CN103146700A - Rice variety 9194 black streaked dwarf restricting locus and molecular marking method thereof - Google Patents
Rice variety 9194 black streaked dwarf restricting locus and molecular marking method thereof Download PDFInfo
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Abstract
The invention relates to a rice variety 9194 black streaked dwarf restricting locus and a molecular marking method thereof. Four rice variety 9194 black streaked dwarf restricting locus are obtained by carrying out genetic linkage analysis on the black streaked dwarf resistance level of the genotype of an F2 single plant obtained from hybridization of the black streaked dwarf restricting rice variety 9194 and susceptible variety suyunuo and the corresponding F2:3 family, wherein qRBSDV3 is positioned between the markers RM282 and RM14898, qRBSDV6 is positioned between the markers RM20069 and RM30, qRBSDV9 is positioned between the markers RM3912 and RM257, and qRBSDV11 is positioned between the markers RM26062 and RM536. The molecular markers of the black streaked dwarf restricting gene are used for detecting whether the resistant variety 9194 and the derived varieties contain the black streaked dwarf restricting locus, so that the resistance level of the black streaked dwarf can be predicated, and the selecting efficiency of the black streaked dwarf restricting rice is greatly improved.
Description
Technical field
The invention belongs to the molecular genetics field, relate to rice varieties 9194 anti-black streak dwarf site and molecule marking method thereof.
Technical background
Paddy rice is the important food crop of China.Black streaked dwarf virus of rice is a kind of virus disease that is caused by rice black-streaked dwarf virus (Rice black-streaked dwarf virus, RBSDV), is mainly propagated without the ovum mode with persistence by small brown rice planthopper.In recent years, along with variation, the winter climate of cropping system and planting type warms and the spread of susceptible variety, pass virus mediator small brown rice planthopper generating capacity rising, black streaked dwarf virus of rice in Jiangsu, Zhejiang, Jiangxi and Fujian occurs on a large scale, and rise to rapidly the upper topmost virus disease of production, bring very large threat for the safety in production of paddy rice.The classical symptom of black streaked dwarf virus of rice is that diseased plant stunts, and the leaf look dark green is stiff, and blade back, leaf sheath and cane have early stage wax, and the later stage is the irregular projection of billet line of chocolate, and the strain of seriously falling ill is not eared.Producing upward at present and there is no specific pesticide for this type of disease, is mainly the early stage control of passing through small brown rice planthopper, alleviate its harm, but prevention effect is unsatisfactory, and contaminate environment, destroys the eubiosis.To prevent and treat the optimal method of virus disease be seed selection and utilize disease-resistant variety, and screening, excavate and innovating disease-resistant germplasm is prerequisite and the basis of carrying out anti-black streak dwarf breeding.
Summary of the invention
The objective of the invention is the above-mentioned deficiency for prior art, rice varieties 9194 anti-black streak dwarf site and molecule marking method thereof are provided.
Purpose of the present invention can be achieved through the following technical solutions:
The anti-black streak dwarf of rice varieties 9194 site, comprise the qRBSDV3 between molecule marker RM282-RM14898, at the qRBSDV6 between molecule marker RM20069-RM30, at the qRBSDV9 between molecule marker RM3912-RM257 and the qRBSDV11 between molecule marker RM26062-RM536; Described molecule marker RM282 primer is SEQ ID NO.1/SEQ ID NO.2, and the amplified band when having qRBSDV3 is the 146bp band; Described molecule marker RM14898 primer is SEQ ID NO.3/SEQ ID NO.4, and the amplified band when having qRBSDV3 is the 176bp band; Described molecule marker RM20069 primer is SEQ ID NO.5/SEQ ID NO.6, and the amplified band when having qRBSDV6 is the 162bp band; Described molecule marker RM30 primer is SEQ ID NO.7/SEQ ID NO.8, and the amplified band when having qRBSDV6 is the 136bp band; Described molecule marker RM3912 primer is SEQ ID NO.9/SEQ ID NO.10, and the amplified band when having qRBSDV9 is the 191bp band; Described molecule marker RM257 primer is SEQ ID NO.11/SEQ ID NO.12, and the amplified band when having qRBSDV9 is the 155bp band; Described molecule marker RM26062 primer is SEQ ID NO.13/SEQ ID NO.14, and the amplified band when having qRBSDV11 is the 149bp band; Described molecule marker RM536 primer is SEQ ID NO.15/SEQ ID NO.16, and the amplified band when having qRBSDV11 is the 242bp band.
The molecule marking method of the anti-black streak dwarf of rice varieties 9194 site qRBSDV3: with molecule marker RM282 primer: SEQ ID NO.1/SEQ ID NO.2 or with molecule marker RM14898 primer: SEQ ID NO.3/SEQ ID NO.4, the anti-black streak dwarf kind of amplifying rice or breeding material DNA, if can amplify the amplified fragments of 146bp with molecule marker RM282 primer, or can amplify the amplified fragments of 176bp with molecule marker RM14898 primer, indicate the existence of Rice Varieties for Resistance black streak dwarf site qRBSDV3.
The molecule marking method of the anti-black streak dwarf of rice varieties 9194 site qRBSDV6: use molecule marker RM20069 primer: SEQ ID NO.5/SEQ ID NO.6, perhaps use molecule marker RM30 primer: the SEQ ID NO.7/SEQ ID anti-black streak dwarf kind of NO.8 amplifying rice or breeding material DNA, if can amplify the amplified fragments of 162bp with molecule marker RM20069 primer, or can amplify the amplified fragments of 136bp with molecule marker RM30 primer, indicate the existence of Rice Varieties for Resistance black streak dwarf site qRBSDV6.
The molecule marking method of the anti-black streak dwarf of rice varieties 9194 site qRBSDV9: use molecule marker RM3912 primer: SEQ ID NO.9/SEQ ID NO.10, perhaps use molecule marker RM257 primer: the SEQ ID NO.11/SEQ ID anti-black streak dwarf kind of NO.12 amplifying rice or breeding material DNA, if can amplify the amplified fragments of 191bp with molecule marker RM3912 primer, or all indicate the existence of Rice Varieties for Resistance black streak dwarf site qRBSDV9 with the amplified fragments that molecule marker RM257 primer can amplify 155bp.
The molecule marking method of the anti-black streak dwarf of rice varieties 9194 site qRBSDV11: use molecule marker RM26062 primer: SEQ ID NO.13/SEQ ID NO.14, perhaps use molecule marker RM536 primer: the SEQ ID NO.15/SEQ ID anti-black streak dwarf kind of NO.16 amplifying rice or breeding material DNA, if can amplify the amplified fragments of 149bp with molecule marker RM26062 primer, or can amplify the amplified fragments of 242bp with molecule marker RM536 primer, indicate the existence of Rice Varieties for Resistance black streak dwarf site qRBSDV11.
Screen the method for the molecule marker in the anti-black streak dwarf of described rice varieties 9194 site, comprise following steps:
(1) take anti-black streak dwarf kind 9194 as maternal, sense black streak dwarf japonica rice variety Suyunuo is male parent, hybridization F
1, built the F that comprises 200 individual plants
2Segregating population, each F
2Individual plant obtains corresponding F by selfing
2:3Family is carried out anti-black streak dwarf and is identified;
(2) extract parent, F with the SDS method
1And F
2The DNA of each strain of colony adopts simple sequence repeat marker SSR to carry out the polymorphism screening to two parents, and PCR carries out on the PTC-200 amplification instrument, and amplified production carries out electrophoretic analysis on 8% polyacrylamide gel, polymorphic primer is arranged at F between the parent
2Analyze in colony, obtain colony's genotype data;
(3) according to chain exchange rule, utilize colony's genotype data to build the genetic map of paddy rice, software used is MAPMARKER/EXP3.0, with the Kosambi function, recombination value is converted to genetic distance;
(4) adopt the field to bring out in conjunction with indoor inoculation and identify the Resistance Identification that carries out black streak dwarf;
(5) utilize MAPMARKER/EXP3.0 software to carry out linkage analysis to colony's genotype of each molecule marker and the black streak dwarf resistance rank of corresponding family, and the Kosambi function is converted to genetic distance.Utilize Windows QTL Cartographer V2.0 software composite interval mapping method, scan in full genome range take 2cM as step-length.5% overall conspicuous level is adopted in the detection of QTL, and the remarkable threshold of corresponding LOD statistic is estimated with the permutation test method, is total to duplicate sampling 1000 times.Additive effect and the contribution rate in each site have been estimated simultaneously;
(6) obtain four of the anti-black streak dwarf gene locuss of anti-black streak dwarf kind 9194, be respectively qRBSDV3, between mark RM282-RM14898; QRBSDV6 is between mark RM20069-RM30; QRBSDV9 is between mark RM3912-RM257; QRBSDV11 is between mark RM26062-RM536.Molecule marker by anti-black streak dwarf key-gene detects whether contain this key-gene site in resistant variety 9194 and derived varieties (being) thereof, and measurable its black streak dwarf resistance level improves the efficiency of selection of anti-black streak dwarf paddy rice greatly.
The application of the anti-black streak dwarf of rice varieties 9194 of the present invention site in rice breeding.
The anti-black streak dwarf of rice varieties 9194 of the present invention site is the application in the anti-black streak dwarf kind of rapid screening or strain preferably.
The application of molecule marker primer in rice breeding in the anti-black streak dwarf of rice varieties 9194 of the present invention site.
The application of the molecule marker primer in the anti-black streak dwarf of rice varieties 9194 of the present invention site in the anti-black streak dwarf kind of rapid screening or strain.
Beneficial effect
In earlier stage, the applicant to derive from 20 countries totally 960 parts of rice varieties carried out anti-black streak dwarf field and bring out with artificial inoculation and identify, screen 4 rice varieties that the black streak dwarf sickness rate is lower, wherein rice varieties 9194, annual three place field tests were all to black streak dwarf performance high resistance in continuous 3 years, further indoor qualification result also shows, this kind has higher black streak dwarf resistance.The excavation in this anti-source is location, clone and the breeding utilization of the water resistant rice black streak dwarf gene material that provides the foundation.The present invention utilizes 9194 to hybridize with the susceptible variety Suyunuo F that obtains
2Colony carries out genetic linkage analysis, obtains four anti-black streak dwarf gene locuss, is respectively qRBSDV3, qRBSDV6, qRBSDV9 and qRBSDV11.By detecting in resistant variety 9194 and derived varieties (being) thereof whether contain anti-black streak dwarf gene locus with the chain molecule marker of above-mentioned four gene locuss, measurable its black streak dwarf resistance level improves the efficiency of selection of anti-black streak dwarf paddy rice greatly.The molecule marking method of the anti-black streak dwarf major gene loci of rice varieties 9194 provided by the present invention has the following advantages:
(1) located first in the world the gene locus of the anti-black streak dwarf in rice varieties 9194 with the SSR mark by the present invention.
(2) by the resistant gene site locality specific of molecule marker of the present invention location, it is convenient to identify.By detecting the chain molecule marker of these and anti-black streak dwarf gene locus, namely can predict the black streak dwarf resistance of rice plant, the genotype detection that is used for rice varieties or strain, judging whether this kind or strain have the black streak dwarf resistance, and then rapid screening disease-resistant variety or strain are used for rice breeding.The resistant gene site easy to detect fast, not affected by environment;
(3) the assistant breeding select target is clear and definite, saves cost.In traditional breeding way, at first to collect parent and Cultivar with anti-source and carry out a series of hybridization, and will carry out individual plant to the resistance of black streak dwarf and select.Black streaked dwarf virus of rice is carried out the phenotypic evaluation complexity, simultaneously affected by environment, because black streak dwarf can not pass poison through ovum, pass the complicated qualification processes such as poison through raising small brown rice planthopper, band poison and inoculation, very difficult, the result reliability of phenotypic evaluation is low.Therefore, anti-black streak dwarf breeding is not only time-consuming, and difficulty is large, and cost is high.By detecting anti-black streak dwarf gene locus, can just identify in seedling stage the individual plant of anti-black streak dwarf, eliminate other plant, not only save production cost but also greatly improve the efficiency of selection of anti-black streak dwarf paddy rice.
Description of drawings
The postvaccinal resistance performance of Fig. 1 9194 and Suyunuo.
Fig. 2 9194 and Suyunuo F
2Colony's anti-black streak dwarf number of times distribution plan.
The distribution of the anti-black-streaked dwarf ospc gene of Fig. 3 rice varieties 9194 on karyomit(e).
The electrophoretogram of the anti-black-streaked dwarf ospc gene of Fig. 4 compact linkage molecule mark.M:Mark; 1:9194; 2: Suyunuo; 3:F
1; 4-13: extremely anti-individual plant; 14-23: utmost point sense individual plant.
Embodiment
Materials and methods:
(1) 9194/ Suyunuo F
2Colony builds and phenotypic evaluation
(1) take anti-black streak dwarf kind 9194 as maternal, sense black streak dwarf japonica rice variety Suyunuo is male parent, and hybridization has built 9194/ Suyunuo F
2Segregating population, each F
2Individual plant obtains corresponding 9194/ Suyunuo F by selfing
2:3Family.
(2) phenotypic evaluation
(2.1) evaluation is brought out in the field
Material is planted respectively in Donghai County Huang Chuan town, soil cities and towns, Ganyu County and experimental plot, hot farm, east, Guanyun County, and selecting surrounding is the plot plantation material to be identified of wheatland, to guarantee to obtain capacity worm source.Material all divided two to repeat sowing in May 10 (front 3 weeks of wheat harvest) in 2010 and 2011, and each repeats single kind and broadcasts 80, uniform broadcasting 1 row, the long 50cm of row, line-spacing 10cm.June 27 transplanted, and single seedling is planted, distance between rows and hills 15cm * 20cm, and each kind is planted 60 caves.Conventional rich water quality management is not used any agricultural chemicals.Identified that material sowed respectively at May 5, May 10 and 3 dates of seeding of bu in Mays 15 in 2012, each kind is broadcast 150, transplanting time is respectively June 20, June 25 and June 30, and each kind is planted 120 caves, and other planting patternss are identical with 2010 and 2011.Black streak dwarf is brought out as follows: at first by dish bat method investigation small brown rice planthopper population density, the enamel basin of 33cm * 45cm * 5cm is aimed at the rice shoot base portion pat plant, small brown rice planthopper is clapped to be fallen within dish, the small brown rice planthopper that then will fall into rapidly dish imports the high 60cm of being about, and diameter is about in the plastic tank of 35cm.Investigate in the experimental plot in early June, fixed 5 samplings of planting material field diagonal lines are clapped 0.30m2 at every, record small brown rice planthopper quantity.At random 100 small brown rice planthoppers that come from the collection of experiment field are carried out the RT-PCR detection of rice black-streaked dwarf virus, determine to take malicious rate.
In rice tillering Sheng phase late July statistics sickness rate.This moment, disease plant showed obvious symptom: extremely significantly stunt, lobus cardiacus is broken through the inferior lobe leaf sheath and go out, the part plant show as from lower pulvinus mouth stretch out in the shape of a spiral, that the top of partial blade occurs revolving shape is curling.And that healthy rice strain performance is tillered is vigorous, and vitality is strong, but jointing not yet, disease plant and healthy plant contrast are very obvious.In addition, the disease plant of this moment seldom has the osculant symptom, adds up as the anti-black streak dwarf phenotypic number of kind with the morbidity percentage mean value of each 2 repetition in test point.
(2.2) artificial inoculation black-streaked dwarf virus
With parent, disease-resistant and susceptible check variety presoaking and germinating, to sow 200 and show money or valuables one carries unintentionally chitting piece in the plastic box of 70.5cm * 50.5cm * 41.5cm, the bottom connects the plastics tubing water flowing, keeps approximately 3cm of water layer.Sick and weak seedling is eliminated in thinning when rice shoot grows to the 2.5-3.0 leaf, keeps the approximately strong seedling of 150 strain neat and consistent, and seal with gauze on each plastic box top.Access was inoculated from the small brown rice planthopper that East Sea Huang Chuan (the test point, small brown rice planthopper is the highest with malicious rate) gathers on June 10th, 2012, drove small brown rice planthopper every day for several times.Take away gauze after 7d, remove the small brown rice planthopper on seedling, postvaccinal transplantation of seedlings to the land for growing field crops, is grown to the Sheng of tillering and investigated the state of an illness during phase.Artificial inoculation each kind same period is done three repetitions, calculates by 20 small brown rice planthoppers of every seedling and connects worm.
(2) 9194/ Suyunuo F
2The molecular marker analysis of colony
(1) extract parent, F with the SDS method
1And F
2The DNA of each strain of colony.
(2) ssr analysis is with reference to the program of Chen et al. (1997).10 μ l reaction systems comprise: 10mM Tris-HCl pH 8.3,50mM KCl, 1.5mM MgCl2,50 μ M dNTPs, 0.2 μ M primer, 0.5U Taq polymerase (TaKaRa, Dalian) and 20ng of DNA profiling.Amplified reaction carries out on PTC-200 (MJ Research Inc.) PCR instrument: 94 ℃ of 4min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, 35 circulations; 72 ℃ of 7min.Amplified production separates with 8% non-sex change PAGE glue, dyes colour developing by silver, and silver dyes program and formulates according to the method for Sanguinetti et al. (1994) and form.The lamp box that luminescent lamp is equipped with in the DNA band utilization of amplification is observed.Record result, polymorphic primer is arranged at F between the parent
2Analyze in colony, obtain colony's genotype data;
(3) according to chain exchange rule, utilize colony's genotype data to build the genetic map of paddy rice, software used is MAPMAKER/EXP3.0, minimum LOD value is made as 2, obtains linkage map;
(4) utilize Windows QTL Cartographer V2.0 software (Wang et al., 2003) composite interval mapping method (Composite interval mapping, CIM), scan in full genome range take 2cM as step-length.5% overall conspicuous level is adopted in the detection of QTL, and the remarkable threshold of corresponding LOD statistic is estimated with permutation test (Permutation test) (Churchill and Doerge, 1994) method, is total to duplicate sampling 1000 times.Additive effect and the contribution rate in each site have been estimated simultaneously.Utilize the MAPMAKER/3.0 analysis software to carry out the compartment analysis of black streak dwarf resistance and SSR mark.And the Kosambi function is converted to genetic distance (cM).
(3) results and analysis:
9194 and Suyunuo through the field of black streak dwarf and its average attack rate of indoor evaluation respectively 8.9% and 78%, show that 9194 have higher black streak dwarf resistance, and Suyunuo is to black streak dwarf extremely sensitive (Fig. 1).200 F
2:3Family is continuous distribution to the fastness frequency distribution of black streak dwarf, shows to have a plurality of anti-black streak dwarf sites (Fig. 2) in this colony.From F
2Select individual plant 10 strains that three places and indoor evaluation all show high resistance or high sense in colony, carry out 12 chromosomal linkage analysises, in paddy rice the 3rd, 6,9 and 11 karyomit(e)s find with objective trait, chain sign is arranged.Further built the 3rd, 6,9 and the linkage map of 11 whole chromosomes, in conjunction with phenotypic data, the QTL that utilizes Windows QTL Cartographer V2.0 software to carry out anti-black streak dwarf detects.Result detects an anti-black streak dwarf QTL qRBSDV3 between paddy rice the 3rd chromosomal marker RM282-RM14898, the LOD value is 2.34, and contribution rate is 5.4%; An anti-black streak dwarf QTL qRBSDV6 detected between paddy rice the 6th chromosomal marker RM20069-RM30, the LOD value is 4.42, and contribution rate is 10.3%; An anti-black streak dwarf QTL qRBSDV9 detected between paddy rice the 9th chromosomal marker RM3912-RM257, the LOD value is 6.63, and contribution rate is 35.5%; An anti-black streak dwarf QTL qRBSDV11 detected between paddy rice the 11st chromosomal marker RM26062-RM536, the LOD value is 3.55, and contribution rate is 31.1%.Above-mentioned four the soluble phenotypic variation 82.3%(of QTLs Fig. 3).
Molecule marker RM282 primer:
Upstream primer: CTGTGTCGAAAGGCTGCAC(SEQ ID NO.1)
Downstream primer: CAGTCCTGTGTTGCAGCAAG(SEQ ID NO.2)
Molecule marker RM14898 primer:
Upstream primer: ATGTTCAACCTTGTCCCGACTAACC(SEQ ID NO.3)
Downstream primer: TAAAGACGGCAGCTATCACTTTGC(SEQ ID NO.4)
With molecule marker RM282 primer: SEQ ID NO.1/SEQ ID NO.2 or with molecule marker RM14898 primer: SEQ ID NO.3/SEQ ID NO.4, the anti-black streak dwarf kind of amplifying rice or breeding material DNA, if can amplify the amplified fragments of 146bp with molecule marker RM282 primer, or can amplify the amplified fragments of 176bp with molecule marker RM14898 primer, indicate exist (Fig. 4) of Rice Varieties for Resistance black streak dwarf site qRBSDV3.
Molecule marker RM20069 primer:
Upstream primer: GCGAGCGAGAGGAGAGATAGACG(SEQ ID NO.5)
Downstream primer: CGAATTCGGCACGAGTAATAGGG(SEQ ID NO.6)
Molecule marker RM30 primer:
Upstream primer: ACACTGTAGCGGCCACTG(SEQ ID NO.7)
Downstream primer: CCTCCACTGCTCCACATCTT(SEQ ID NO.8)
With molecule marker RM20069 primer: SEQ ID NO.5/SEQ ID NO.6, perhaps use molecule marker RM30 primer: the SEQ ID NO.7/SEQ ID anti-black streak dwarf kind of NO.8 amplifying rice or breeding material DNA, if can amplify the amplified fragments of 162bp with molecule marker RM20069 primer, or can amplify the amplified fragments of 136bp with molecule marker RM30 primer, indicate exist (Fig. 4) of Rice Varieties for Resistance black streak dwarf site qRBSDV6.
Molecule marker RM3912 primer:
Upstream primer: GCGAGCGAGAGGAGAGATAGACG(SEQ ID NO.9)
Downstream primer: CGAATTCGGCACGAGTAATAGGG(SEQ ID NO.10)
Molecule marker RM257 primer:
Upstream primer: ACACTGTAGCGGCCACTG(SEQ ID NO.11)
Downstream primer: CCTCCACTGCTCCACATCTT(SEQ ID NO.12)
With molecule marker RM3912 primer: SEQ ID NO.9/SEQ ID NO.10, perhaps use molecule marker RM257 primer: the SEQ ID NO.11/SEQ ID anti-black streak dwarf kind of NO.12 amplifying rice or breeding material DNA, if can amplify the amplified fragments of 191bp with molecule marker RM3912 primer, or all indicate exist (Fig. 4) of Rice Varieties for Resistance black streak dwarf site qRBSDV9 with the amplified fragments that molecule marker RM257 primer can amplify 155bp.
Molecule marker RM26062 primer:
Upstream primer: TTTGCGTTTGCGTTTGCGTAGG(SEQ ID NO.13)
Downstream primer: ACCCGTACACCTCCACACAGTGC(SEQ ID NO.14)
Molecule marker RM536 primer:
Upstream primer: TCTCTCCTCTTGTTTGGCTC(SEQ ID NO.15)
Downstream primer: ACACACCAACACGACCACAC(SEQ ID NO.16)
With molecule marker RM26062 primer: SEQ ID NO.13/SEQ ID NO.14, perhaps use molecule marker RM536 primer: the SEQ ID NO.15/SEQ ID anti-black streak dwarf kind of NO.16 amplifying rice or breeding material DNA, if can amplify the amplified fragments of 149bp with molecule marker RM26062 primer, or can amplify the amplified fragments of 242bp with molecule marker RM536 primer, indicate exist (Fig. 4) of Rice Varieties for Resistance black streak dwarf site qRBSDV11.
By detecting in resistant variety 9194 and derived varieties (being) thereof whether contain anti-black streak dwarf gene locus with the chain molecule marker of above-mentioned four gene locuss, measurable its black streak dwarf resistance level improves the efficiency of selection of anti-black streak dwarf paddy rice greatly.
Claims (10)
1. the anti-black streak dwarf of rice varieties 9194 site, it is characterized in that comprising the qRBSDV3 between molecule marker RM282-RM14898, at the qRBSDV6 between molecule marker RM20069-RM30, at the qRBSDV9 between molecule marker RM3912-RM257 and the qRBSDV11 between molecule marker RM26062-RM536; Described molecule marker RM282 primer is SEQ ID NO.1/SEQ ID NO.2, and the amplified band when having qRBSDV3 is the 146bp band; Described molecule marker RM14898 primer is SEQ ID NO.3/SEQ ID NO.4, and the amplified band when having qRBSDV3 is the 176bp band; Described molecule marker RM20069 primer is SEQ ID NO.5/SEQ ID NO.6, and the amplified band when having qRBSDV6 is the 162bp band; Described molecule marker RM30 primer is SEQ ID NO.7/SEQ ID NO.8, and the amplified band when having qRBSDV6 is the 136bp band; Described molecule marker RM3912 primer is SEQ ID NO.9/SEQ ID NO.10, and the amplified band when having qRBSDV9 is the 191bp band; Described molecule marker RM257 primer is SEQ ID NO.11/SEQ ID NO.12, and the amplified band when having qRBSDV9 is the 155bp band; Described molecule marker RM26062 primer is SEQ ID NO.13/SEQ ID NO.14, and the amplified band when having qRBSDV11 is the 149bp band; Described molecule marker RM536 primer is SEQ ID NO.15/SEQ ID NO.16, and the amplified band when having qRBSDV11 is the 242bp band.
2. the molecule marking method of the anti-black streak dwarf of rice varieties 9194 site qRBSDV3, it is characterized in that: with molecule marker RM282 primer: SEQ ID NO.1/SEQ ID NO.2 or with molecule marker RM14898 primer: SEQ ID NO.3/SEQ ID NO.4, the anti-black streak dwarf kind of amplifying rice or breeding material DNA, if can amplify the amplified fragments of 146bp with molecule marker RM282 primer, or can amplify the amplified fragments of 176bp with molecule marker RM14898 primer, indicate the existence of Rice Varieties for Resistance black streak dwarf site qRBSDV3.
3. the molecule marking method of the anti-black streak dwarf of rice varieties 9194 site qRBSDV6, it is characterized in that: use molecule marker RM20069 primer: SEQ ID NO.5/SEQ ID NO.6, perhaps use molecule marker RM30 primer: the SEQ ID NO.7/SEQ ID anti-black streak dwarf kind of NO.8 amplifying rice or breeding material DNA, if can amplify the amplified fragments of 162bp with molecule marker RM20069 primer, or can amplify the amplified fragments of 136bp with molecule marker RM30 primer, indicate the existence of Rice Varieties for Resistance black streak dwarf site qRBSDV6.
4. the molecule marking method of the anti-black streak dwarf of rice varieties 9194 site qRBSDV9, it is characterized in that: use molecule marker RM3912 primer: SEQ ID NO.9/SEQ ID NO.10, perhaps use molecule marker RM257 primer: the SEQ ID NO.11/SEQ ID anti-black streak dwarf kind of NO.12 amplifying rice or breeding material DNA, if can amplify the amplified fragments of 191bp with molecule marker RM3912 primer, or all indicate the existence of Rice Varieties for Resistance black streak dwarf site qRBSDV9 with the amplified fragments that molecule marker RM257 primer can amplify 155bp.
5. the molecule marking method of the anti-black streak dwarf of rice varieties 9194 site qRBSDV11, it is characterized in that: use molecule marker RM26062 primer: SEQ ID NO.13/SEQ ID NO.14, perhaps use molecule marker RM536 primer: the SEQ ID NO.15/SEQ ID anti-black streak dwarf kind of NO.16 amplifying rice or breeding material DNA, if can amplify the amplified fragments of 149bp with molecule marker RM26062 primer, or can amplify the amplified fragments of 242bp with molecule marker RM536 primer, indicate the existence of Rice Varieties for Resistance black streak dwarf site qRBSDV11.
6. screen the method for the molecule marker in the anti-black streak dwarf of rice varieties 9194 claimed in claim 1 site, it is characterized in that comprising following steps:
(1) take anti-black streak dwarf kind 9194 as maternal, sense black streak dwarf japonica rice variety Suyunuo is male parent, hybridization F
1, built the F that comprises 200 individual plants
2Segregating population, each F
2Individual plant obtains corresponding F by selfing
2:3Family is carried out anti-black streak dwarf and is identified;
(2) extract parent, F with the SDS method
1And F
2The DNA of each strain of colony adopts simple sequence repeat marker SSR to carry out the polymorphism screening to two parents, and PCR carries out on the PTC-200 amplification instrument, and amplified production carries out electrophoretic analysis on 8% polyacrylamide gel, polymorphic primer is arranged at F between the parent
2Analyze in colony, obtain colony's genotype data;
(3) according to chain exchange rule, utilize colony's genotype data to build the genetic map of paddy rice, software used is MAPMARKER/EXP3.0, with the Kosambi function, recombination value is converted to genetic distance;
(4) adopt the field to bring out in conjunction with indoor inoculation and identify the Resistance Identification that carries out black streak dwarf;
(5) utilize MAPMARKER/EXP3.0 software to carry out linkage analysis to colony's genotype of each molecule marker and the black streak dwarf resistance rank of corresponding family, and the Kosambi function is converted to genetic distance.Utilize Windows QTL Cartographer V2.0 software composite interval mapping method, scan in full genome range take 2cM as step-length.5% overall conspicuous level is adopted in the detection of QTL, and the remarkable threshold of corresponding LOD statistic is estimated with the permutation test method, is total to duplicate sampling 1000 times.Additive effect and the contribution rate in each site have been estimated simultaneously;
(6) obtain four of the anti-black streak dwarf gene locuss of anti-black streak dwarf kind 9194, be respectively qRBSDV3, between mark RM282-RM14898; QRBSDV6 is between mark RM20069-RM30; QRBSDV9 is between mark RM3912-RM257; QRBSDV11 is between mark RM26062-RM536.Molecule marker by anti-black streak dwarf key-gene detects whether contain this key-gene site in resistant variety 9194 and derived varieties (being) thereof, and measurable its black streak dwarf resistance level improves the efficiency of selection of anti-black streak dwarf paddy rice greatly.
7. the application of the anti-black streak dwarf of rice varieties 9194 claimed in claim 1 site in rice breeding.
8. application according to claim 7 is characterized in that the application of the anti-black streak dwarf of rice varieties 9194 claimed in claim 1 site in the anti-black streak dwarf kind of rapid screening or strain.
9. the application of molecule marker primer in rice breeding in the anti-black streak dwarf of rice varieties 9194 claimed in claim 1 site.
10. application according to claim 9 is characterized in that the application of molecule marker primer in the anti-black streak dwarf kind of rapid screening or strain in the anti-black streak dwarf of described rice varieties 9194 site.
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