CN103555718A - SSR (simple sequence repeat) markers on No.3 chromosome, closely linked to RBSDV (rice black-streaked dwarf virus) resistant QTL (quantitative trait locus) and application thereof - Google Patents
SSR (simple sequence repeat) markers on No.3 chromosome, closely linked to RBSDV (rice black-streaked dwarf virus) resistant QTL (quantitative trait locus) and application thereof Download PDFInfo
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Abstract
The invention discloses molecular markers closely linked to an RBSDV (rice black-streaked dwarf virus) major QTL (quantitative trait locus). The essence of the invention is that according to the linkage and segregation rules, the F2 population of the hybrid generation of Tetep and RBSDV susceptible varieties is taken as the mapping population and the SSR (simple sequence repeat) markers RM5626 and RM7097 on the No.3 chromosome, which are closely linked to the RBSDV resistant major QTL are obtained through analysis of linkage between the results of evaluation of disease resistance and the SSR marker data. Whether a plant has resistance to RBSDV can be judged by detecting the SSR marker banding pattern of the single rice plant of the hybrid combination generation of Tetep and derived varieties (lines) thereof and the RBSDV susceptible varieties. After the SSR markers are applied to assistant selection breeding of the RBSDV resistant varieties of Tetep and derived varieties (lines) thereof/RBSDV susceptible varieties, the defects of poor stability and repeatability of RBSDV resistance evaluation, time and labor waste, high technical threshold and the like in conventional breeding can be overcome and the results can conduce to simplifying the selection method and improving the breeding efficiency and then accelerating the breeding process of RBSDV resistant varieties.
Description
Technical field:
The molecule marker RM5626 and the RM7097 that the present invention relates to a kind of on No. 3 karyomit(e)s and black streaked dwarf virus of rice resistance main effect QTL compact linkage, can be applicable to molecular mark, belongs to paddy disease-resistant breeding and biology field.
Background technology:
Black streaked dwarf virus of rice, cause of disease is rice black-streaked dwarf virus (Rice black-streaked dwavf virus, RBSDV), is a kind of malignant diseases viral disease of being propagated without ovum mode with persistence by small brown rice planthopper.Infect RBSDV paddy rice diseased plant and mostly can not ear, the underproduction is serious, except paddy rice, also endangers the crops such as wheat, corn, barley and Chinese sorghum.20th century 60, this disease of the later stage nineties are broken out and are caused disaster on North China, East China and other places, in recent years, the variation of the envrionment conditionss such as the popularization of the extensive plantation of susceptible variety and rice wheat crop rotation method and winter climate warm is constantly risen the generating capacity that passes virus mediator small brown rice planthopper, causes this disease to occur in East China Dao Qu big area.Up to now, almost do not have effectively to prevent and treat the chemical agent of virus disease, the emergency schedule of controlling worm diseases prevention cannot be as a long term policy, and utilizing kind self disease resistance is to prevent and treat virus disease economy, effective means the most.At present, less for the research report of black streaked dwarf virus of rice genetics of resistance, result of study (Acta Agronomica Sinica, 2009,35 (12): 2213-2217; Acta Agronomica Sinica, 2010,36 (8): 1258-1264; Molecular Breeding, 2012,29 (4): the 925-938) feature of the existing quantitative character of multilist, but the resistant gene site of different researchs is not identical, also there is research (Treat ofCrop Res, 2007 (8): 105-115) discovery common wild-rice introgressive line is controlled by a pair of dominant nuclear gene the resistance of black streaked dwarf virus of rice, and its resistance is qualitative character.This shows in the paddy rice resource of different sources black streaked dwarf virus of rice resistance mechanism that may there are differences, and genetic mechanism is comparatively complicated.
Black streaked dwarf virus of rice grave illness district field identification method is convenient and swift, but because it is subject to envrionment conditions, year and interregional small brown rice planthopper a situation arises the repeatability that the factors such as (generating capacity and with malicious rate) inconsistent have affected result, cause Resistance Identification result between different research to greatly differ from each other, grave illness district field test is very easily subject to the interference of the stripe disease propagated by small brown rice planthopper simultaneously in addition.Artificial inoculation authentication method (plant protection journal; 2011; 38 (4): 301-305) be a kind of Resistance Identification of carrying out under controlled condition; utilize it to evaluate the resistance level of rice varieties to black streaked dwarf virus of rice; except overcoming well two problems that are difficult to evade in field test; can also widen qualification time, but because RBSDV passes without small brown rice planthopper ovum, the method be wasted time and energy relatively.Cause Resistance Identification to become one of major reason that black streaked dwarf virus of rice breeding work makes slow progress.
Molecular mark technology has effectively solved this problem, by building genetic linkage maps and quantitative trait locus (the Quantitative Trait Locus in important anti-source, QTL) analyze, can effectively find the molecule marker with black streaked dwarf virus of rice resistance main effect QTL compact linkage, use these marks to tie up to and to carry out the morning of screening from generation to generation seedling stage the offspring in this anti-source and derived prods thereof, eliminate disease plant, not only saved cost, also improved breeding efficiency.
Summary of the invention:
The present invention is directed to above-mentioned research background, black streaked dwarf virus of rice disease-resistant variety Te Tebo and the susceptible main breed Huaihe River rice screening of take is for No. 5 material, 842, screen 160 polymorphism SSR marks in to (http://www.gramene.org) microsatellite marker, therefrom select 127 SSR marks to No. 5/Te Tebo of Huaihe River rice F
2colony analyzes, and builds rice genetic linkage map, by itself and F
2:3the Disease Resistance Identification result of family is carried out linkage analysis, has obtained on No. 3 karyomit(e)s molecule marker RM5626 and RM7097 with black streaked dwarf virus of rice resistance main effect QTL compact linkage, can be applicable to the assisted selection of black streaked dwarf virus of rice disease-resistant variety.
On No. 3 karyomit(e)s provided by the present invention, with molecule marker RM5626 and the RM7097 of black streaked dwarf virus of rice resistance main effect QTL compact linkage, obtain by the following method:
1) susceptible variety Huaihe River rice No. 5 (♀) obtains hybrid F with disease-resistant variety Te Tebo (♂) hybridization
1, F
1selfing obtains F
2mapping population, individual plant results F
2:3family seed is for Disease Resistance Identification;
2) CTAB method is extracted parent, F
1and F
2the DNA of colony;
3) utilize 127 couples of SSR primer pair parents, the F selecting
1and F
2colony carries out pcr amplification, and amplified production, by Polyacrylamide Gel Electrophoresis, obtains molecular marker data, builds genetic linkage map;
4) adopt artificial inoculation authentication method to measure F
2:3the disease resistance of family, divides into disease-resistant and susceptible whether to fall ill;
5) according to marker bands, by Mapmaker/Exp3.0 computed in software, go out linkage inheritance distance between mark, and draw according to the position being respectively marked on karyomit(e) with Mapdraw software, build rice genetic linkage map;
6) adopt the Resistance QTL of the software Windows QTL Cartographer V2.5 detection black streaked dwarf virus of rice based on composite interval mapping method.The threshold value of LOD value is decided to be 2.0, presses the number of P=0.005 probable value detection Resistance QTL and the position on karyomit(e) thereof.
On No. 3 karyomit(e)s, comprise with the molecule marker RM5626 of black streaked dwarf virus of rice resistance main effect QTL compact linkage and the application of RM7097: its derived varieties of Te Tebo (being) of take is object with RBSDV susceptible variety cross combination offspring's single rice plant, by detecting the banding pattern data of RM5626 and RM7097 mark on its No. 3 karyomit(e)s, can predict the resistance of this plant to black streaked dwarf virus of rice.
The present invention can overcome the shortcomings such as black streaked dwarf virus of rice Resistance Identification stability in conventional breeding, repeatability is poor and waste time and energy, and its result can be simplified system of selection and improve breeding efficiency, and then accelerates the breeding process of disease-resistant variety.
Accompanying drawing explanation:
Fig. 1 utilizes No. 5/Te Tebo of Huaihe River rice F
2the Molecular Markers Linkage Map that colony builds
Black streaked dwarf virus of rice Resistance QTL genetic linkage group positioning analysis schematic diagram in Fig. 2 Te Tebo
Wherein: the linkage group section that comprises QTL is shown, and left side is genetic distance between mark (cM); Right side is mark title.
Embodiment:
In embodiment, method therefor is ordinary method if no special instructions below.
Embodiment 1, with the acquisition of the molecule marker of black streaked dwarf virus of rice resistance main effect QTL compact linkage
1, vegetable material
2008 field planting parent Huaihe River, academy of agricultural sciences, Nian Jiangsu Province rice No. 5 and Te Tebo, and hybridization obtains hybrid F
1, next year F
1selfing obtains F
2seed, 2009 at Hainan breeding F
2colony, as mapping population.F
2individual plant numbering, gets parent, hybrid F tillering phase
1and F
2the partial blade of each individual plant of colony ,-70 ℃ of Refrigerator stores are for ssr analysis, F
2colony's individual plant sowing, in order to phenotypic evaluation.2010 to susceptible main breed Huaihe River rice No. 5, disease-resistant variety Te Tebo, F
1and F
2:3family is carried out artificial inoculation evaluation (plant protection journal, 2011,38 (4): 301-305).
2, pass the screening of virus mediator
Black streaked dwarf virus of rice grave illness district gathers performance RBSDV symptom (Zhejiang Agriculture science in Yancheng, Jiangsu Province, Xuzhou, Lianyun Harbour etc., 1984, (4): paddy rice diseased plant 185-192.), by RT-PCR method (Jiangsu agricultural journal, 2009,25 (6): 1263-1267) detect diseased plant and whether carry RBSDV, utilize primer (CP-F:ATGGGTACCAACAAGCC corresponding to oryza virus 3 (RSV) coat protein gene, CP-R:CTAGTCATCTGCACCTT) detect diseased plant, choose do not carry RSV RBSDV diseased plant as malicious source.By 1-2 age nontoxic small brown rice planthopper nymph on malicious source, raise malicious 2-3d, then move in the beaker of giving birth to Wu-Yu-Geng 3 rice shoot and raise and make it spend the phase of walking around to, after 12-13d, from every glass, take out at random 30 through DIBA method (Agricultural University Of Nanjing's Master's thesis, 2012, Du Linlin) measure it and be with malicious rate, for calculating effective inoculation worm amount [effectively inoculate worm amount (worm)=inoculation worm amount (worm) * be with malicious rate (%)].
3, DNA extraction (CTAB method)
1) take 500mg rice leaf and be placed in 2.0mL Eppendorf pipe, centrifuge tube is placed in to liquid nitrogen cooling, fill after liquid nitrogen and take out rapidly, with grinding rod, be ground into powder;
2) 65 ℃ of Water Unders are bathed 1-1.5h, 15min concussion 1 time;
3), under 4 ℃ of conditions 13, the centrifugal 10min of 000rpm, gets upper strata water;
4) add 900 μ L chloroforms: primary isoamyl alcohol solution (24: 1), fully mixes to shake to solution colour and become white from green;
5), under 4 ℃ of conditions 13, the centrifugal 10min of 000rpm, gets upper strata water;
6) add equal-volume Virahol (or 2 times of volume dehydrated alcohols), standing 20-30min under-20 ℃ of conditions, is settled out cotton-shaped DNA;
7) under 4 ℃ of conditions 13, the centrifugal 10min of 000rpm, abandons supernatant, adds 1mL70% washing with alcohol, and under 4 ℃ of conditions 13, the centrifugal 5min of 000rpm, abandons supernatant and blot with filter paper, is placed on super clean bench and dries.
8) add 30 μ L deionized water dissolving DNA, 4 ℃ save backup.
With Eppendorf BioPhotometer Plus nucleic acid-protein determinator, measure OD value and concentration, each sample DNA is diluted to 20ng/ μ L standby.
4, SSR labeled analysis
(1) pcr amplification
Adopt the PCR reaction system of 10 μ L, as table 1;
Table 1PCR reaction system
PCR response procedures: 95 ℃ of sex change 5min; 95 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ of extension 1min, carry out 35 circulations; 72 ℃ are extended 10min.Product adds loading buffer stopped reaction, stand-by.
(2) polyacrylamide gel electrophoresis
Adopt 8% polyacrylamide gel to detect above-mentioned PCR reaction product, electrophoresis apparatus used is two plate holder core Vertial electrophorestic tank DYCZ-30 types (Liuyi Instruments Plant, Beijing).Concrete operation step is as follows:
1) with liquid detergent sheet glass scrub repeatedly, by distilled water flushing, oven for drying, available alcohol wipe before assembling sheet glass.
2) two sheet glass are put in rubber strip of paper used for sealing, sepharose seals bottom and is mounted on electrophoresis chamber.
3) by table 2 order, add each liquid storage preparation acrylamide gel, fully shake up and with pipettor, the glue preparing is evenly injected between two sheet glass afterwards, then insert comb, notice preventing that comb bottom from producing bubble.Standing 1h treats that it solidifies in left and right.
Table 2 is prepared the volume of polyacrylamide gel agents useful for same
4) in electrophoresis chamber, add the electrophoretic buffer of 1 * TAE, extract comb, PCR product is joined in point sample hole.
5) 200V constant voltage electrophoresis 1-2h.
(3) dyeing of polyacrylamide gel (argentation)
1) with spirituosity 10%, the solution of glacial acetic acid 0.5% is fixed 2 times, 6 minutes/time.
2) abandon fixed solution, add 0.2%AgNO
3solution infiltration 10-12 minute.
3) after infiltration, use ddH
2o cleans.
4) with 0.002% hypo solution, put about 30s of generation.
5) put Dai Houyong containing 1.5%NaOH, the colour developing of the solution of 0.4% formaldehyde.
6) band clear after, distilled water flushing, color development stopping reaction.
7) with preservative film encapsulate, preserve, and on white light, record experimental result.
5, the structure of genetic map and qtl analysis
According to the synthetic 842 pairs of SSR primers of micro-satellite database (http://www.gramene.org), between parent, carry out polymorphic detection, between selected parent, there are 127 polymorphic SSR labeled analysis F2 colonies.
Utilize MAPMAKER/EXP3.0 software to carry out linkage analysis.Wherein the assignment identical with No. 5 banding patterns of Huaihe River rice is " A ", and the assignment identical with Te Tebo banding pattern is " B ", and the banding pattern assignment of heterozygous is " H ", and banding pattern assignment special or disappearance is "-".According to the linkage relationship between each mark, with " group " order, in LOD value, be greater than at 3.0 o'clock, mark is divided into groups.Again with Kosambi function by each group between mark recombination fraction convert genetic distance (Centimorgan, cM) to.For little linkage group, directly adopt " compare " order, select to have putting in order of maximum likelihood value and build locking frame figure; For larger linkage group, first use the method for 2 tests, calculate any LOD value between mark between two, adopt " compare " order to obtain the optimal arrangement order of its suggestion, build basic framework figure, " try " order, finds out the most suitable position of each mark (maximum likelihood value method) again.Use " ripple " checking simultaneously.Finally use " map " order to obtain genetic linkage maps.Built the paddy rice collection of illustrative plates that comprises 12 genetic linkage groups, covered rice genome 2179.6cM, between 127 SSR marks, average headway is 17.16cM (Fig. 1)
Employing is genetic map and the F to structure based on composite interval mapping method (Windows QTL Cartographer V2.5)
2:3phenotypic data is analyzed, and the threshold value of LOD value is decided to be 2.0, presses the number of P=0.005 probable value detection Resistance QTL and the position on karyomit(e) thereof.The QTL of 1 water resistant rice black streak dwarf on No. 3 karyomit(e)s, detected, (name is with reference to the principle Theor Appl Genet of McCouch for called after qRBSDV-3,1988,76 (6): 815-829), between the 3rd chromosomal RM5626-RM7097, LOD value is 4.07, and contribution rate is 17.5% (Fig. 2).
On 2, No. 3 karyomit(e)s of embodiment with the closely linked molecule marker of black streaked dwarf virus of rice Resistance QTL in the application of take in the cross combination offspring that Huaihe River rice No. 5 and Te Tebo be parent
On No. 3 karyomit(e)s, obtain with the closely linked SSR mark of black streaked dwarf virus of rice Resistance QTL, the F to Huaihe River rice No. 5 with Te Tebo hybridization
2:3the part individual plant of family has carried out black streaked dwarf virus of rice resistance prediction, extract respectively each individual plant DNA, then with the primer of SSR mark RM5626 and RM7097, carry out pcr amplification analysis, by band type analysis, determine whether to exist corresponding mark, exist this strain black streaked dwarf virus of rice of explanation of mark to reach anti-level, it is susceptible not existing.Utilize subsequently method that artificial inoculation is identified measure the actual resistance of tested strain black streaked dwarf virus of rice and compare with predicting the outcome, result demonstration (table 3), predicts the outcome and matches with actual detected result.
No. 5/Te Tebo of the Molecular Prediction Huaihe River rice filial generation F of table 3 and Te Tebo main effect QTL compact linkage
2:3black streaked dwarf virus of rice resistance
Above-mentioned enforcement does not limit the present invention in any form.
Claims (2)
1. with the molecule marker of black streaked dwarf virus of rice resistance main effect QTL compact linkage, it is characterized in that: with RBSDV susceptible variety/Te Tebo filial generation F
2colony is mapping population, by the linkage analysis between simple sequence repeat marker (SSR) data and Disease Resistance Identification result, obtains on No. 3 karyomit(e)s molecule marker RM5626 and RM7097 with black streaked dwarf virus of rice resistance main effect QTL linkage.
2. according to right 1, obtain the application of the molecule marker of black streaked dwarf virus of rice resistance main effect QTL linkage in a kind of and RBSDV susceptible variety/Te Tebo combination, it is characterized in that: the single rice plant of Te Tebo and derived varieties thereof (being) and RBSDV susceptible variety filial generation of take is object, by detecting the banding pattern data of its molecule marker RM5626 and RM7097, can judge whether this plant has black streaked dwarf virus of rice resistance.
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