CN104450694A - q SRBSDV6 (Southern rice black-streaked dwarf virus 6) and molecular marker method thereof - Google Patents
q SRBSDV6 (Southern rice black-streaked dwarf virus 6) and molecular marker method thereof Download PDFInfo
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Abstract
The invention provides a q SRBSDV6 (Southern rice black-streaked dwarf virus 6) and a molecular marker method thereof. BC3F7 is obtained through hybridization, backcross and selfing of Guangxi common wild rice material y11 and the susceptible variety Guanghui 998 and then is subjected to association analysis and genetic linkage analysis to obtain the q SRBSDV6, and the q SRBSDV6 is positioned between RM20148 and RM 20297; when the molecular marker RM20148 primer has q SRBSDV6, the amplification band is 129 bp band; when the molecular marker RM 20297 primer has q SRBSDV6, the amplification band is 172 bp band. According to the invention, the molecular markers of q SRBSDV6 genes can be adopted to detect whether the resistant material y11 and the derived varieties (series) contain the q SRBSDV6, the resistance level of the SRBSDV6 can be predicted, and the selective efficiency of the q SRBSDV6 rice can be greatly improved.
Description
Technical field
The invention belongs to molecular genetics field, relate to the sick site qSRBSDV6 of the anti-southern rice black-streaked dwarf of Guangxi common wild-rice material y11 and molecule marking method thereof.
Background technology
Paddy rice is one of most important food crop in the world, and whole world population over half take rice as main food.Southern rice black-streaked dwarf was found first from calendar year 2001, and between short several years, the state of an illness spreads rapidly, caused extreme loss to the vast rice district of south China and North Vietnam Rice Production.Different from common rice black streak dwarf, the Virus Diseases of Rice of southern rice black-streaked dwarf disease to be a kind of with white backed planthopper be primary vehicle, its pathogenic virus is southern rice black-streaked dwarf virus (Southern rice black-streaked dwarf virus, be called for short SRBSDV), be the novel species that Reoviridae Fijivirus belongs to the 2nd group.Current production there is no specific pesticide for this type of disease, and mainly plant is in early days by the control of Whitebacked Planthopper, alleviate its harm, but prevention effect is undesirable, and contaminate environment, destroy the ecosystem.Cultivate and utilize disease-resistant variety to be acknowledged as the most economical effective and viral measure of eco-friendly control always, screening, excavation and innovation Resistant gerplasm are prerequisite and the basis of the sick breeding of anti-southern rice black-streaked dwarf.But about the research of anti-southern rice black-streaked dwarf ospc gene molecule mechanism is few, the Resistence research of host is also at the early-stage, only have the report of the relevant resistant material screening of a several section at present, there is not been reported for resistant gene excavation and breeding for disease resistance research.
Summary of the invention
For the above-mentioned deficiency of prior art, the invention discloses the sick site qSRBSDV6 of anti-southern rice black-streaked dwarf and molecule marking method thereof.
The sick site qSRBSDV6 of anti-southern rice black-streaked dwarf, between molecule marker RM20148-RM20297; Described molecule marker RM20148 primer, amplified band when there is qSRBSDV6 is 129bp band; Described molecule marker RM20297 primer, amplified band when there is qSRBSDV6 is 172bp band.
The molecule marking method of the sick site qSRBSDV6 of described anti-southern rice black-streaked dwarf: with molecule marker RM20148 primer, or with the sick kind of the anti-southern rice black-streaked dwarf of molecule marker RM20297 primer amplification or breeding material DNA, if 129bp amplified fragments can be amplified with molecule marker RM20148 primer, or 172bp amplified fragments can be amplified with molecule marker RM20297 primer, then indicate the existence of the sick site qSRBSDV6 of the anti-southern rice black-streaked dwarf of rice varieties (strain).
The method of the molecule marker of the sick site qSRBSDV6 of the anti-southern rice black-streaked dwarf described in screening, comprises the following steps:
Step 1: with the common wild-rice material y11 of anti-southern rice black-streaked dwarf disease for donor, the cultivated rice kind of sense southern rice black-streaked dwarf disease extensive 998 be extensively acceptor, by hybridizing, backcrossing and selfing constructs a set of rice chromosome fragment introgressive line BC comprising 280 strains
3f
7;
Contriver has carried out the artificial inoculation of southern rice black-streaked dwarf disease to 648 parts of common wild-rices and cultivated rice material and qualification is brought out in field, screen material-common wild-rice y11 that the sick sickness rate of 1 part of southern rice black-streaked dwarf is lower, this common wild-rice material y11 continuous three years 3 field tests are all to southern rice black-streaked dwarf disease performance high resistance, carry out indoor inoculation qualification further, the result of three repetitive identified shows that this kind has the sick resistance of higher southern rice black-streaked dwarf.The excavation in this anti-source is the location of anti-southern rice black-streaked dwarf ospc gene, clone and breeding utilization provide material foundation.
Step 2: adopt indoor inoculation qualification to bring out in conjunction with field the Resistance Identification that southern rice black-streaked dwarf disease is carried out in qualification;
Step 3: get high resistance and each 30 strains of high sense in introgressive line, CTAB method (cetyl trimethylammonium bromide method) is utilized to extract the DNA of rice plant, the high and low pond of constructed dna, utilizes the paddy rice Super BSA method antagonism southern rice black-streaked dwarf ospc gene based on simplifying genomic sequencing technique SLAF-seq to carry out key-gene location;
Step 4: extensively with the sick introgressive line of high resistance southern rice black-streaked dwarf and receptor parent extensive 998 hybridize and obtain F2:3 family, Resistance Identification is carried out to wherein 300 familys, extract DNA, by simple sequence repeat marker ssr analysis, utilize the southern rice black-streaked dwarf sick resistance rank of MAPMARKER/EXP3.0 software to polymorphism mark genotype and corresponding family to carry out linkage analysis, and Kosambi function is converted to genetic distance.Utilize Windows QTL Cartographer V2.0 software composite interval mapping method, be that step-length scans in genome with 2cM, the detection of QTL adopts 5% global significance level, and the remarkable threshold values permutation test method of corresponding LOD statistic is estimated, altogether duplicate sampling 1000 times;
Step 5: obtain the sick site qSRBSDV6 mono-of the anti-southern rice black-streaked dwarf of anti-southern rice black-streaked dwarf sick wild-rice material y11, between mark RM20148-RM20297, whether detected in resistant material y11 and derived varieties (being) thereof containing this key-gene site by the molecule marker of the sick key-gene of anti-southern rice black-streaked dwarf, predict the sick resistance level of its southern rice black-streaked dwarf, greatly improve the efficiency of selection of the sick paddy rice of anti-southern rice black-streaked dwarf.
Wherein, key-gene described in step 3 is positioned on the 6th karyomit(e); Step 3 adopts CTAB method, can Polysaccharide removing very well, is more conducive to the DNA of Isolation and purification rice plant.
The application of the sick site qSRBSDV6 of anti-southern rice black-streaked dwarf of the present invention in rice breeding.
The application of the sick site qSRBSDV6 of anti-southern rice black-streaked dwarf of the present invention in the sick kind of the anti-southern rice black-streaked dwarf of rapid screening or strain.
The molecule marker primer of the sick site qSRBSDV6 of anti-southern rice black-streaked dwarf of the present invention is in the application in rice breeding.
The application of molecule marker primer in the sick kind of the anti-southern rice black-streaked dwarf of rapid screening or strain of the sick site qSRBSDV6 of anti-southern rice black-streaked dwarf of the present invention.
Compared with prior art, beneficial effect of the present invention is:
The present invention utilizes the common wild-rice material y11 of anti-southern rice black-streaked dwarf disease and susceptible variety extensively extensive 998 to hybridize, backcross and BC that selfing obtains
3f
7carry out association analysis and genetic linkage analysis, obtain a sick site qSRBSDV6 of anti-southern rice black-streaked dwarf, whether detected in resistant material y11 and derived varieties (being) thereof containing anti-southern rice black-streaked dwarf ospc gene site by the molecule marker chain with qSRBSDV6, the resistance level of its southern rice black-streaked dwarf disease can be predicted, greatly improve the efficiency of selection of the sick paddy rice of anti-southern rice black-streaked dwarf.The molecule marking method of the sick major gene loci of anti-southern rice black-streaked dwarf provided by the present invention, has the following advantages:
(1) the present invention located the sick site of Rice Resistance southern rice black-streaked dwarf with utilizing based on the paddy rice Super BSA method and SSR molecular marker that simplify genomic sequencing technique SLAF-seq in the world first.
(2) clear and definite by the resistant gene site location of Molecular mapping of the present invention, identify convenient and swift.By detecting the chain molecule marker in these and anti-southern rice black-streaked dwarf ospc gene site, namely the sick resistance level of the southern rice black-streaked dwarf of rice plant can be predicted, for the genotype detection of rice varieties or strain, to judge whether this kind or strain have the sick resistance of southern rice black-streaked dwarf, and then rapid screening disease-resistant variety or strain are used for rice breeding, resistant gene site fast easy to detect, not easily affected by environment.
(3) assistant breeding select target is clear and definite, and efficiency is high.Traditional breeding technology in the past, in raising resistance breeding method, utilizes the donor parents containing disease-resistant gene to carry out with receptor parent hybridizing, repeatedly backcrossing or be polymerized and reestablish diplomatic relations usually.And Single-plant selection to be carried out to the resistance of southern rice black-streaked dwarf disease; But because southern rice black-streaked dwarf disease can not pass poison through the worm's ovum of white backed planthopper, pass through and raise white backed planthopper, raise poison and inoculate the qualification process passing the complexity such as poison, very greatly, the result reliability of phenotypic evaluation is low for qualification difficulty; Therefore, anti-southern rice black-streaked dwarf is sick, and breeding is not only time-consuming takes a lot of work, and difficulty is large, cost is high.By utilizing the Markers for Detection southern rice black-streaked dwarf ospc gene site in the present invention, the individual plant of anti-southern rice black-streaked dwarf disease can be just identified in seedling stage, eliminate other plant, not only cost-saving, and substantially increase the efficiency of selection of the sick paddy rice of anti-southern rice black-streaked dwarf.
Accompanying drawing explanation
Fig. 1 is the anti-southern rice black-streaked dwarf ospc gene position on chromosome of common wild-rice material y11.
Fig. 2 is the electrophoretogram of anti-southern rice black-streaked dwarf ospc gene compact linkage molecule mark.
In figure: M:Mark; 1:y11; 2: extensively extensive 998; 3-12: extremely anti-individual plant; 13-22: pole sense individual plant.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Materials and methods:
Step 1: the structure of genetic analysis segregating population;
With anti-southern rice black-streaked dwarf sick wild-rice material y11 for male parent, the sick kind of sense southern rice black-streaked dwarf extensive 998 be extensively maternal, prepares F
1hybrid, then extensive 998 to be extensively maternal, 998/y11 hybrid is that male parent carries out backcrossing until BC
3f
1, carry out afterwards from accompanying each other for breeding acquisition BC
3f
7colony.Utilize the sick introgressive line agriculture 14 of high resistance southern rice black-streaked dwarf to hybridize selfing again with receptor parent, obtain Resistant segregation colony F2:3 family.
Step 2: adopt indoor inoculation qualification to bring out in conjunction with field the Resistance Identification that southern rice black-streaked dwarf disease is carried out in qualification;
(1) qualification is brought out in field
Material is planted in field that paddy rice is tested, academy of agricultural sciences, Guangxi, Nanning City respectively, that susceptible rice field, Na Xia village, shuttle town, Fangchenggang City Fangcheng District, Xingan County, Guilin City Hunan Li Zhen point susceptible rice field, pool village.Within 2012-2014 years continuous 3 years, carry out 3 qualifications, the qualification of Nanning City and Fangchenggang City is made in evening and is carried out, the qualification of Guilin City in make and carry out.36 strains inserted by each material, if two repetitions, conventional water and fertilizer management, does not use any agricultural chemicals, and expert evidence side plantation susceptible variety TN1 is as bringing out kind.Early tillering stage is detected the TR-PCR that 50 white backed planthoppers come from the collection of test field carry out southern rice black-streaked dwarf virus at random, determines to be with malicious rate.
Phase statistics sickness rate is contained in rice tillering.Now disease plant shows obvious symptom: extremely significantly stunt, and blade is dark green, stiff, and lobus cardiacus stretches out from inferior lobe leaf sheath, and the vein of blade back and cane occur that wax or short strip shape knurl are dashed forward, stipes has angry fibrous root.And healthy plant growth is vigorous, highly normally, disease plant and healthy plant contrast are clearly.Add up using the percent incidence mean value that each experimental point 2 repeats as the sick phenotypic number of anti-southern rice black-streaked dwarf of kind.
(2) artificial inoculation southern rice black-streaked dwarf virus qualification
By parent, BC
3f
7colony's strain, disease-resistant and susceptible check variety presoaking and germinating, sow about 100 and show money or valuables one carries unintentionally chitting piece in the enamel basin of 20cm × 30cm × 3cm, if 2 repetitions.When rice shoot grows to 1.5-2 leaf, eliminate weak seedling, retain the healthy seedling of 50 strain neat and consistent.Put into insect protected net cage, access from Nanning dan Bu Zhen collection and pass through the white backed planthopper raising poison, every plant inoculates in the ratio of 2-3 cephalont.Expelling parasite 3-5 time every day, guarantees that every strain rice seedling all has white backed planthopper to stop.The plant hopper on seedling is removed after 3d.Postvaccinal rice seedling is transplanted to insect-proof net chamber, and tillering regularity to be grown to carries out Disease investigation.
The sick key-gene location of anti-southern rice black-streaked dwarf of wide extensive 998 segregating populations of step 3:y11/
(1) parent, F1 and BC is extracted by CTAB method
3f
7the DNA of colony's strain, gets high resistance, each 30 strains of high sense, utilizes CTAB method to extract the DNA of rice plant, the high and low pond of constructed dna in introgressive line;
(2) utilize the paddy rice Super BSA method enantiopathy gene based on simplifying genomic sequencing technique SLAF-seq to carry out analyzing and the assignment of genes gene mapping, experimental procedure is as follows:
sLAF-seq experiment flow: according to the enzyme butt case of previous information method design, carry out enzyme to each sample genomic dna to cut, flat end is carried out to endonuclease bamhi, 5 ' phosphorylation and 3 ' end add A process, connect sequence measuring joints, build the SALF-seq library of each sample, cut glue and choose target fragment, finally carry out pcr amplification and high-flux sequence;
bioinformatic analysis: the reads data of each sample obtained that checks order are assessed, after similarity cluster, genotype error correction, obtain SLAF label, polymorphism analysis is carried out to SLAF label, obtain the polymorphism mark of sample room, carry out the assignment of genes gene mapping according to the polymorphism mark obtained, obtain the difference mark that proterties is relevant.To the SLAF label obtained, carry out polymorphism analysis according to the difference between number of alleles and gene order, obtain the SLAF label of polymorphism, i.e. Marker.According to the positioning result of Marker, the quantity of statistics Marker on each karyomit(e), draws Marker distribution plan on chromosome;
the assignment of genes gene mapping: to obtain 8,526 Marker, carry out parent's data normalization, determine allelic parental source according to parent's degree of depth that checks order, choose a kind of genotype and derive from 1-2, another kind of genotype derives from the Marker totally 6,403 of 2-1.To these 6,403 Marker, SNP-index correlating method is adopted to test analysis.
Step 4: utilize the 6th chromosomal SSR marker to analyze F2:3 segregating population and enantiopathy gene is analyzed.Ssr analysis program is as follows:
described 12 μ lPCR reaction systems comprise: DNA (10ng/ μ l), 2 μ l; Primer (4pmol/ μ l), 1.5 μ l; 2 × Taq PCR Master Mix (middle Ke Ruitai), 6 μ l; DdH2O, 2.5 μ l;
amplified reaction carries out on Dongsheng dragon ETC811 amplification instrument, and described PCR reaction conditions is: 95 DEG C of denaturation 5min, 95 DEG C of sex change 35s, 55 DEG C of renaturation 35s, and 72 DEG C extend 1min, 35 circulations, and last 72 DEG C extend 7min.The amplified production Native PAGE glue of 8% is separated, by the colour developing of silver dye.There is polymorphic primer to analyze in F2:3 colony between parent, obtain sample genotype data;
according to chain exchange rule, utilize colony's genotype data to build the 6th map of paddy rice, software used is MARKER/EXP3.0, and minimum LOD value is set to 2, obtains linkage map;
utilizing Windows QTL Cartographer V2.5 software composite interval mapping method (Composite interval mapping, CIM), is that step-length scans in genome with 2cM.The detection of QTL adopts 5% global significance level, and the remarkable threshold permutation test method of corresponding LOD statistic is estimated, its duplicate sampling 1000 times.Have estimated additive effect and the contribution rate in each site simultaneously.Utilize MARMAKER/EXP3.0 analysis software to carry out the compartment analysis of the sick resistance of southern rice black-streaked dwarf and SSR marker, and Kosambi function is converted to genetic distance (cM).
Step 5: results and analysis
Y11 and extensively extensive 998 through the field of southern rice black-streaked dwarf disease and indoor qualification, its average attack rate is respectively 4.2% and 82.2%, show that y11 has the sick disease resistance of higher southern rice black-streaked dwarf, and extensively extensive 998 high sense southern rice black-streaked dwarfs is sick.Association analysis result shows, No. 6 karyomit(e) has and one section of region being obviously positioned at below threshold line detected, and this section of region is the region associated with anti-southern rice black-streaked dwarf ospc gene found.This site is positioned on the 6th chromosome long arm, and hereditary physical distance is 2Mb.Analyzed by SSR molecular marker, the F2:3 family of high resistance introgressive line and receptor parent is utilized to construct the 6th chromosomal genetic map, in conjunction with phenotypic data, the QTL utilizing Windows QTL Cartographer V2.5 software to carry out anti-southern rice black-streaked dwarf disease detects.As shown in Figure 1, result marks a QTL qSRBSDV6 anti-southern rice black-streaked dwarf disease being detected between RM20148 and RM20297 on the 6th karyomit(e) of paddy rice, and LOD value is 7.93, and contribution rate is 22.7%, 22.7% of soluble phenotypic variation.
Molecule marker RM20148 primer:
Upstream primer: GAGCCGAACTGACCTCCACTGC
Downstream primer: CACTGCCTCCGGGTCATTGC
Molecule marker RM20297:
Upstream primer: TTGGCACGGCCATATAACAAGC
Downstream primer: AAGTTGATGGCCTTTGGTTTGC
With molecule marker RM20148 primer or with RM20297 primer amplification anti-south rice black streaked dwarf virus of rice kind or breeding material DNA, as shown in Figure 2, the amplified fragments of 129bp can be amplified with molecule marker RM20148, or the amplified fragments of 172bp can be amplified with molecule marker RM20297 primer, then indicate the existence of the site qSRBSDV6 of Rice Varieties for Resistance southern rice black-streaked dwarf disease.
Whether detected in resistant material y11 and derived varieties (being) thereof containing anti-southern rice black-streaked dwarf ospc gene site by the molecule marker chain with said gene site, the sick resistance level of its southern rice black-streaked dwarf measurable, improves the efficiency of selection of the sick paddy rice of anti-southern rice black-streaked dwarf greatly.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.
Claims (8)
1. the sick site qSRBSDV6 of anti-southern rice black-streaked dwarf, is characterized in that: it is between molecule marker RM20148-RM20297; Described molecule marker RM20148 primer, amplified band when there is qSRBSDV6 is 129bp band, and described molecule marker RM20297 primer, amplified band when there is qSRBSDV6 is 172bp band.
2. the molecule marking method of the sick site qSRBSDV6 of anti-southern rice black-streaked dwarf according to claim 1, it is characterized in that: with molecule marker RM20148 primer, or with the sick kind of molecule marker RM20297 primer amplification Rice Resistance southern rice black-streaked dwarf or breeding material DNA, if 129bp amplified fragments can be amplified with molecule marker RM20148 primer, or 172bp amplified fragments can be amplified with molecule marker RM20297 primer, then indicate the existence of the sick site qSRBSDV6 of the anti-southern rice black-streaked dwarf of rice varieties (strain).
3. the method for the molecule marker of the sick site qSRBSDV6 of screening anti-southern rice black-streaked dwarf as claimed in claim 1, is characterized in that: comprise the following steps:
Step 1: with the common wild-rice material y11 of anti-southern rice black-streaked dwarf disease for donor, the cultivated rice kind of sense southern rice black-streaked dwarf disease extensive 998 be extensively acceptor, by hybridizing, backcrossing and selfing constructs a set of rice chromosome fragment introgressive line BC comprising 280 strains
3f
7;
Step 2: adopt indoor inoculation qualification to bring out in conjunction with field the Resistance Identification that southern rice black-streaked dwarf disease is carried out in qualification;
Step 3: get high resistance and each 30 strains of high sense in introgressive line, CTAB method is utilized to extract the DNA of rice plant, the high and low pond of constructed dna, utilizes the paddy rice Super BSA method antagonism southern rice black-streaked dwarf ospc gene based on simplifying genomic sequencing technique SLAF-seq to carry out key-gene location;
Step 4: extensively with the sick introgressive line of high resistance southern rice black-streaked dwarf and receptor parent extensive 998 hybridize and obtain F2:3 family, Resistance Identification is carried out to wherein 300 familys, extract DNA, by simple sequence repeat marker ssr analysis, utilize the black streak dwarf resistance rank of MAPMARKER/EXP3.0 software to polymorphism mark genotype and corresponding family to carry out linkage analysis, and Kosambi function is converted to genetic distance; Utilize Windows QTL Cartographer V2.0 software composite interval mapping method, be that step-length scans in genome with 2cM, the detection of QTL adopts 5% global significance level, and the remarkable threshold values permutation test method of corresponding LOD statistic is estimated, altogether duplicate sampling 1000 times;
Step 5: obtain the sick site qSRBSDV6 mono-of the anti-southern rice black-streaked dwarf of anti-southern rice black-streaked dwarf sick wild-rice material y11, between mark RM20148-RM20297, whether detected in resistant variety y11 and derived varieties (being) thereof containing this key-gene site by the molecule marker of the sick key-gene of anti-southern rice black-streaked dwarf, predict the sick resistance level of its southern rice black-streaked dwarf, greatly improve the efficiency of selection of the sick paddy rice of anti-southern rice black-streaked dwarf.
4. molecule marking method according to claim 3, is characterized in that: key-gene described in step 3 is positioned on the 6th karyomit(e).
5. the application of the sick site qSRBSDV6 of anti-southern rice black-streaked dwarf according to claim 1 in rice breeding.
6. application according to claim 5, is characterized in that: the application of the sick site qSRBSDV6 of described anti-southern rice black-streaked dwarf in the sick kind of the anti-southern rice black-streaked dwarf of rapid screening or strain.
7. the application of molecule marker primer in rice breeding of the sick site qSRBSDV6 of anti-southern rice black-streaked dwarf according to claim 1.
8. application according to claim 7, is characterized in that: the application of molecule marker primer in the sick kind of the anti-southern rice black-streaked dwarf of rapid screening or strain of the sick site qSRBSDV6 of described anti-southern rice black-streaked dwarf.
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| CN105925574B (en) * | 2016-06-29 | 2019-05-07 | 中国农业科学院郑州果树研究所 | A kind of and the associated molecular labeling VMC2B1-1 of fruit white rot of grape resistance and its application |
| CN108913795A (en) * | 2018-06-11 | 2018-11-30 | 广西壮族自治区农业科学院水稻研究所 | Anti- southern rice black-streaked dwarf disease site qSRBSDV9 and its molecule labelling method |
| CN108913795B (en) * | 2018-06-11 | 2021-09-10 | 广西壮族自治区农业科学院水稻研究所 | Southern rice black-streaked dwarf resistant locus qSRBSDV9 and molecular marking method thereof |
| CN110184374A (en) * | 2019-05-07 | 2019-08-30 | 扬州大学 | Black streaked dwarf virus of rice Resistance QTL qRBSDV-4 close linkage label and its application |
| CN114164298A (en) * | 2022-01-25 | 2022-03-11 | 广西壮族自治区农业科学院 | Southern rice black-streaked dwarf resistant locus qSRBSDV10 and molecular marking method thereof |
| CN114164298B (en) * | 2022-01-25 | 2023-04-14 | 广西壮族自治区农业科学院 | Southern rice black-streaked dwarf resistant locus qSRBSDV10 and molecular marking method thereof |
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