CN101560566A - Molecular marker auxiliary selection method for TYLCV breeding - Google Patents
Molecular marker auxiliary selection method for TYLCV breeding Download PDFInfo
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Abstract
The invention discloses a molecular marker auxiliary selection method for TYLCV breeding, and belongs to the technical field of vegetable disease resistance breeding. The method comprises the following steps: (1) extracting DNA from breeding separation generation materials; (2) sieving SCAR molecular markers of anti-TYLCVD genes; (3) performing double PCR amplification and electrophoretic analytic detection on the DNA; and (4) evaluating the resistance of the breeding separation generation materials according to the detection result. The method can accurately and quickly detect whether any generation material in the breeding process is polymerized with the anti-TYLCV genes in a laboratory so as to obviously shorten the period of disease resistance breeding and reduce the breeding scale. Moreover, the method has the advantages of simple and convenient operation, time conservation, labor conservation, cost reduction and the like, reduces the workload, improves the sieving efficiency of resistant materials, accelerates the disease resistance breeding process, and can be popularized and applied to tomato breeding units.
Description
Technical field
The invention belongs to the vegetable disease-resistant breeding technical field, especially belong to a kind of method that is used for the molecular marker assisted selection of TYLCV breeding.
Background technology
Tomato (Lycopersicon esculentum Mill.) is important in the world vegetable crop.It is wide in variety, and the output height is nutritious, of many uses.The major cause of development of restriction tomato production and output is the popular harm of disease and the restriction of adverse environmental factor.Tomato yellow leaf curl virus disease (TYLCVD) is one of important disease of restriction tomato production, this disease is tomato yellow leaf curl virus (the Tomato yellow leaf curl virus that is belonged to (Begomovirus) by geminivirus infection section (Geminiviridae) bean golden mosaic virus, TYLCV) cause, and by Bemisia tabaci (Bemisia tobaci) propagation, as far back as the sixties in 19th century, just there is report in states such as Israel.Along with the variation of global climate, the change of agricultural tillage system, the rapid reinforcement and the Bemisia tabaci amboceptor of international trade activity are unprecedentedly being expanded all over the world, TYLCVD is the big area eruption and prevalence worldwide, on tomato production, cause serious harm, according to the preliminary statistics, the tomato of existing at least 39 countries is endangered by the destructiveness of this viroid.From the nineties in 20th century, ground such as the Zhejiang of China, Shandong, Shanghai, Guangxi, Yunnan, Jiangsu, Henan, Guangdong, Fujian, Hainan and Taiwan also find to have the harm of geminivirus infection in succession on tomato.The tomato yellow leaf curl virus disease is after breaking out first, in several years just rapid spread come and cause breaking out greatly, so must take immediate steps and carries out active and effective prevention and control, wherein preventing and treating the valid approach of this tomato yellow leaf curl virus disease is exactly to cultivate good disease-resistant variety.Traditional breeding for disease resistance mainly is to identify with the plant Phenotypic Selection by resistance to carry out, but this method not only needs breeding man to have abundant breeding experience, yet have simultaneously big such as workload, the cycle long, be subject to low etc. the limiting factor of influence, the breeding efficiency of envrionment conditions.
(marker-assisted selection is a kind ofly to exist by analyzing not have with the closely linked molecule marker of this disease-resistant gene MAS), thereby determines a kind of method whether this gene exists molecular marker assisted selection.This indirect system of selection is not subjected to the restriction of envrionment conditions, and can realize that in the stage in seedling stage of breeding early generation material this had both significantly reduced workload, has accelerated the process of breeding again, thereby has obviously improved the breeding efficiency of anti-this disease kind.
Studies show that in the past is not have anti-TYLCV gene in tomato common cultivation kind (Lycopersicon esculentum), only has a spot of strain that TYLCV is shown certain patience, and contain the gene of anti-TYLCV in the wild-type tomato material.Therefore, in the tomato breeding system, finding and the closely linked mark of anti-TYLCV gene, all is necessary and useful for disease-resistant plant screening and gene clone.Wherein, Hanson etc. (2002) (Hanson P M, Bernacchi D, Green S, Tanksley S D, Muniyappa V, Padmaja A S, Chen H M, Kuo G, Fang D, Chen J T.Mapping of awild tomato introgression associated with tomato yellow leaf curl virus resistance ina cultivated tomato line.Journal of the American Society of Horticultural Science, 2000,125:15-20) utilize 92 RFLP marks the resistant gene from wild-type tomato resisting etiolation curve leaf disease virus to be positioned analysis as probe, be located within the scope of about 14.6cM between long-armed TG36 (84cM) of No. 11 karyomit(e) and TG393 (103cM) mark, this gene was named as Ty-2 in 2006, and was positioned between TG36 (84cM) and TG26 (92cM) mark.Garcia etc. (2007) (GarciaB E, Martin C T, Maxwell D P.Detection methods for the Ty-1 gene for resistance tobegomoviruses on chromosome 6 of tomato.2007, http://www.plantpath.wisc.edu/GeminivirusResistantTomatoes/Mark ers/MAS-Protocols/IntroTy1.pdf) the 2 couples of primer T0302F/T0302R and the T0302F/TY2R1 according to T0302 (89cM) indicia designs can effectively detect genotype ty2/ty2 and Ty2/Ty2, but owing to do not know the linkage degree of this mark and Ty-2 gene, so this mark perhaps can not detect the material that all contain the Ty-2 gene.Ji etc. (2007) (Ji Y, Schuster D J, Scott J W.Ty-3, a begomovirus resistance locus nearthe Tomato yellow leaf curl virus resistance locus Ty-1 on chromosome 6 of tomato.Molecular Breeding, 2007,20:271-284) utilize disease-resistant self-mating system 021108 of susceptible kind 7781x (from Lycopersicon chilense LA2779) and the disease-resistant self-mating system 034611 of susceptible kind 8248x (from Lycopersicon chilense LA1932) hybridization after F
2Segregating population carries out linkage inheritance analysis and QTL positioning analysis; Come from the F of LA2779
2Colony, the resistance site be positioned at No. 6 karyomit(e)s of tomato long-armed on, come from the F of LA1932
2Colony, Ty-3 site, location, resistance site.Maxwell in 2007 etc. further find that the G8 gene order that comes from LA2779 and LA1932 is different, and are the unnamed gene from LA2779 Ty-3, are Ty-3a from the unnamed gene of LA1932.
Research on above-mentioned resistance molecular genetic basis confirms, on No. 6 karyomit(e)s and No. 11 karyomit(e)s, there is the resistance site, and found corresponding molecule marker, as being marked with of Ty-2: TG36, TG393, C2_At4g32930, TG105A, T0302, C2_At5g25760, Hba78A16T7, T0386A etc.; Ty-3 is marked with: T1079, TG590, cLET-1-I13, P169C, C2_At3g11210, C2_At5g05690, T0507, C2_At5g41480, TG118, C2_At4g27700, FLUW25, P6-25, FER-G8, UBC621, UBC697, UBC169 etc., this is that the application of molecular marker assisted selection (MAS) method on TYLCV breeding lays the foundation.A direction from now on will be the concrete function in each site of research, carry out the pyramiding breeding of different effect gene.If Ty-2 and Ty-3 are aggregated in the same strain, will produce and stablize persistent resistance more.
Summary of the invention
The present invention seeks to, identify the defective that restriction, breeding efficiency are low, cost is high that combines with the plant phenotype that the existing workload of method is big, process is slow, the cycle is long, is subject to envrionment conditions at the conventional resistance that adopts in the tomato breeding for disease resistance, provide a kind of in the TYLCV breeding process, be used for early stage segregating generation and disease resistance evaluation in seedling stage, the method that energy is accurate, quick, high efficiency selected goes out to contain the molecular marker assisted selection of resisting etiolation curve leaf disease virus ospc gene material.
The object of the invention is achieved by the following technical programs:
The molecular marker-assisted selection method of TYLCV breeding, this method is carried out according to the following steps:
(1) from breeding segregating generation material extraction DNA: with the seed of TYLCV breeding segregating generation material, sowing; When treating that seedling grows to 3～4 true leaves, every plant is got 1 spire, extracts DNA with improved CTAB method;
(2) the SCAR molecule marker of the anti-TYLCVD gene of screening: according to delivering (Hanson P M, Bernacchi D, Green S, Tanksley S D, Muniyappa V, Padmaja A S, Chen H M, Kuo G, Fang D, Chen J T.Mapping of a wild tomato introgression associated with tomatoyellow leaf curl virus resistance in a cultivated tomato line.Journal of the AmericanSociety of Horticultural Science, 2000,125:15-20; Ji Y, Betteray B V, Smeets J, Jensen K S, Mej í a L, Scott J W, Havey M J, Maxwell D P.Co-dominant SCARMarker, P6-25, for Detection of Ty-3, Ty-3a, and Ty-3b introgressions from threeSolanum chilense accessions at 25 cM of Chromosome 6 of Begomovirus-ResistantTomatoes.http: //www.plantpath.wisc.edu/GeminivirusResistantTomatoes/Mark ers/MAS-Protocols/P6-25-locus.pdf) documents and materials, just select the molecule marker of anti-tomato yellow leaf curl virus ospc gene, the laggard performing PCR amplification of synthetic primer; And, therefrom filter out the anti-tomato yellow leaf curl virus ospc gene Ty-2 that is fit to own breeding material and the SCAR molecule marker of Ty-3 according to the isozygotying and heterozygosis banding pattern and inoculate qualification result artificial seedling stage of pcr amplification; The primer sequence of Ty-2 and Ty-3 SCAR mark is: primer 1:5 '-TGG CTC ATC CTG AAG CTG ATA GCG C-3 ', primer 2: 5 '-AGTGTA CAT CCT TGC CAT TGA CT-3 ', primer 3:5 '-GGT AGT GGA AAT GAT GCTGCT C-3 ', primer 4:5 '-GCT CTG CCTATT GTC CCATATATAACC-3 ';
(3) foundation of double PCR reaction system and electrophoretic analysis detect: the double PCR reaction system of Ty-2 and Ty-3 gene is: the cumulative volume of amplified reaction is 10 microlitres, 0.7 μ L dna profiling, 0.2 μ L 10mmolL
-1DNTPs, 50ng μ L
-1Each 0.125 μ L of Ty-2 upstream and downstream primer, 50ng μ L
-1Each 0.125 μ L of Ty-3 upstream and downstream primer, 1 μ L contains 20mmolL
-1Mg
2+10 * reaction buffer, 2U μ L
-1Taq enzyme 0.25 μ L, sterile pure water 7.35 μ L; The PCR response procedures is: after 94 ℃ of pre-sex change of 2min, follow 94 ℃ of sex change 30s, and 54 ℃ of renaturation 1min, 72 ℃ are extended 1min, 40 amplification cycles, last 72 ℃ are extended 10min; The PCR product carries out electrophoretic separation analysis, EB dyeing, automated imaging on the Bio-RAD gel imaging system on 1.2% sepharose;
(4) according to the evaluation of detected result: breeding segregating generation material resistance is estimated according to imaging to breeding segregating generation material resistance, if contain the DNA band of 900bp and/or 450bp size in the amplified band, then contain the Ty-2 and/or the Ty-3 gene of anti-tomato yellow leaf curl virus disease in this material; Otherwise if then do not have Ty-2 and/or Ty-3 gene in this material.
Described breeding segregating generation material is the F of high separation
2The BC of progeny population or high separation
1Progeny population.
Described improved CTAB method is extracted DNA, preparation CTAB damping fluid: 100mL 1molL
-1TrispH 7.5,140mL 5molL
-1NaCl, 20mL 0.5molL
-1EDTA pH 8.0,740mL MiliQH
2O, 20g CTAB; The fresh blade of 10mg is put into mortar, adds 150 μ L CTAB damping fluids, grinds gently with the alms bowl pestle, and then adds 150 μ L CTAB damping fluid mixings; 65 ℃ of water-bath 40min; Add 24: 1 chloroform/primary isoamyl alcohol of 300 μ L, mixing 5min makes sample and chloroform fully mixed up and down; 13000rmin
-1Centrifugal 5min; Get supernatant liquor 150 μ L, be added in the Virahol 200 μ L of-20 ℃ of precoolings, mixing gently turns upside down; 10000～12000rmin
-1Centrifugal 3～4min abandons supernatant liquor; Make the Virahol volatilization clean, add the ddH that 50～100 μ L contain RNase
2The O dissolving DNA; Detect with 1% agarose electrophoresis then.
Described double PCR reaction system, its Mg
2+Concentration is 2mmolL
-1, primer concentration is 0.625ng μ L
-1, annealing temperature is 54 ℃, cycle number is 40;
The invention has the beneficial effects as follows:
(1) the present invention has adopted the double PCR technology of target material DNA, and experimental implementation has been simplified in two pairs of primer amplifications simultaneously in same reaction system so widely.The reagent that consumes in the double PCR amplification and the time ratio of preparation use the single PCR of 2 test tubes to lack half, therefore this technology has high-level efficiency and characteristics cheaply, be fit to the great amount of samples material is analyzed and identified, shorten the time greatly and reduce workload, the screening efficiency of marker material is doubled.
(2) adopt the improved CTAB method of the present invention to extract DNA, can extract tomato DNA simple and easy, apace, do not need again to handle, and in leaching process, also do not utilize to become privileged and learn medicine such as mercaptoethanol and PVP etc., reduced cost through liquid nitrogen;
(3) the present invention can resist the material of any generation in the sick breeding process of tomato yellow leaf curl virus in the laboratory, all can to its whether the gene of the anti-TYLCV of polymerization directly detect accurately and rapidly, this neither is subjected to the influence of envrionment conditions, again can be in office when the phase carries out, and do not influence the normal growth of plant.
(4) molecular marker assisted selection polymerization TYLCV breeding method of the present invention is compared with conventional breeding for disease resistance, authentication method, have cycle weak point, small scale, easy and simple to handle, save time, advantage such as laborsaving, cost-saving.
Description of drawings
Fig. 1 utilizes the double PCR technology to (2698BC
1-6 * 07-027) F
2The electrophoresis detection of part segregating generation material
Annotate: M:Marker; The F of 1:(2698BC1-6 * 07-027)
2-1; The F of 2:(2698BC1-6 * 07-027)
2-6; The F of 3:(2698BC1-6 * 07-027)
2-8; The F of 4:(2698BC1-6 * 07-027)
2-12; The F of 5:(2698BC1-6 * 07-027)
2-13; The F of 6:(2698BC1-6 * 07-027)
2-18; The F of 7:(2698BC1-6 * 07-027)
2-22; The F of 8:(2698BC1-6 * 07-027)
2-27; The F of 9:(2698BC1-6 * 07-027)
2-29; The F of 10:(2698BC1-6 * 07-027)
2-30; The F of 11:(2698BC1-6 * 07-027)
2-41; The F of 12:(2698BC1-6 * 07-027)
2-52; The F of 13:(2698BC1-6 * 07-027)
2-53; The F of 14:(2698BC1-6 * 07-027)
2-55; The F of 15:(2698BC1-6 * 07-027)
2-61; The F of 16:(2698BC1-6 * 07-027)
2-67; The F of 17:(2698BC1-6 * 07-027)
2-68; The F of 18:(2698BC1-6 * 07-027)
2-71; The F of 19:(2698BC1-6 * 07-027)
2-72; The F of 20:(2698BC1-6 * 07-027)
2-79; The F of 21:(2698BC1-6 * 07-02/)
2-80; The F of 22:(2698BC1-6 * 07-027)
2-83; The F of 23:(2698BC 1-6 * 07-027)
2-84; The F of 24:(2698BC1-6 * 07-027)
2-89;
Fig. 2 utilizes the double PCR technology to ((9179 * 07-027) * 07-026) F
2The electrophoresis detection of part segregating generation material
Annotate: M:Marker; The F of 1:((9179 * 07-027) * 07-026)
2-12; The F of 2:((9179 * 07-027) * 07-026)
2-54; The F of 3:((9179 * 07-027) * 07-026)
2-88; The F of 4:((9179 * 07-027) * 07-026)
2-67; The F of 5:((9179 * 07-027) * 07-026)
2-112; The F of 6:((9179 * 07-027) * 07-026)
2-193; The F of 7:((9179 * 07-027) * 07-026)
2-13; The F of 8:((9179 * 07-027) * 07-026)
2-105; The F of 9:((9179 * 07-027) * 07-026)
2-5; The F of 10:((9179 * 07-027) * 07-026)
2-48;
Embodiment
Also the present invention is described in further detail in conjunction with the accompanying drawings by following examples, but should be appreciated that the present invention is not limited by these contents.
Embodiment 1:(utilizes molecular marker-assisted selection method that the resistance of filial generation material is detected 1)
The concrete implementation step of this example is:
1, parent material and hybridizing method:
1. parent material:
Tomato elite plant strain T01-198: bred and preserved by academy of agricultural sciences, Zhejiang Province Vegetable Research Institute, this strain system is unlimited, bright red, middle fruit, and storage tolerance, anti-leaf mold, sense tomato chrysanthemum curve leaf disease virus disease is located away from Holland and introduces first-filial generation ' Tomato Serrei ';
07-026: contain the sick resistant gene Ty-3 of anti-tomato yellow leaf curl virus, provide by Vegetable Research centre of development, Asia (AVRDC);
07-027: contain the sick resistant gene Ty-2 of anti-tomato yellow leaf curl virus, provide by Vegetable Research centre of development, Asia (AVRDC);
2. hybridizing method:
With T07-026 is maternal, and T01-198 is a male parent, obtains F
1Cross combination 07-026 * T01-198; Be that male parent is to F again with T01-198
1Backcross, BC from generation to generation obtains to backcross
1To BC
1Separate the offspring and carry out Markers for Detection, filter out 07-026 * T01-198BC
1-6, called after 2698BC
1-6;
With 2698BC
1-6 is maternal, and 07-027 is that male parent is carried out Ty-3 and the polymerization of Ty-2 resistant gene, obtains F
1Cross combination 2698BC
1-6 * 07-027 by selfing, obtains the F of high separation
2Progeny population.
2, utilize the SCAR molecular marker-assisted selection method, from above-mentioned breeding segregating generation material, detect, filter out resistant material:
(1) from breeding segregating generation material extraction DNA: with TYLCV breeding segregating generation material B C
1And F
2Seed, being seeded in matrix is the peat composed of rotten mosses: in the seedling culture hole plate of vermiculite=2: 1 (volume ratio), when treating that seedling grows 3～4 true leaves, each plant is got 1 spire respectively, extracts DNA with improved CTAB method then; At first prepare the CTAB damping fluid: 100mL 1molL
-1Tris pH 7.5,140mL 5molL
-1NaCl, 20mL 0.5molL
-1EDTApH 8.0,740mL MiliQ H
2O, 20g CTAB.DNA extraction is that the fresh blade of getting 10mg is put into mortar, adds 150 μ LCTAB damping fluids, grinds gently with the alms bowl pestle, and then adds 150 μ L CTAB damping fluid mixings; 65 ℃ of water-bath 40min; Add 24: 1 chloroform/primary isoamyl alcohol of 300 μ L, mixing 5min makes sample and chloroform fully mixed up and down; 13000rmin
-1Centrifugal 5min; Get supernatant liquor 150 μ L, be added in the Virahol 200 μ L of-20 ℃ of precoolings, mixing gently turns upside down; 10000～12000rmin
-1Centrifugal 3～4min abandons supernatant liquor; Make the Virahol volatilization clean, add the ddH that 50～100 μ L contain RNase
2The O dissolving DNA; Detect with 1% agarose electrophoresis then;
(2) the SCAR molecule marker of the anti-TYLCVD gene of screening: according to delivering (Hanson P M, Bernacchi D, Green S, Tanksley S D, Muniyappa V, Padmaj a A S, Chen H M, Kuo G, Fang D, Chen J T.Mapping of a wild tomato introgression associated with tomatoyellow leaf curl virus resistance in a cultivated tomato line.Journal of the AmericanSociety of Horticultural Science, 2000,125:15-20; Ji Y, Betteray B V, Smeets J, Jensen K S, Mej í a L, Scott J W, Havey M J, Maxwell D P.Co-dominant SCARMarker, P6-25, for Detection of Ty-3, Ty-3a, and Ty-3b introgressions from threeSolanum chilense accessions at 25 cM of Chromosome 6 of Begomovirus-ResistantTomatoes.
Http:// www.plantpath.wisc.edu/GeminivirusResistantTomatoes/Mark ers/MAS-Protocols/P6-25-locus.pdf) documents and materials, select the molecule marker of 18 anti-tomato yellow leaf curl virus ospc genes, synthetic primer carries out pcr amplification; Isozygotying and heterozygosis banding pattern and inoculate qualification result artificial seedling stage according to pcr amplification again, filter out the SCAR molecule marker of sick Ty-2 of the anti-tomato yellow leaf curl virus that is fit to own breeding material and Ty-3 gene: the primer sequence of Ty-2 and Ty-3SCAR mark: primer 1:5 '-TGG CTC ATC CTG AAG CTG ATA GCG C-3 ', primer 2: 5 '-AGT GTA CATCCT TGC CAT TGA CT-3 ', primer 3:5 '-GGT AGT GGA AAT GAT GCT GCT C-3 ', primer 4:5 '-GCT CTG CCTATT GTC CCATATATAACC-3 '.
(3) foundation of dual-PCR method and detection: set up single stage method and can detect Ty-2 and Ty-3 gene simultaneously, optimizing reaction system and condition obtain best amplification and detect effect, and the mix primer of Ty-2 and Ty-3 gene is amplification simultaneously in same reaction system,
The double PCR reaction system: the amplified reaction cumulative volume is 10 microlitres; 0.7 μ L dna profiling, 0.2 μ L10mmolL
-1DNTPs, 50ng μ L
-1Each 0.125 μ L of Ty-2 upstream and downstream primer, 50ng μ L
-1Each 0.125 μ L of Ty-3 upstream and downstream primer, 1 μ L contains 20mmolL
-1Mg
2+10 * reaction buffer, 2U μ L
-1Taq enzyme 0.25 μ L, sterile pure water 7.35 μ L.The PCR response procedures is: after 94 ℃ of pre-sex change of 2min, follow 94 ℃ of sex change 30s, and 54 ℃ of renaturation 1min, 72 ℃ are extended 1min, 40 amplification cycles, last 72 ℃ are extended 10min; In this reaction system, best Mg
2+Concentration is 2mmolL
-1, primer concentration is 0.625ng μ L
-1, annealing temperature is 54 ℃, cycle number is 40;
The PCR product carries out the electrophoretic separation analysis on 1.2% sepharose, electrophoresis finishes the back with EB dyeing, and on the Bio-RAD gel imaging system automated imaging, can estimate the resistance of breeding segregating generation material according to this analyzing and testing result;
Through checking repeatedly, reliable results can be used in same PCR reaction system Ty-2 and Ty-3 gene being detected simultaneously.
3, result and analysis:
Elite plant strain T01-198 in used tomato material does not amplify the DNA band of 900bp and/or 450bp size, and illustrating does not have Ty-2 and/or Ty-3 gene in this material; In the 07-026 material, amplify the band of a 450bp size, illustrate that this material is the Ty-3/Ty-3 genotype; In the 07-027 material, amplify the band of a 900bp size, illustrate that this material is the Ty-2/Ty-2 genotype.The target intermediate materials 07-026 * T01-198 and the 07-026 * T01-198BC that obtain
1-6, all amplify two DNA bands of 320bp and 450bp size, illustrate that these materials are Ty-3/ty-3 genotype; Intermediate materials 2698BC
1-6 * 07-027 amplifies 4 DNA bands of 320bp, 450bp, 800bp and 900bp, illustrates that this material is a Ty-2 Ty-3/ty-2ty-3 genotype.To 2698BC
1The F of-6 * 07-027 high separation
2Progeny population carries out Ty-2 and Ty-3 Markers for Detection (Fig. 1), wherein 18 parts is Ty-2 Ty-3/ty-2ty-3 genotype, 7 parts is the Ty-3/Ty-3 genotype, 12 parts is the Ty-3ty-2/ty-3ty-2 genotype, 10 parts of Ty-2ty-3/ty-2ty-3 genotype, other be the ty-2ty-3/ty-2ty-3 genotype.
Embodiment 2:(utilizes molecular marker-assisted selection method that the resistance of filial generation material is detected 2)
The concrete implementation step of this example is:
1, parent material and hybridizing method:
1. parent material:
The 9179th, after material 9132 hybridization are introduced in the marketing fresh first-filial generation kind 9502 of the high anti-leaf mold of introducing from Europe and Vegetable Research centre of development, Asia, the stable strain that in the high separation offspring, forms through the selection of 8 generations strain system, this strain system is unlimited, pink, big fruit, anti-leaf mold, sense tomato chrysanthemum curve leaf disease virus disease;
07-026 and 07-027 material are with embodiment 1;
2. hybridizing method:
9179 to be female parent, 07-027 is a male parent, obtains F
1Cross combination 9179 * 07-027; With 07-026 is that male parent is to this F
1Hybridize, acquisition three friendship combinations " (9179 * 07-027) * 07-026 "; Utilizing Ty-2 to hand over the combined altitudes selfing to separate the offspring with the Ty-3 molecule marker to three detects.
2, utilize the SCAR molecular marker-assisted selection method, from above-mentioned breeding segregating generation material, detect, filter out resistant material: specifically from the double PCR amplification of the SCAR molecule marker of breeding segregating generation material extraction DNA, the anti-TYLCVD gene of screening and DNA and the method that detects with embodiment 1;
3, result and analysis:
Elite plant strain 9179 in used tomato material does not amplify the DNA band of 900bp and/or 450bp size, and illustrating does not have Ty-2 and/or Ty-3 gene in this material; In the 07-027 material, amplify the band of a 900bp size, illustrate that this material is the Ty-2/Ty-2 genotype; In the 07-026 material, amplify the band of a 450bp size, illustrate that this material is the Ty-3/Ty-3 genotype; The target intermediate materials 9179 * 07-027 that obtains amplifies two DNA bands of 800bp and 900bp size, illustrates that these materials are Ty-2/ty-2 genotype; Three hand over composition materials " (9179 * 07-027) * 07-026 ", amplify 4 DNA bands of 320bp, 450bp, 800bp and 900bp, illustrate that this material is a Ty-2 Ty-3/ty-2ty-3 genotype (see figure 2); Hand over the combined altitudes selfing to separate the offspring to three and carry out Ty-2 and Ty-3 Markers for Detection, wherein 2 parts is the Ty-2/Ty-2 genotype, 8 parts is the Ty-3/Ty-3 genotype, 30 parts is the Ty-2ty-3/ty-2ty-3 genotype, 5 parts is the Ty-3ty-2/ty-3ty-2 genotype, 25 parts is the Ty-2Ty-3/ty-2ty-3 genotype, other be the ty-2ty-3/ty-2ty-3 genotype.
Embodiment 3:(using artificial is inoculated the checking of identification method to the molecular marker assisted selection resistant material seedling stage)
The resistance of using selection markers is isozygotied and heterozygosis banding pattern selection resistant plant, in the TYLCV breeding process, adopt double PCR Technique on T y-2 and Ty-3 gene to detect simultaneously, therefrom filter out the material 07-026 * T01-198BC that contains resistant gene
1-6,07-026 * T01-198BC
1-86,07-026 * T01-198BC
1-112, (2698BC
1-6 * 07-027) F
2-53, (2698BC
1-6 * 07-027) F
2-72, ((9179 * 07-027) * 07-026) F
2-88, ((9179 * 07-027) * 07-026) F
2-105 etc., further using artificial is inoculated identification method the molecular marker assisted selection resistant plant is verified (table 1) seedling stage subsequently.
Table 1 resistant material TYLCV Bemisia tabaci infects postvaccinal incidence
| Resistant material | The Markers for Detection situation | Sickness rate % | Conclusion |
| 07-026×T01-198BC 1-6 | Ty-3 | 0 | Disease-resistant |
| 07-026×T01-198BC 1-86 | Ty-3 | 4％ | Disease-resistant |
| 07-026×T01-198BC 1-112 | Ty-3 | 3％ | Disease-resistant |
| (2698BC 1-6×07-027)F 2-53 | Ty-3、Ty-2 | 0 | Disease-resistant |
| (2698BC 1-6×07-027)F 2-72 | Ty-3、Ty-2 | 1％ | Disease-resistant |
| ((9179×07-027)×07-026)F 2-88 | Ty-3、Ty-2 | 0％ | Disease-resistant |
| ((9179×07-027)×07-026)F 2-105 | Ty-3、Ty-2 | 2％ | Disease-resistant |
Sequence table
＜110〉Zhejiang Academy of Agricultural Science
＜120〉molecular marker-assisted selection method of TYLCV breeding
<160>4
<210>1
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design according to known array, synthetic by Shanghai Bioisystech Co., Ltd, as the upstream primer of Ty-2 molecule marker
<400>1
tggctcatcc tgaagctgat agcgc 25
<210>2
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design according to known array, synthetic by Shanghai Bioisystech Co., Ltd, as the downstream primer of Ty-2 molecule marker
<400>2
agtgtacatc cttgccattg act 23
<210>3
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design according to known array, synthetic by Shanghai Bioisystech Co., Ltd, as the upstream primer of Ty-3 molecule marker
<400>3
ggtagtggaa atgatgctgc tc 22
<210>4
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design according to known array, synthetic by Shanghai Bioisystech Co., Ltd, as the downstream primer of Ty-3 molecule marker
<400>4
gctctgccta ttgtcccata tataacc 27
Claims (4)
1, the molecular marker-assisted selection method of TYLCV breeding is characterized in that this method carries out according to the following steps:
(1) from breeding segregating generation material extraction DNA: with the seed of TYLCV breeding segregating generation material, sowing; When treating that seedling grows to 3～4 true leaves, every plant is got 1 spire, extracts DNA with improved CTAB method;
(2) the SCAR molecule marker of the anti-TYLCVD gene of screening: according to the documents and materials of having delivered, just select the molecule marker of anti-tomato yellow leaf curl virus ospc gene, the laggard performing PCR amplification of synthetic primer; And, therefrom filter out the anti-tomato yellow leaf curl virus ospc gene Ty-2 that is fit to own breeding material and the SCAR molecule marker of Ty-3 according to the isozygotying and heterozygosis banding pattern and inoculate qualification result artificial seedling stage of pcr amplification; Shown in SEQ ID NO.1 and SEQ ID NO.2, the sequence of Ty-3 upstream and downstream primer is respectively shown in SEQ ID NO.3 and SEQ ID NO.4 respectively for the sequence of Ty-2 upstream and downstream primer;
(3) foundation of double PCR reaction system and electrophoretic analysis detect: the double PCR reaction system of Ty-2 and Ty-3 gene is: the cumulative volume of amplified reaction is 10 microlitres, 0.7 μ L dna profiling, 0.2 μ L 10mmolL
-1DNTPs, 50ng μ L
-1Each 0.125 μ L of Ty-2 upstream and downstream primer, 50ng μ L
-1Each 0.125 μ L of Ty-3 upstream and downstream primer, 1 μ L contains 20mmolL
-1Mg
2+10 * reaction buffer, 2U μ L
-1Taq enzyme 0.25 μ L, sterile pure water 7.35 μ L; The PCR response procedures is: after 94 ℃ of pre-sex change of 2min, follow 94 ℃ of sex change 30s, and 54 ℃ of renaturation 1min, 72 ℃ are extended 1min, 40 amplification cycles, last 72 ℃ are extended 10min; The PCR product carries out electrophoretic separation analysis, EB dyeing, automated imaging on the Bio-RAD gel imaging system on 1.2% sepharose;
(4) according to the evaluation of detected result: breeding segregating generation material resistance is estimated according to imaging to breeding segregating generation material resistance, if contain the DNA band of 900bp and/or 450bp size in the amplified band, then contain the Ty-2 and/or the Ty-3 gene of anti-tomato yellow leaf curl virus disease in this material; Otherwise if then do not have Ty-2 and/or Ty-3 gene in this material.
2, by the described molecular marker-assisted selection method of claim 1, it is characterized in that described breeding segregating generation material is the F of high separation
2The BC of progeny population or high separation
1Progeny population.
3,, it is characterized in that it is to prepare the CTAB damping fluid: 100mL 1molL that described improved CTAB method is extracted DNA by the described molecular marker-assisted selection method of claim 1
-1Tris pH 7.5,140mL 5molL
-1NaCl, 20mL 0.5molL
-1EDTA pH 8.0,740mL MiliQ H
2O, 20g CTAB; The fresh blade of 10mg is put into mortar, adds 150 μ L CTAB damping fluids, grinds gently with the alms bowl pestle, and then adds 150 μ L CTAB damping fluid mixings; 65 ℃ of water-bath 40min; Add 24: 1 chloroform/primary isoamyl alcohol of 300 μ L, mixing 5min makes sample and chloroform fully mixed up and down; 13000rmin
-1Centrifugal 5min; Get supernatant liquor 150 μ L, be added in the Virahol 200 μ L of-20 ℃ of precoolings, mixing gently turns upside down; 10000～12000rmin
-1Centrifugal 3～4min abandons supernatant liquor; Make the Virahol volatilization clean, add the ddH that 50～100 μ L contain RNase
2The O dissolving DNA; Detect with 1% agarose electrophoresis then.
4, by the described molecular marker-assisted selection method of claim 1, it is characterized in that described double PCR reaction system, its Mg
2+Concentration is 2mmolL
-1, primer concentration is 0.625ng μ L
-1, annealing temperature is 54 ℃, cycle number is 40.
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