name for Sichuan Province is extensive
538, good extensive 99, become extensive 3203, sky extensive 198, happy extensive 188, become that extensive 19, good fortune is extensive 673, become extensive 761, become extensive 425, the extensive 3T in Lu, one-tenth
Extensive 2115, river extensive 9348, the most extensive 4245, refined extensive 907, continuous extensive 663, anti-65, raise extensive 136, raise extensive 507, R6547, raise extensive 818,
Anti-98, China accounts for, Zhenhui 084, the most extensive 128, cloud are extensive 310, town is extensive 41, it is extensive 210 to become, another name for Sichuan Province is extensive 527, town is extensive 62, town is extensive 46, river is extensive
047, good fortune is extensive 7185, cloud is extensive 68, cloud is extensive 23, brocade is extensive 907, cloud is extensive 26, cloud is extensive 808, cloud is extensive 40, prosperous extensive 121, interior extensive 182, bright extensive
62, interior extensive 3105, dual anti-bright account for, the most extensive 66, middle group 14, become extensive 157, extensive 728, extensive 118, the town of reviving of reviving is extensive 10003, D3745, peaceful
Extensive wide anti-account for, town is extensive 100, continuous extensive 146, Hunan R702, third 4114, Lu extensive 602, continuous extensive 3724, Margarita is glutinous, R9368, Liu Fengxiang
Account for, China Resources 2, R476, R900, R6326, another name for Sichuan Province are extensive 498, continuous extensive 523, continuous extensive 662, become extensive 178, the most extensive 116, the most extensive 290, wide
Extensive 710, the most extensive 138, Shan is extensive 721, Guanghui 122, the most extensive 169, the most extensive 428, bright extensive 70, bright extensive 2088, bright extensive 1259, purple extensive
100, extensive No. 10 of drought, expensive extensive 2190, expensive extensive 5832, expensive extensive 168, red extensive 98, the most extensive 207, the most extensive 89, the most extensive 90, bright extensive 69, bright
Extensive 70, gold is extensive 161, river is the most extensive 909, town is extensive 832, town Xian 1B, Y58S, Guangzhan 63S)；Wherein Pi9, Pi2, Piz, Pi-zt,
Donor kind DNA of Pi40 is provided by Inst. of Agricultural Science, Lixiahe Prefecture, Jiangsu Prov., and other seed rice materials are collected by our unit.
Donor kind DNA is used the extraction of SDS method to obtain by rice leaf.
2 test methods
2.1 sequencings and marker development
According to (Yiwen Deng, Xudong Zhu, Ying Shen, 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, at http: //
The DNA sequence obtaining the fine respective section of rice varieties Japan on www.ncbi.nlm.nih.gov/ website (is positioned at Japanese fine gene
Organize No. 6 chromosome 10360000-10440000bp), utilize the software Primer 5 relatively uniform distribution of software (left every 8kb
Right) design 10 to fragment length expected product at the amplification label of 500-1200bp.Respectively with paddy prunus mume (sieb.) sieb.et zucc. No. 4 and Japan fine,
9311 is template amplification, product order-checking comparison, develops specific molecular marker further according to comparison result.
2.2 detecting step
Step one, DNA extraction (SDS method):
1, the blade of 2cm length is shredded in the centrifuge tube putting 2ml, add steel ball, be then placed in the thermos flask of dress liquid nitrogen
Quick-freezing, quickly fishes for and is placed on 48 punch dies, builds lid and is placed on sample grinding machine vibrations 30s, takes off 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, place in 65 DEG C of water-baths, 30min.
3, add 150 μ l KAc (PH 4.8), place-20 DEG C of refrigerators, 30min.
4, add and the isopyknic chloroform of SDS: isoamyl alcohol (volume ratio 24:1) solution, place agitator
Fully shake up, 20min.
5, centrifugal, 12000rpm, 4min, transferase 12 00 μ l supernatant is put in 1.5ml centrifuge tube.
6, in supernatant, add the dehydrated alcohol of-20 DEG C of pre-coolings of 2 times of volumes, be placed in-20 DEG C of refrigerators, 20min.
7, centrifugal, 12000rpm, 4min, abandon supernatant, air-dry, add 200 μ l ddH2O dissolves, and this is DNA mother solution.
8, mother solution dilution 10 times is DNA working solution.
9,1.5 μ l are taken for pcr amplification reaction.
Step 2, PCR expand:
Pcr amplification reaction is carried out in PCR amplification instrument.
1, reaction system is as follows:
Cumulative volume: 10 μ l.DNA 1.5 μ l, 2mM SEQ ID NO:1 forward primer 0.5 μ l, under 2mM SEQ ID NO:2
Trip primer 0.5 μ l, 10 × Taq Buffer (GENERAY, JaRa company) 1.2 μ l, 1mM dNTP0.3 μ l, 1000U Taq DNA
Polymerase (GENERAY) 0.1 μ l, adds ddH2O complements to 10 μ l.
2, the amplification program of PCR is as follows:
The detection of step 3, PCR primer and order-checking:
Detect by 8% polyacrylamide gel electrophoresis, take the PCR primer 2 μ l expanded, with DNA Marker
(100bp-I DNA ladder) compares as molecular weight, electrophoresis 1 hour under 240V constant voltage.Silver staining display DNA band, passes through
Comparison DNA Marker finds target stripe, for needing the product of order-checking, is sent by the residue amplified production containing purpose band
To Nanjing Genscript Biotechnology Co., Ltd.'s sequence verification；For the detection of target gene, then judge anti-according to clip size
Sense genotype.
3 result of the tests
According to sequencing result we find in the 4th pair of product paddy prunus mume (sieb.) sieb.et zucc. No. 4 have with long-grained nonglutinous rice 9311 and japonica rice Japan fine all
Have significant DNA sequence difference, show with japonica rice Japan fine compared with, Gu Mei 4 lacks 74 bases (bp), deletion sites position
At No. 6 chromosome 10436119bp～10436192bp of Japanese fine rice genome, and lack 45bp compared with 9311
(Fig. 1).According to this difference, we devise specific marker Pigm-4, and its primer sequence is SEQ ID NO:2 and SEQ ID
Shown in NO:3, analyze material to be tested with Pigm-4, find that only having paddy prunus mume (sieb.) sieb.et zucc. No. 4 in altogether 250 parts of materials can amplify disappearance
The specific band of 74bp, thus demonstrating Pigm-4 is the special labellings (Fig. 3) of No. 4 genomes of paddy prunus mume (sieb.) sieb.et zucc., and with
InDel587 compares, and Pigm-4 is only 14kb, chain tightr (Fig. 2) with the distance of target zone.
Table 1 is for detecting the PCR labelling of screening Pigm
Universal method design is utilized to obtain the marker upstream Pigm-2 (position of Pigm according to known Xian round-grained rice sequence difference afterwards
Setting in No. 6 chromosome 10366681bp～10366821bp of Japanese fine rice genome), Pigm-2 expands donor paddy prunus mume (sieb.) sieb.et zucc. 4
Number extensive with receptor parent force 2674, town 82, town rice No. 18, finds all to there is polymorphism between donor and 3 parts of acceptor materials, permissible
For further molecular marker assisted selection.
Embodiment 2 utilizes genome-specific markers Pigm-4 and marker upstream Pigm-2 transduction Pigm (t) gene, cultivates
Wide spectrum rice blast resistant rice new varieties
The test material of the present embodiment and test method are substantially with embodiment 1, and donor kind used is paddy prunus mume (sieb.) sieb.et zucc. No. 4, receptor
But kind (has participated in the examination of Estimation of Paddy In Jiangsu Province district the new lines being eliminated because rice blast is the most up to standard, also for one for force fortune 2674
Claiming military 2674, those skilled in the art can obtain by conventional route, it addition, receptor kind is not limited in force 2674), town extensive
82, detection is labeled as both wings labelling Pigm-2 and Pigm-4, and the method for rice blast inoculated identification and judge is with reference 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).)。
Force fortune 2674//military fortune No. 4 derivative BC1F2 of 2674/ paddy prunus mume (sieb.) sieb.et zucc. 652 individual plants altogether are detected, finds
Pigm-2 labelling 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 strain, and Pigm-4 is marked with Pigm/Pigm (i.e. AA type), Pigm/pigm (i.e. Aa type), pigm/pigm
The individuality of (i.e. aa type) is 162:330:160 strain respectively, the ratio of the 1:2:1 of equal coincidence statistics meaning, but has 6 exchanges
Individual plant (individual plant that i.e. two marker genetypes are inconsistent), we carry out inoculated identification to 102 individual plants randomly selected afterwards,
Finding have 73 individualities to show as blast resisting in 75 individualities altogether of AA and Aa, 2 individualities show as susceptible, and these 2
Susceptible individual plant just 6 exchange individual plant in 2 (one is Aa type for Pigm-2, and Pigm-4 is aa type；Another for for
Pigm-2 is aa type, and Pigm-4 is Aa type), and 27 individualities that two labellings are aa type all show as susceptible (Fig. 4).
The extensive 82/ paddy prunus mume (sieb.) sieb.et zucc. in above-mentioned two labelling 82//town extensive to town No. 4 derivative BC1F1 24 individual plants altogether are utilized to examine
Surveying, Pigm/pigm (i.e. Aa type), the individuality of pigm/pigm (i.e. aa type) are 13:11 strain respectively, do not find to exchange individual plant, symbol
Closing the ratio of the 1:1 of statistical significance, we carry out the inoculated identification equal table of all individualities to 13 heterozygotes (i.e. Aa type) individual 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 is permissible
Distinguishing the anti-sense difference of Pigm gene, the screening accuracy of single marking reaches 99.01%, and Pigm-4 Yu Pigm-2 two mark
Remember with the use of time screening accuracy reach 100%.
Claims (10)
1. the molecular marker of No. 4 blast resistant gene Pigm of a paddy prunus mume (sieb.) sieb.et zucc., it is characterised in that include molecular marker Pigm-4, institute
The molecular marker Pigm-4 stated is to comprise to be positioned at No. 6 chromosome 10436119bp～10436192bp of rice genome
DNA fragmentation, No. 4 this section of base deletions of Gu Mei.
Molecular marker the most according to claim 1, it is characterised in that the fragment length of described molecular marker Pigm-4 is
150～500bp.
Molecular marker the most according to claim 1, it is characterised in that described molecular marker Pigm-4 is for comprising such as SEQ
The DNA fragmentation of sequence shown in ID NO.1.
Molecular marker the most according to claim 3, it is characterised in that the sequence such as SEQ of described molecular marker Pigm-4
Shown in ID NO.1.
Molecular marker the most according to claim 1, it is characterised in that also include coordinating with described molecular marker Pigm-4 making
Molecular marker Pigm-2, described molecular marker Pigm-2 be positioned at No. 6 chromosome of rice genome for comprising
DNA fragmentation at 10366681bp～10366821bp.
6. an amplimer, it is characterised in that primer is Pigm-4 primer to or/and Pigm-2 primer pair, wherein Pigm-4
The upstream and downstream primer of primer pair is respectively as shown in SEQ ID NO:2 and SEQ ID NO:3, and the upstream and downstream of Pigm-2 primer pair are drawn
Thing is respectively as shown in SEQ ID NO:4 and SEQ ID NO:5.
7. according to the molecular marker described in any one of Claims 1 to 5 in molecular marker assisted selection anti-rice blast rice breeding
In application.
Application the most according to claim 7, it is characterised in that including: blast resistant gene Pigm is proceeded to receptor parent,
The Marker-assisted selection described in the utilization rice material containing disease-resistant gene Pigm.
Application the most according to claim 8, it is characterised in that during Marker-assisted selection rice material described in utilization, adopt
With Pigm-4 primer to or/and Pigm-2 primer is to expanding, wherein the upstream and downstream primer of Pigm-4 primer pair is the most such as
Shown in SEQ 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
Shown in ID NO:5.
10. improving water according to the molecular marker described in any one of Claims 1 to 5 by the method for molecular marker assisted selection
Application in the blast resisting infection ability of rice.
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| CN106929585A (en) * | 2016-12-29 | 2017-07-07 | 深圳兴旺生物种业有限公司 | The detection method of blast resistant gene Pigm and its application |
| 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 |
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