CN102766626A - Molecular marker closely linked to resistance gene Ty-2 for tomato yellow leaf curl virus - Google Patents
Molecular marker closely linked to resistance gene Ty-2 for tomato yellow leaf curl virus Download PDFInfo
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Abstract
The invention provides a molecular marker closely linked to tomato yellow leaf curl virus resistance gene Ty-2. Nucleotide sequences of the molecular marker are shown in SEQ ID No.1. The scar marker linked to the gene Ty-2 is effective in marker assisted selection of the gene Ty-2. The distance of close linkage between the molecular marker and the tomato yellow leaf curl virus resistance gene Ty-2 is 2.2cm. The gene Ty-2 can be precisely located or approached by the chromosome walking method by using the molecular as a signpost, and foundation for cloning the gene Ty-2 is laid.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of and closely linked molecule marker of tomato yellow leaf curl virus resistance gene and application thereof.
Background technology
Tomato yellow leaf curl virus (TYLCV) belongs to geminivirus infection section (Geminiviridae) bean golden mosaic virus and belongs to (Begomovirus), mainly propagates through Bemisia tabaci (Bemisia tabaci).TYLCV originates from Middle East and Mediterranean basin, mainly is distributed in western part, Mediterranean Sea, Japan, southeastern US and area, the Caribbean Sea, is that a kind of torrid zone, subtropical zone have destructive tomato virus disease.From 1964 by definite designation since (Cohen and Harpaz 1964), in the Middle East, numerous countries and regions such as Europe, South East Asia, East Asia and Africa work the mischief in succession (Czosnek and Laterrot, 1997; Polston and Anderson1997; Moriones and Navas-Castillo, 2000).From the nineties in 20th century, also on tomato, find to have in succession on the ground such as Zhejiang, Shanghai, Guangxi, Yunnan, Jiangsu, Henan, Guangdong, Fujian, Hainan and Taiwan of China TYLCV harm (Cai Jian and etc., 2006; He Zifu etc., 2007; Wang Dongsheng etc., 2007; Zhao Tongmin etc., 2007).
TYLCV exists serious threat to tomato production, and breeding resistant variety is the effective means of this disease of control.Research shows, has the gene of anti-TYLCV in the wild-type tomato, and wherein disease-resistant gene Ty-2 particularly all shows the higher resistance to TYLCV in the area, Asia on Vietnam, India, Israel and China mainland and other places.Ty-2 is a single dominant gene that comes from the crinosity tomato, is positioned at first on the fragment of the chromosomal long-armed about 19cM of o.11, further is positioned at (Hanson et al., 2006 in the 6.5cM zone in 2009; Ji et al., 2007c; Ji et al., 2009).Given this, the Ty-2 gene is carried out Fine Mapping, obtain with Ty-2 close linkage even isolating molecule marker altogether, will lay the foundation for the clone of Ty-2, for the breeding for disease resistance of Ty-2 gene more closely linked molecule marker is provided simultaneously.
Summary of the invention
In order to address the above problem, the object of the present invention is to provide a kind of and the closely linked molecule marker of tomato yellow leaf curl virus Ty-2 resistant gene.
Another object of the present invention is to provide the application of this molecule marker in the assignment of genes gene mapping of tomato yellow leaf curl virus resistance or detection and marker-assisted breeding.
In order to realize the object of the invention, the invention provides a kind of and the closely linked molecule marker of tomato yellow leaf curl virus Ty-2 resistant gene, the nucleotide sequence of this molecule marker is shown in SEQID No.1.
The present invention also provides a kind of amplification right with the primer of the closely linked molecule marker of tomato yellow leaf curl virus Ty-2 resistant gene, and this primer is following to sequence:
P1:5’-CACACATATCCTCTATCCTATTAGCTG-3’；
P2:5’-CGGAGCTGAATTGTATAAACACG-3’。
The present invention also provides a kind of carrier that contains above-mentioned molecule marker.
The present invention also provides a kind of and the detection method closely linked molecule marker of tomato yellow leaf curl virus resistance gene; This method is that template is through pcr amplification with the tomato dna group DNA of anti-sense tomato yellow leaf curl virus; Obtain to identify simultaneously the genotypic specific spectruming belt of male parent, female parent and heterozygote thereof; This specific spectruming belt comprises plant bands of a spectrum that carry resistant gene and the plant bands of a spectrum that do not carry resistant gene, and the said plant bands of a spectrum that carry resistant gene are the molecule marker of nucleotide sequence shown in SEQ ID No.1.
The nucleotide sequence of the said plant bands of a spectrum that do not carry resistant gene is shown in SEQ ID No.2.
In the aforesaid method, the PCR reaction system is:
10 μ L reaction systems comprise each 0.4 μ M of forward and reverse primer, 0.8mM dNTPs, 2.5mMMgCl
2, 1 μ l 10X buffer, and 0.25units Taq polysaccharase (New England Biolabs, Ipswich, MA), dna profiling 15ng.
In the aforesaid method, the pcr amplification program is: 94 ℃ become 5min in advance; 94 ℃ of sex change 30s, 55 ℃ or 60 ℃ of annealing 45s, 72 ℃ are extended 30-60s, 37 circulations; 72 ℃ are extended 5-10min.
The present invention also provides above-mentioned molecule marker and the application of detection method in the assignment of genes gene mapping of tomato yellow leaf curl virus resistance or detection or marker-assisted breeding.
The beneficial effect that the present invention possesses is:
1) molecule marker of the present invention has further been located tomato yellow leaf curl virus resistance gene Ty-2.The high specificity of mark, stability are high, and the screening method of mark is simple and efficient to handle, and be less demanding to test set and primer template quality, and it is few to have a test reagent consumption, and speed is fast, and cost is low, suitable big batch, the advantage of high-throughput, robotization.Be fit to very much the realization of modern agriculture molecular breeding;
2) the present invention provides important molecular genetics information for the map based cloning of tomato yellow leaf curl virus resistance gene Ty-2;
3) tomato yellow leaf curl virus resistance gene Ty-2 molecule marker primer of the present invention is applied in the breeding work; With reducing the popular financial loss that tomato production is caused of tomato yellow leaf curl virus disease greatly; Reduce the agricultural chemicals usage quantity simultaneously; Be of value to and reduce production costs, have very big application potential and higher economic value.
4) the present invention filters out the SCAR mark with Ty-2 gene linkage, can effectively be used for the marker assisted selection of Ty-2 gene, and the close linkage distance of said molecule marker and tomato yellow leaf curl virus resistance gene Ty-2 is 2.2cM.Be labeled as road sign with this, can carry out the Fine Mapping of Ty-2 gene or pass through the chromosome walking method, thereby improve the accuracy of selecting, shortening the breeding cycle, also lay the first stone simultaneously for the clone of Ty-2 gene near the Ty-2 gene.
Description of drawings
Fig. 1 is that Ty-2 gene of the present invention is on the differing materials H24 (left side; Hanson et al.; 2000), (screening from the F3 of H9205 for strain is for F1 hybrid H9205 (the 2nd), E959; Ji et al., 2009) and the infiltration fragment among the F11.E504 (the reorganization individual plant that the right, this research filter out).Dash area is represented resistance infiltration fragment, and each collection of illustrative plates left-hand digit is represented the genetic distance of each molecule marker, and F11.E504 is the new genetic map that makes up of this research, and it is blank not permeate segmental zone.
Fig. 2 is phenotypic evaluation for 26 reorganization of the present invention individual plant molecular marker gene type with the reorganization individual plant selfing.Wherein, the resistance osmotic tablets stage mode black disease-resistant individual plant of representing to isozygoty, the grey colour specification susceptible individual plant that isozygotys, hatched example areas representes that the molecular genotype of that section shows heterozygosis.Each numeral above the pillar contains this genotypic reorganization individual plant strain number.R representes that all reorganization individual plant selfing are that all individual plants are disease-resistant; S representes that all individual plants are susceptible.
Fig. 3 for the present invention and the closely linked molecule marker of anti-tomato yellow leaf curl virus resistance gene Ty-2 to the disease-resistant parent Tyqueen of heterozygosis, disease-resistant parent material E951-7, susceptible parent material Horizon detected result.Among the figure, M:100bp DNA Marker, 1: the resistance of isozygotying parent E951-7,2: susceptible parent Horizon, 3: the disease-resistant parent Tyqueen of heterozygosis.
Fig. 4 carries out molecular marker assisted selection detected result to the F6 of the transformation Ty-2 of seminar gene for population material for Ty-2 genetic marker P1-16 of the present invention and the Ty-2 genetic marker T0302 that reported.Among the figure, M is 100bp DNAMarker.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to protection scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The screening of embodiment 1 and Ty-2 gene close linkage mark
Vegetable material: used disease-resistant heterozygote (Ty-2/ty-2), disease-resistant homozygote (Ty-2/Ty-2) and the susceptible homozygote (ty-2/ty-2) of this experiment is respectively TyQueen, E951-7 and Horizon.TyQueen is a commercial cross-fertilize seed.E951-7 (screening for strain be from the F4 of H9205) is near isogenic line (NILs) material from Chilean tomato, only contains the resistance fragment (Ji et al., 2009) of Ty-2 gene at No. 11 karyomit(e).Horizon is a common cultivation tomato material.
The population material that the mark checking is used:
1) F4 that from Heinz F1 hybrid H9205, makes up of Ji etc. (2009) screened 7,000 colony's individual plants in spring in 2011 for colony.Wherein, filter out between C2 At1g07960 and M1 26 of the individual plants that reorganization takes place, molecular marker gene type and phenotypic evaluation result such as Fig. 2 of 26 reorganization individual plants.26 reorganization individual plants were transplanted in the ground at the beginning of 2011 4 months, and the results seed is to be used for phenotypic evaluation.
2) for the accuracy of further verification mark, utilize the F6 of the transformation Ty-2 of seminar gene to verify that for the cherry tomato population material colony's qualification result is as shown in Figure 4.To be F6 hybridized for 6 generations for the cherry tomato population material by cherry tomato kind gold prince wife and material C LN3022F2-154-22-5-0 that contains the TY2 disease-resistant gene of Taiwan inferior vegetables center introduction obtains for it.
Inoculation is identified: with reference to the method for Griffiths and Scott (2001).Behind 2 ~ 3 leaf periods of young plant or the rooting of cuttings, it is positioned in the growth room that is placed with band toxic smoke aleyrodid, kills the Bemisia tabaci of band poison after two weeks, afterwards young plant is transplanted to greenhouse or big Tanaka, observe state of an illness result.
Molecule marker: used molecule marker all is based on the SCAR or the CAPS mark of PCR reaction in this test; Molecule marker C2_At1g07960, TG36, T0386A, cLEN-11-F24 and T0302 are from SGN website (http://www.solgenomic.org), and the labeled primer of newly designing and developing out according to tomato dna group sequence has P1-16,2-1,5-3, cL1, cL2, M1, M2 and M3.C2_At1g07960 (82.5cM) (Ji et al., 2009) and T0302 (89cM) (Garcia et al., 2007) are used as the flank mark of screening reorganization individual plant.
This research and utilization a pair of FOD 3 molecule marker TG36 (84cM) and TG105 (90cM) calculate tomato dna group 1cM ≈ 100kb, the genetic distance to all molecule markers of using in this research has carried out estimating (Fig. 1) then.
Polymerase chain reaction: used tomato material in this test; The F4 that comprises Tyqueen, E951-7, Horizon, H9205 is for 7; 000 population material and (cherry tomato kind gold prince wife and the material C LN3022F2-154-22-5-0 that contains the TY2 disease-resistant gene of Taiwan inferior vegetables center introduction hybridized 6 generations obtained) F6 are for the cherry tomato population material; The genomic dna of these materials extracts (Fulton et al., 1995) with the CTAB method from the blade of 2-3 week young plant.10 μ L reaction systems comprise each 0.4 μ M of forward and reverse primer, 0.8mM dNTPs, 2.5mM MgCl
2, 1 μ l 10X buffer, and 0.25units Taq polysaccharase (New England Biolabs, Ipswich, MA), dna profiling 15ng.PCR is reflected at Perkin-Elmer GeneAmp PCR 9700, and (PerkinElmer Inc., Waltham carry out on WA).Response procedures is 94 ℃ and becomes 5min in advance; 94 ℃ of sex change 30s, differing temps (being 55 ℃ basically) annealing 45s, 72 ℃ are extended 30-60s, 37 circulations; 72 ℃ are extended 5-10min.For the CARS mark, TV 10 μ L enzymes are cut and are contained 4uLPCR product and the corresponding enzyme of 1.5units, reaction 30to60min in the system.PCR or enzyme are cut product and are all separated being added with in 2.0% sepharose of EB, and uv lamp is colour developing down.
The chromosomal localization of genetic distance estimation, linkage analysis and tomato yellow leaf curl virus resistance gene:
According to the pcr amplification result; Find the primer that between parents, disease-resistant pond and susceptible pond, has polymorphum; Through reorganization individual plant colony and offspring's RIL thereof polymorphism primer is verified, utilized the locating information enantiopathy gene of this primer to position and verify; Then, in conjunction with the physical map of tomato dna group sequence, the Fine Mapping of this disease-resistant gene is further analyzed.
Result and analysis
The screening of mark: carry out pcr amplification, screening at disease-resistant parent E951-7, susceptible parent Horizon; Discovery has severally can amplify polymorphum to primer between parents; Through electrophoretic analysis and disease resistance qualification result, therefrom select a safety, stable best and do not need SCAR primer that enzyme cuts selective marker as tomato yellow leaf curl virogene Ty-2 to reorganization individual plant offspring segregating population.The amplification of this primer is as shown in Figure 3, and concrete primer sequence is following:
Seq?ID?No.3:5’-CACACATATCCTCTATCCTATTAGCTG-3’
Seq?ID?No.4:5’-CGGAGCTGAATTGTATAAACACG-3’。
Reclaim above-mentioned primer and expand the bands of a spectrum that and order-checking, in the sequence in the disease-resistant variety shown in SEQ ID No.1, in the sequence in the susceptible variety shown in SEQ ID No.2.
Further calculate linkage distance, the close linkage distance of molecule marker P1-16 of the present invention and tomato yellow leaf curl virus resistance gene Ty-2 is 2.2cM.
Selected closely linked molecule marker P1-16 carries out molecular marker assisted selection to the F6 of the transformation Ty-2 of seminar gene for population material 48 strains (cherry tomato kind gold prince wife and the kind H9205 that contains the TY2 disease-resistant gene hybridized 6 generations obtained) among the mark T0302 that utilizes the Ty-2 gene reported simultaneously and the embodiment 1.The result shows: in 48 individual plants; Mark P1-16 (nucleotide sequence length is the positive of 300bp) detects positive individual plant 26 strains, and numbering is respectively 11,12,13,14,15,16,17,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45 and No. 47 individual plants; T0302 (nucleotide sequence length is the positive of 700bp) detects positive individual plant 26 strains, and numbering is respectively 11,12,13,14,15,16,17,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45 and No. 47 individual plants; The individual plant of test positive, the field is disease-resistant to be accredited as anti-ly, and wherein No. 39 individual plant T0302 mark genotype are heterozygous, and the P1-16 mark is homozygous, and further selfing separates and can obtain the shorter breeding for disease resistance material of resistance infiltration fragment.The molecule marker of this experiment invention and the close linkage (Fig. 4) of resistant gene Ty-2 have been proved.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from know-why of the present invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (10)
- One kind with the closely linked molecule marker of tomato yellow leaf curl virus Ty-2 resistant gene, the nucleotide sequence of this molecule marker is shown in SEQ ID No.1.
- 2. the primer of amplification claim 1 said molecule marker.
- 3. primer as claimed in claim 2, this primer sequence is following:P1：5’-CACACATATCCTCTATCCTATTAGCTG-3’；P2：5’-CGGAGCTGAATTGTATAAACACG-3’。
- 4. carrier that contains the said molecule marker of claim 1.
- 5. use the detection method of the said molecule marker of claim 1; It is characterized in that; This method is that template is through pcr amplification with the tomato dna group DNA of anti-sense tomato yellow leaf curl virus; Obtain identifying simultaneously the genotypic specific spectruming belt of male parent, female parent and heterozygote thereof, this specific spectruming belt is the plant bands of a spectrum that carry the plant bands of a spectrum of resistant gene and do not carry resistant gene, and the said plant bands of a spectrum that carry resistant gene are the described molecule marker of claim 1.
- 6. detection method as claimed in claim 5 is characterized in that, the nucleotide sequence of the said plant bands of a spectrum that do not carry resistant gene is shown in SEQ ID No.2.
- 7. method as claimed in claim 5 is characterized in that, said PCR system is following: 10 μ L reaction systems comprise each 0.4 μ M of forward and reverse primer, 0.8mM dNTPs, 2.5mMMgCl 2, 1 μ l 10X buffer, 0.25units Taq polysaccharase, dna profiling 15ng.
- 8. method as claimed in claim 5 is characterized in that, the program of said pcr amplification is: 94 ℃ become 5min in advance; 94 ℃ of sex change 30s, 55 ℃ or 60 ℃ of annealing 45s, 72 ℃ are extended 30-60s, 37 circulations; 72 ℃ are extended 5-10min; 4 ℃ of storage temperatures.
- 9. the application of the said molecule marker of claim 1 in the assignment of genes gene mapping of tomato yellow leaf curl virus resistance, detection or marker-assisted breeding.
- 10. the application of any said detection method of claim 5-8 in the assignment of genes gene mapping of tomato yellow leaf curl virus resistance, detection or marker-assisted breeding.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103999767A (en) * | 2014-06-05 | 2014-08-27 | 青岛农业大学 | Breeding method of tomato breeding material with resistance to root knot nematode disease and yellow leaf curl virus disease |
| CN106811462A (en) * | 2015-11-30 | 2017-06-09 | 中国农业科学院蔬菜花卉研究所 | Gray leaf spot gene Sm anti-with tomato chain Indel marks and its amplimer and application |
| CN109777883A (en) * | 2018-02-26 | 2019-05-21 | 中国农业科学院蔬菜花卉研究所 | The specific primer of the codominant marker of tomato yellow leaf curl virus Ty-2 resistant gene and its application |
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| US20100132067A1 (en) * | 2004-08-23 | 2010-05-27 | Pioneer Hi-Bred International, Inc. | Marker mapping and resistance gene associations in soybean |
| CN101899509A (en) * | 2010-07-06 | 2010-12-01 | 王有福 | Primers and probe used for real-time fluorescent PCR assay of apricot chlorotic leafroll phtoplasma and method thereof |
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| US20100132067A1 (en) * | 2004-08-23 | 2010-05-27 | Pioneer Hi-Bred International, Inc. | Marker mapping and resistance gene associations in soybean |
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Non-Patent Citations (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103999767A (en) * | 2014-06-05 | 2014-08-27 | 青岛农业大学 | Breeding method of tomato breeding material with resistance to root knot nematode disease and yellow leaf curl virus disease |
| CN106811462A (en) * | 2015-11-30 | 2017-06-09 | 中国农业科学院蔬菜花卉研究所 | Gray leaf spot gene Sm anti-with tomato chain Indel marks and its amplimer and application |
| CN109777883A (en) * | 2018-02-26 | 2019-05-21 | 中国农业科学院蔬菜花卉研究所 | The specific primer of the codominant marker of tomato yellow leaf curl virus Ty-2 resistant gene and its application |
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