CN101948918A - Molecular marker auxiliary selection method for tomato yellow leaf curl viral disease and knot nematode - Google Patents
Molecular marker auxiliary selection method for tomato yellow leaf curl viral disease and knot nematode Download PDFInfo
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Abstract
The invention discloses a molecular marker auxiliary selection method for tomato yellow leaf curl viral disease and knot nematode. The method is characterized by comprising the following steps of: (1) extracting DNA from a tomato material F1 and a selfed separation offspring F2 individual; (2) performing multiple PCR amplification on the tomato material F1 and the selfed separation offspring F2; and (3) performing resistance evaluation on the breeding separation generation material according to the detection result. The multiple PCR amplification method can amplify Ty-1 genes and Mi genes in the same reaction system at the same time, so the method greatly simplifies experiment operation and experiment expense, is suitable for identifying and analyzing a large amount of sample material, greatly shortens the time and reduces the workload.
Description
Technical field
The present invention relates to farm crop molecular marking technique field, especially a kind of molecular marker-assisted selection method that detects anti-Ty-1 of tomato and root knot nematode gene simultaneously.
Background technology
Tomato is the high worldwide crop of a kind of wide adaptability, output.(Tomato yellow leaf curl virus TYLCV) belongs to geminivirus infection section bean golden mosaic virus and belongs to tomato yellow leaf curl virus, mainly propagates by Bemisia tabaci.In recent years, because the unprecedented expansion of global warming and Bemisia tabaci, this disease also increases the weight of year by year more than 40 national big area outbursts, has become the critical limitation factor on the many regional tomato production in the world.(institute thinks the acquisition breeding parent for Tomato yellow leaf curl disease, resistance TYLCD), and a large amount of research concentrates on from wild relatives screens because the cultivation tomato does not have the tomato yellow leaf curl virus disease.Can avoid complicated resistance authentication step if the utilization molecule marker carries out assistant breeding, also help shortening the breeding cycle.Based on this, the molecule marking research work in resistance site is carried out also early.The resistance of TYLCD is controlled by 1 major gene, and called after Ty-1 is partial dominance heredity, in addition, also has the modifying factor more than 2 at least.
Zamir etc. (1994) are used to come from BC2S1 and the BC2S2 colony of L.esculentum (M82-1-8) * L.Chilense (LA1969), and the RFLP labeling technique, the major gene Ty-1 of 1 anti-tomato yellow leaf curl virus disease is positioned on No. 6 karyomit(e) of tomato.Wherein the Ty-1 on the karyomit(e) 6 is the resistance oligogene, and be modifying factor (Zamir D on karyomit(e) 3 and 7, Michelson I E, Zakay Y, Navot N, Zeidan M, Sarfatti M, Eshed Y, Harel E, Pleban T, van-Oss H, Kedar N, Rabinowitch H D, Czosnek H.Mapping and introgression of a Tomato yellow leaf curl virus tolerance gene, Ty-1.Theoretical and Applied Genetics, 1994,88,141-146).
Having studies confirm that on karyomit(e) 6,3 and 7 of above-mentioned molecular genetic can be found corresponding resistance site and molecule marker.SSR molecule marker as Nizio screenings such as (2008) can be used as mark (the Nizio D AD that tomato Ty-1 allelotrope is identified, Maluf W R, Figueira A D, Nogueira D W, Silva V D, Neto A C G.Fingerprinting of tomato genotypes resistant to begomovirus by a codominant molecular marker linked to Ty-1gene.Pesquisa Agropecuaria Brasileira, 2008,43 (12): 1699-1705), and CAPS mark (the Ana P C of Ana etc. (2007) screening, Jos é M B, Mara J D, Fernando N V.Identification of a CAPSmarker tightly linked to the tomato yellow leaf curl disease resistance gene Ty-1 in tomato.European Journal of Plant Pathology, 2007,117 (4): 347-356), Zhang etc. (2002) screen at long-armed TG153 of the 6th karyomit(e) and QTL (the Zhang L P of the anti-TYLCV between the CT83, Khan A, Nino L D, Foolad M R.A molecular linkage map of tomato displaying chromosomal locations of resistance gene analogs based on a Lycopersicon esculentum * L.hirsutum cross, Genome, 2002,45:133-146), the RFLP molecule marker that comes from the resistance site of S.habrochaites is positioned between the Sub_clause 11 chromosomal long-armed TG393 and TG36, just said Ty-2 site, and come from S.chilense equally and be located in the resistance site of the 6th karyomit(e) on long-armed, this resistance site has comprised Ty-1.These researchs are laid a good foundation for molecular marker assisted selection.
(2007) such as P é rez de Castro discover that the Ty-1 gene is a resistant gene that is positioned at " hot-spot ".This " hot-spot " also has (the Seah S such as resistant gene Mi of tomato root-knot eelworm, Telleen A C, Williamson V M.Introgressed and endogenous Mi-1 gene clusters in tomato differ by complex rearrangements in flanking sequences and show sequence exchange and diversifying selection among homologues.Theor.Appl.Genet., 2007,114:1289-1302).Mi is an independent major gene, the main kind of Turbatrix all there is resistance (Gilbert J C.Some linkage studies with the Mi gene for resistance to root-knot nematodes.Report of the Tomato Genetics Cooperative, 1958,8:15-17).Since tomato gene against meloidogyne Mi and Ty-1 gene are positioned in " hot-spot ", next have much studies have shown that, these two genes are closely linked.In tomato breeding, find to have simultaneously the tomato species (Mej í a et al., 2005, unpublished data) in Mi and Ty-1 site as Sheng Kaluosi university.
According to (1991) such as Messeguer, and the gene of Mi gene-correlation gradually to ooze be site between TG297 and TG231, this report gradually oozes identical with the gene about the Ty-1 gene of (1994) report such as Zamir basically.P é rezde Castro et al. (2007) thinks on karyomit(e) 6 and to have a CAPs mark, this mark utilizes the PCR product behind JB1F and the JB1R primer amplification to cut with the TaqI enzyme to carry out enzyme and cut, thereby found a mark (Ana P é rez de Castro with the closely linked TY of Ty-1, Jos é Miguel Blanca, Mar í a Jos é D í ez, Fernando Nuez
-als.Identification of a CAPS marker tightly linked to the Tomato yellow leaf curl disease resistance gene Ty-1 in tomato.Eur J Plant Pathol.2007,117:347-356).Use the report that this mark carries out the evaluation of tomato resistant gene type but up to the present lack.
Summary of the invention
The objective of the invention is, the restriction of condition such as big at the workload that occurs in the conventional breeding for disease resistance offspring of the tomato screening process, that the cycle is long and breeding efficiency is low, defect of high cost, provide a kind of and be used for identifying simultaneously in early days TYLCV and the genotypic molecule marking method of root knot nematode disease, thereby be used for molecular mark.
The present invention adopts following technical scheme:
The molecular marker-assisted selection method of a kind of TYLCV and root knot nematode is characterized in that, may further comprise the steps: (1) is from tomato material F
1Separate offspring F with selfing
2Individual plant extracts DNA; (2) to tomato material F
1Separate offspring F with selfing
2Carry out the multiplex PCR amplification; (3) enzyme of multiplex PCR amplified production is cut and the electrophoretic analysis detection; (4) according to detected result breeding segregating generation material is carried out evaluation of resistance.
Described molecular marker-assisted selection method, the method that described step (1) is extracted DNA is: with tomato material F
1With selfing segregating generation material F
2Sowing, when treating that seedling grows to 3～4 true leaves, every plant is got 1 spire, extracts DNA with improved CTAB method.
Described molecular marker-assisted selection method, the concrete grammar of described step (2) is: synthetic primer, carry out pcr amplification, and carry out enzyme with the taqI enzyme and cut; Cut shown the isozygotying and the heterozygosis banding pattern of result according to pcr amplification and enzyme, inoculate qualification result with artificial seedling stage and compare, determine the molecule marker of anti-Ty-1 and root knot nematode gene; The upstream and downstream primer sequence that wherein detects the Ty-1 gene is respectively JB-1 upper:5 '-AAC CAT TAT CCG GTT CAC TC-3 ' and JB-1 lower:5 '-TTT CCATTC CTT GTT TCT CTG-3 ', and the upstream and downstream primer sequence of root knot nematode is respectively Mi upper:5 '-TCG GAG CCT TGG TCT GAA TT-3 ' and Mi lower:5 '-GCC AGA GATGAT TCG TGA GA-3 '.
Described molecular marker-assisted selection method, the concrete grammar of described step (3) is: multi-PRC reaction system 20 μ l, 1 times of reaction buffer, 2.0mM MgCl
2, 0.1 μ M upstream and downstream primer, 0.2mM dNTPs, the Taq polysaccharase of 0.04U and the template DNA of 20ng replenish ddwater to 20 μ l; The pcr amplification program be 94 ℃ 5 minutes, then 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 2 minutes, 35 circulations, 72 ℃ were extended 10 minutes; The enzyme system of cutting is 20 μ l, wherein contains 7 μ l pcr amplification products, 10U taqI, 2 μ l reaction buffers; 65 ℃ of 1.5h; Pcr amplification and enzyme are cut product and are carried out the electrophoretic separation analysis with 2% agarose gel electrophoresis, EB dyeing, imaging on the gel imaging instrument of Bio-rad then.
Described molecular marker-assisted selection method, the evaluation method of described step (4) is: if enzyme is cut the specific band that only contains 400bp in the banding pattern, for sense Ty-1 homozygous genotype material,, be anti-Ty-1 material if enzyme is cut the specific band that contains 500bp and 400bp in the banding pattern; If contain the specific band of 570bp and 180bp, be root-knot nematode resistant homozygous genotype material; If contain 750bp, 570bp, the specific band of 180bp is root-knot nematode resistant heterozygous genes section bar material; If only produce the specific band of 750bp, no TaqI restriction enzyme site is sense root knot nematode disease homozygous genotype material.Described molecular marker-assisted selection method, the filial generation colony that described segregating generation material is a high separation or the backcross progeny colony of high separation.
Described molecular marker-assisted selection method, the DNA that described CTAB method is extracted is that preparation CTAB damping fluid: the plant leaf that (1) takes a morsel is in 1.5ml Eppendorf pipe, and quick-frozen is 15 seconds in the tapping nitrogen, add 100 μ l CTAB damping fluids and a small amount of quartz sand, grind to form homogenate; (2) add 500 μ l CTAB damping fluids, 65 ℃ of insulation 35-40min, mixing is rocked in the centre; (3) add isopyknic 24: 1 chloroform: primary isoamyl alcohol (v/v), put upside down mixing 5min gently, 4 ℃ of centrifugal 10min of 15000rpm forward supernatant in the new pipe to, add the Virahol of 2/3 volume; (4) place 5min under the room temperature, 4 ℃ of 1500rpm, centrifugal 10min abandons supernatant, the washing with alcohol precipitation with 70% 2-3 time; Abandon supernatant, carry out vacuum-drying, in each sample, add 50 μ l sterilization distilled water and an amount of Rnase, 37 ℃ of digestion 1h, be stored in-20 ℃ standby.
The invention has the beneficial effects as follows:
(1) the present invention adopts the amplification method of multiplex PCR, Ty-1 gene and Mi gene simultaneously can increase in same reaction system, simplified experimental implementation and experiment cost so greatly, be suitable for the great amount of samples material is identified and analyzed, shortened the time greatly and reduce workload.
(2) the present invention can resist the tomato yellow leaf curl virus disease in the laboratory and root knot nematode disease resistant gene type detects accurately and rapidly and analyzes, in conventional breeding, play very important booster action, the link of artificial inoculation evaluation and the time of step and wait have been reduced, be not subjected to the influence of envrionment conditions, be not subjected to the influence of plant growth condition yet.
At the multiplex PCR molecule marking method that the invention discloses about Ty-1 and Mi gene, and cut the disease-resistant gene type situation of judging material, to realize to carry out two disease-resistant proterties the target of while Screening and Identification with a reaction system by carrying out the TaqI enzyme.This method will be for screening TYLCV and the application of root knot nematode disease in breeding laid a good foundation simultaneously.This will lay the foundation for the pyramiding breeding that carries out the different effect gene.
Description of drawings
Fig. 1 utilizes the electrophoresis detection of multiple CAPs labeling technique to 7 tomato first generations of hybrid;
Annotate: M:marker;
Fig. 2 utilizes the part selfing F of multiple CAPs labeling technique to tomato first generation of hybrid E13
2The electrophoresis detection of segregating generation material; Annotate: M:marker; The F of 1～23:E13
21～23;
Fig. 3 utilizes the part selfing F of multiple CAPs labeling technique to first generation of hybrid A10
2The electrophoresis detection of segregating generation material; Annotate: M:marker; 1～19: the F of first generation of hybrid A10
21～19.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
Embodiment: utilize the part selfing F of multiple CAPs labeling technique to 7 the tomato first generations of hybrid, E13 and A10
2The electrophoresis detection of segregating generation material
The concrete implementation step of this example is:
1, material and method:
B08,109,314,198,288 is a tomato first-filial generation.Wherein B08,109,314,198 is the sick kind of sense tomato yellow leaf curl virus, and 288 is the sick kind of anti-tomato yellow leaf curl virus.E13: the good first generation of hybrid of tomato, draw from Holland, this strain haw, storage tolerance, anti-tomato yellow leaf curl virus disease.A10: the good first generation of hybrid of tomato, draw from Korea S.This kind haw, storage tolerance, sense tomato yellow leaf curl virus disease.To E13 and the selfing of A10 bagging, obtain the F of high separation respectively
2Progeny population perhaps is the backcross progeny colony of E13 and A10 high separation.
2, utilize the CAPs molecular marker-assisted selection method, from above-mentioned breeding segregating generation material, detect its anti-sense genotype.
(1) from above-mentioned tomato material, extracts DNA.Part selfing F with first generation of hybrid material, E13 and A10
2The segregating generation material is seeded in the bed of growing seedlings, and when treating that seedling grows 3～4 true leaves, each plant is got a slice spire respectively, extracts DNA with the method for CTAB.Be transplanted to the serious plot of morbidity in autumn then and carry out the disease resistance evaluation.The extracting method of CTAB is as follows: the plant leaf that (1) takes a morsel is in 1.5ml Eppendorf pipe, and quick-frozen is 15 seconds in the tapping nitrogen, adds 100 μ lCTAB damping fluids and a small amount of quartz sand, grinds to form homogenate.(2) add 500 μ l CTAB damping fluids, 65 ℃ of insulation 35-40min, mixing is rocked in the centre.(3) add isopyknic 24: 1 chloroform: primary isoamyl alcohol (v/v), put upside down mixing 5min gently, 4 ℃ of centrifugal 10min of 15000rpm forward supernatant in the new pipe to, add the Virahol of 2/3 volume.(4) place 5min under the room temperature, 4 ℃ of 1500rpm, centrifugal 10min abandons supernatant, the washing with alcohol precipitation with 70% 2-3 time.Abandon supernatant, carry out vacuum-drying, in each sample, add 50 μ l sterilization distilled water and an amount of Rnase, 37 ℃ of digestion 1h, be stored in-20 ℃ standby.
(2) the tomato material is carried out the multiplex PCR amplification;
According to the documents and materials of having delivered (Ana P é rez de Castro, Jos é Miguel Blanca, Mar í a Jos é D í ez, Fernando Nuez
-als.Identification of a CAPS marker tightly linked to the Tomato yellow leaf curl disease resistance gene Ty-1 in tomato.Eur J Plant Pathol.2007,117:347-356), the laggard performing PCR amplification of synthetic primer.Multi-PRC reaction system 20 μ l, 1 times of reaction buffer, 2.0mM MgCl
2, 0.1 μ M upstream and downstream primer, 0.2mM dNTPs, the Taq polysaccharase of 0.04U and the template DNA of 20ng replenish ddwater to 20 μ l.The pcr amplification program be 94 ℃ 5 minutes, then 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 2 minutes, 35 circulations, 72 ℃ were extended 10 minutes.The upstream and downstream primer sequence that detects the Ty-1 gene locus is respectively shown in JB-1upper:5 '-AAC CAT TAT CCG GTT CAC TC-3 ' and the JB-1 lower:5 '-TTT CCA TTC CTT GTT TCTCTG-3 ', and the upstream and downstream primer sequence that detects root knot nematode resistance site is respectively as Mi upper:5 '-TCG GAGCCT TGG TCT GAA TT-3 ' and Mi lower:5 '-GCC AGA GAT GAT TCG TGA GA-3 '.
Described tomato material is tomato first-filial generation F
1, E13 and A10 part selfing F
2The segregating generation material perhaps is the backcross progeny colony of E13 and A10 high separation.
(3) the multiplex PCR amplified production enzyme of DNA is cut and the electrophoretic analysis detection;
The multiplex PCR amplification is carried out enzyme with the taqI enzyme to be cut.Cut shown the isozygotying and heterozygosis banding pattern and inoculate qualification result artificial seedling stage of result according to pcr amplification and enzyme, determine the molecule marker of anti-Ty-1 and Mi gene.The enzyme system of cutting is 20 μ l, wherein contains 7 μ l pcr amplification products, 10U taqI, 2l reaction buffer.65℃1.5h。Pcr amplification and enzyme are cut product and are carried out the electrophoretic separation analysis with 2% agarose gel electrophoresis, EB dyeing, imaging on the gel imaging instrument of Bio-rad then.
(4) according to detected result material is carried out evaluation of resistance.
Cut result's banding pattern estimates the anti-sense of breeding segregating generation material genotype according to enzyme.If enzyme is cut the specific band that only contains 400bp in the banding pattern,,, be the sick material of anti-tomato yellow leaf curl virus if enzyme is cut the specific band that contains 500bp and 400bp in the banding pattern for the sick homozygous genotype material of sense tomato yellow leaf curl virus; If contain the specific band of 570bp and 180bp, be root-knot nematode resistant homozygous genotype material; If contain 750bp, 570bp, the specific band of 180bp is root-knot nematode resistant heterozygous genes section bar material; If only produce the specific band of 750bp, no TaqI restriction enzyme site is sense root knot nematode disease homozygous genotype material.
3, interpretation of result
As shown in Figure 1, the B08 of first-filial generation, 109,314,198, A10 only produce the specific band of about 400bp, are the gene pure section bar material of sense tomato yellow leaf curl virus disease; E13 and 288 produces the specific band of 500bp and 400bp, is the material of anti-tomato yellow leaf curl virus disease.Concerning the resistance of Mi, the enzyme of Fig. 1 is cut the result and is shown that E13 and 198 produces the specific band of 570bp and 180bp, is root-knot nematode resistant homozygous genotype material; 288 and A10 produce 750bp, 570bp, the specific band of 180bp is root-knot nematode resistant heterozygous genes section bar material; B08,109 and 314 specific bands that produce 750bp, no TaqI restriction enzyme site is sense root knot nematode disease homozygous genotype material.
The JB-1 mark can be used for detecting the anti-sense genotype of the Ty-1 gene of tomato plant.As shown in Figure 2, the enzyme of JB-1PCR product is cut the result and is shown, and 2,3,4,7,8,9,10,11,12,13,15,16, the enzyme of 18,23 individual plants is cut the specific band that the result has produced about 400bp and 500bp; 6,14,20,21,22 enzymes are cut the specific band (Fig. 2) that has produced about 400bp.This result shows, JB-1PCR product enzyme is cut the material promptly the 6th, 14,20 that the result only produces the specific band of about 400bp, 21,22 individual plants belong to the sick homozygous genotype material of sense tomato yellow leaf curl virus, and JB-1PCR product enzyme is cut the material promptly the 2nd, 3 that the result produces the specific band of 400bp and 500bp, 4,7,8,9,10,11,12,13,15,16,18,23 individual plants belong to the sick material of anti-tomato yellow leaf curl virus.
F from E13
2Enzyme for Mi is cut the result, and E13 is totally 23 materials, all produces the specific band of 570bp and 180bp, for containing the disease-resistant homozygous genotype material (Fig. 2) of Mi.
F at A10
2In colony, PCR product enzyme is cut the material that the result only produces the specific band of about 400bp and is belonged to susceptible homozygous genotype material, and offspring's individual plant does not produce separation (Fig. 3) in this site.
A10 is totally 19 materials, and wherein 1,4,5,6,7,8,9,10,11,13,16,19 individual plants produce 750bp, and 570bp, the individual plant of the specific band of 180bp are the disease-resistant heterozygous genes section bar material that contains Mi; 3,15,17,18 plant that produce the specific band of 570bp and 180bp are the disease-resistant homozygous genotype material that contains Mi; 2,12,14 individual plants do not have Taq I restriction enzyme site, only produce the specific band of 750bp, for not containing the susceptible homozygous genotype material (Fig. 3) of Mi.
Through checking repeatedly, reliable results.
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (7)
1. the molecular marker-assisted selection method of TYLCV and root knot nematode is characterized in that may further comprise the steps: (1) is from tomato material F
1Separate offspring F with selfing
2Individual plant extracts DNA; (2) to tomato material F
1Separate offspring F with selfing
2Carry out the multiplex PCR amplification; (3) enzyme of multiplex PCR amplified production is cut and the electrophoretic analysis detection; (4) according to detected result breeding segregating generation material is carried out evaluation of resistance.
2. molecular marker-assisted selection method according to claim 1 is characterized in that, the method that described step (1) is extracted DNA is: with tomato material F
1With selfing segregating generation material F
2Sowing, when treating that seedling grows to 3～4 true leaves, every plant is got 1 spire, extracts DNA with improved CTAB method.
3. molecular marker-assisted selection method according to claim 1 is characterized in that, the concrete grammar of described step (2) is: synthetic primer, carry out pcr amplification, and carry out enzyme with the taqI enzyme and cut; Cut shown the isozygotying and the heterozygosis banding pattern of result according to pcr amplification and enzyme, inoculate qualification result with artificial seedling stage and compare, determine the molecule marker of anti-Ty-1 and root knot nematode gene; The upstream and downstream primer sequence that wherein detects the Ty-1 gene is respectively JB-1 upper:5 '-AACCAT TAT CCG GTT CAC TC-3 ' and JB-1 lower:5 '-TTT CCA TTC CTT GTT TCT CTG-3 ', and the upstream and downstream primer sequence of root knot nematode is respectively Mi upper:5 '-TCG GAG CCT TGG TCT GAA TT-3 ' and Mi lower:5 '-GCC AGA GAT GAT TCG TGA GA-3 '.
4. molecular marker-assisted selection method according to claim 1 is characterized in that, the concrete grammar of described step (3) is: multi-PRC reaction system 20 μ l, 1 times of reaction buffer, 2.0mM MgCl
2, 0.1 μ M upstream and downstream primer, 0.2mM dNTPs, the Taq polysaccharase of 0.04U and the template DNA of 20ng replenish ddwater to 20 μ l; The pcr amplification program be 94 ℃ 5 minutes, then 94 ℃ 1 minute, 54 ℃ 1 minute, 72 ℃ 2 minutes, 35 circulations, 72 ℃ were extended 10 minutes; The enzyme system of cutting is 20 μ l, wherein contains 7 μ l pcr amplification products, 10U taqI, 2 μ l reaction buffers; 65 ℃ of 1.5h; Pcr amplification and enzyme are cut product and are carried out the electrophoretic separation analysis with 2% agarose gel electrophoresis, EB dyeing, imaging on the gel imaging instrument of Bio-rad then.
5. molecular marker-assisted selection method according to claim 1, it is characterized in that, the evaluation method of described step (4) is: if enzyme is cut the specific band that only contains 400bp in the banding pattern, be sense Ty-1 homozygous genotype material, if enzyme is cut the specific band that contains 500bp and 400bp in the banding pattern, be anti-Ty-1 material; If contain the specific band of 570bp and 180bp, be root-knot nematode resistant homozygous genotype material; If contain 750bp, 570bp, the specific band of 180bp is root-knot nematode resistant heterozygous genes section bar material; If only produce the specific band of 750bp, no TaqI restriction enzyme site is sense root knot nematode disease homozygous genotype material.
6. molecular marker-assisted selection method according to claim 1 is characterized in that, the filial generation colony that described segregating generation material is a high separation or the backcross progeny colony of high separation.
7. molecular marker-assisted selection method according to claim 2, it is characterized in that, the DNA that described CTAB method is extracted is, preparation CTAB damping fluid: the plant leaf that (1) takes a morsel is in 1.5ml Eppendorf pipe, quick-frozen is 15 seconds in the tapping nitrogen, add 100 μ l CTAB damping fluids and a small amount of quartz sand, grind to form homogenate; (2) add 500 μ lCTAB damping fluids, 65 ℃ of insulation 35-40min, mixing is rocked in the centre; (3) add isopyknic 24: 1 chloroform: primary isoamyl alcohol (v/v), put upside down mixing 5min gently, 4 ℃ of centrifugal 10min of 15000rpm forward supernatant in the new pipe to, add the Virahol of 2/3 volume; (4) place 5min under the room temperature, 4 ℃ of 1500rpm, centrifugal 10min abandons supernatant, the washing with alcohol precipitation with 70% 2-3 time; Abandon supernatant, carry out vacuum-drying, in each sample, add 50 μ l sterilization distilled water and an amount of Rnase, 37 ℃ of digestion 1h, be stored in-20 ℃ standby.
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| CN102604941A (en) * | 2012-03-20 | 2012-07-25 | 江苏省农业科学院 | Molecular marker method linked with tomato yellow leaf curl disease resistance gene Ty-2 |
| CN102604941B (en) * | 2012-03-20 | 2013-08-14 | 江苏省农业科学院 | Molecular marker method linked with tomato yellow leaf curl disease resistance gene Ty-2 |
| CN103993067A (en) * | 2014-01-21 | 2014-08-20 | 舟山出入境检验检疫局综合技术服务中心 | Multiplex PCR detection method for three nematodes of Anisakidae |
| CN103966201A (en) * | 2014-05-15 | 2014-08-06 | 中国科学院武汉植物园 | Method for extracting aquatic plant DNA based on high-efficiency sample preservation |
| 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 |
| CN106939339A (en) * | 2017-03-24 | 2017-07-11 | 上海交通大学 | The Multiplex PCR of tomato Ty 1, Ty 3 and Mi genes is detected simultaneously |
| CN113699273A (en) * | 2021-09-30 | 2021-11-26 | 中国农业科学院深圳农业基因组研究所 | SNP locus combination for detecting resistance of tomato root-knot nematode and application thereof |
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