CN104830849A - Molecular markers of soybean mosaic virus resistant gene (RSC8) and applications thereof - Google Patents
Molecular markers of soybean mosaic virus resistant gene (RSC8) and applications thereof Download PDFInfo
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Abstract
The invention discloses molecular markers of soybean mosaic virus resistant gene (RSC8) and applications thereof. The invention discloses two pairs of novel primers for screening resistant gene (RSC8) related with soybean anti-soybean mosaic virus in the soybean molecular breeding field, a preparation method of molecular markers and applications. The analysis results show that the provided molecular markers (ZL-39 and ZL-52) can precisely trace the resistant gene (RSC8) of a soybean disease-resistant species (Kefeng No.1), and can predict the resistance of soybean to soybean mosaic virus, so that researchers can more conveniently identify and screen anti-soybean mosaic virus materials under the laboratory conditions. The molecular marker assisted breeding technology can avoid the influences of environmental factors and artificial factors on phenotype; and the provided two pairs of molecular markers (ZL-39 and ZL-52), which are tightly interlocked with the resistant gene (RSC8), can improve the accuracy in selection of anti-soybean mosaic virus breeding materials so as to achieve the goals of breeding soybean species which can resist soybean mosaic virus.
Description
Technical field
The present invention relates to soybean molecular breeding field, particularly relate to a kind of qualification and screening soybean mosaic virus resistant gene R
sC8primer pair, molecule marker, molecule marking method and application.
Background technology
Soybean mosaic virus (Soybean mosaic virus, SMV) disease is a kind of worldwide disease, and in the world, all there is generation in each soybean producing region.Within 1899, at China's northeast first meeting report, the harm in succession reporting this disease is economized in Shandong, Jiangsu and Hubei etc. subsequently, and SMV all has generation in China from north to south.Generally, the production loss that SMV causes is at 15-35%, and in the repeating transmission raw time of certain areas, the production loss caused can reach more than 70%, even has no harvest (Ren etc., 1997).In SMV and host and the long-term common evolutionary process of environment, there occurs Differentiation of Pathogenicity, create different Pathogenic Types, be referred to as strain.At present, China utilizes a set of unified differential host's system, and national SMV is divided into 22 strain: SC1-SC22 (Li etc., 2010 by unification; King is firm greatly, and 2013).
Wherein SC8 is the SMV strain extensively distributed at China's the Yellow River and Huai He River and soybean producing region, Zhong Xia basin, the Changjiang river.Screen that anti-source, cultivation and plantation disease-resistant variety are that control SMV is most economical, safety and effective means.Rich No. 1 of section is the anti-source of a kind of wide spectrum, except to except SC8 strain performance high resistance, all shows high resistance to 17 strains in other 21 strains.Genetic analysis shows, section rich No. 1 to SC8 strain by 1 to dominant resistant gene (R
sC8) control.So seed selection is containing R
sC8the soybean varieties of resistant gene be control that soybean Mosaic harm is most economical, one of effective means.
To after deliberation clearly resistant gene effectively utilized, can breeding cycle be shortened, meet domestic at present to SMV resistance high quality soybean kind in the urgent need to.But, utilize the method for traditional field or indoor inoculation qualification to soybean mosaic virus resistant gene R
sC8carry out offspring's tracking not only to take time and effort, and there is many uncertain factors, utilize molecular marker-assisted selection method can effectively solve this difficult problem.Wang etc. (2010) utilize genome SSR marker by soybean mosaic virus resistant gene R
sC8be limited on soybean No. 2 karyomit(e)s less than in the region of 200kb.This is screening further and soybean mosaic virus resistant gene R
sC8close linkage be even divided into from SSR marker lay a good foundation.Based on this, contriver, by developing new mark, utilizes the method for linkage analysis to soybean mosaic virus resistant gene R
sC8carry out Fine Mapping, further by soybean mosaic virus resistant gene R
sC8to be locked in the region of 30.8kb on soybean No. 2 karyomit(e)s, and to find and R
sC8closely linked molecule marker, effectively can improve efficiency and the accuracy of enantiopathy Material selec-tion in the breeding of soybean SMV resistance, accelerates the utilization of marker assisted selection technology in SMV resistant variety Breeding Process.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of and soybean mosaic virus resistant gene R
sC8closely linked molecule marker.
The another one technical problem that the present invention will solve is to provide the primer pair identifying above-mentioned molecule marker.
The present invention also has a technical problem that will solve to be to provide a kind of qualification and screening soybean mosaic virus resistant gene R
sC8the preparation method of molecule marker.
The present invention also has a technical problem that will solve to be to provide the application of above-mentioned molecule marker.
Above-mentioned purpose of the present invention can be achieved through the following technical solutions:
Resistant gene R of the present invention
sC8from soybean resistant variety section rich No. 1, this resistant gene is positioned on No. 2 karyomit(e)s of soybean, has resistance of wide spectrum.
The present invention is first according to resistant gene R
sC8first positioning result, utilize the sequences Design Auele Specific Primer in target gene section, excavate for the identification of resistant gene R
sC8molecule marker, and be applied to screen in the F2 colony built by section rich No. 1 × Nan Nong 1138-2, associative list type analysis, find that strong resistance weakly heterogeneous that different banding pattern is corresponding reaches significantly or pole conspicuous level.
The present invention for the identification of with screening soybean mosaic virus resistant gene R
sC8molecule marker, comprise molecule marker ZL-39 and ZL-52; Molecule marker ZL-39 and ZL-52 is for template carries out the fragment of pcr amplification gained with the genomic dna of large pulse family rich No. 1 and southern agriculture 1138-2; Molecule marker ZL-39 and ZL-52 and resistant gene R
sC8between genetic distance be respectively 2.2cM and 1.6cM.
The present invention is used for the primer pair of amplifier molecule mark ZL-39, comprises the upstream primer with the nucleotide sequence shown in SEQ ID NO:1 and the downstream primer with the nucleotide sequence shown in SEQ ID NO:2.
The present invention is used for the primer pair of amplifier molecule mark ZL-52, comprises the upstream primer with the nucleotide sequence shown in SEQ ID NO:3 and the downstream primer with the nucleotide sequence shown in SEQ ID NO:4.
The invention provides a kind of qualification and screening soybean mosaic virus resistant gene R
sC8the preparation method of molecule marker, comprise the following steps:
(1) extraction of soybean material genomic dna:
With seedling stage fresh blade for material, adopt CTAB method to extract DNA, detailed step is as follows:
1) get soybean tender leaf 2-3g, grinding powder in liquid nitrogen, pour the filling in the centrifuge tube of 650 μ L CTAB Extraction buffers of 65 DEG C of preheating about 30min into, then add 20 μ L beta-mercaptoethanols, 65 DEG C of water-bath about 60min, period jog 3-5 time;
2) add the chloroform of 624 μ L and the isoamyl alcohol of 26 μ L, and fully shake up, be placed on jog 20min on shaking table;
3) through the centrifugal 15min of 10000rpm, supernatant liquor is proceeded in a new centrifuge tube;
4) add the Virahol that 200 μ L are freezing, shake up up and down;
5) through the centrifugal 5min of 8000rpm, outwell liquid, add 1000 μ L TE damping fluids, until completely dissolved, extracting is once again for the isoamyl alcohol adding the chloroform of 624 μ L and 26 μ L;
6) through the centrifugal 5min of 8000rpm, Aspirate supernatant forwards in a new centrifuge tube;
7) add the freezing dehydrated alcohol of 1000 μ L, at-20 DEG C, leave standstill 20min to precipitate DNA;
8) the centrifugal 5min of 10000rpm, abandoning supernatant;
9) 70% ethanol 800 μ L cleans twice, and Vacuumdrier dries up;
10) be dissolved in TE by DNA, the agarose gel electrophoresis with 1% detects; Be placed in-20 DEG C stand-by;
(2) pcr amplification:
The primer pair being used for amplifier molecule mark ZL-39 or the primer pair that is used for amplifier molecule mark ZL-52 are added PCR reaction system, and the DNA of soybean breeder colony plant to be increased; Amplification system is 4.0 μ LTaq × Master PCR Mix, and 2.0 μ L concentration are 1.0ppmol/L upstream and downstream primer, 20.0ng/ μ L template DNA 3.0 μ L, ddH
2o 1.0 μ L; Reaction total amount 10.0 μ L; PCR program: 94 DEG C of denaturation 5min, each circulation 95 DEG C of sex change 60s, 55 DEG C of annealing 50s, 72 DEG C extend 60s, totally 30 circulations, and last 72 DEG C extend 10min, 12 DEG C of preservations;
(3) PCR primer detects:
PCR reaction product is on mass ratio 8% non-denaturing polyacrylamide gel electrophoresis, observes under gel imaging system with cma staining, records.
(4) qualification result:
The plant that can amplify with section rich No. 1 same clip or comprise its fragment is for containing soybean mosaic virus resistant gene R
sC8plant; Otherwise, only can amplify with the plant of southern agriculture 1138-2 same clip as not containing soybean mosaic virus resistant gene R
sC8plant.
Present invention also offers for the identification of with screening soybean mosaic virus resistant gene R
sC8molecule marker ZL-39 and ZL-52, and for amplifier molecule mark ZL-39 primer pair and for amplifier molecule mark ZL-52 primer pair Soybean Resistance soybean mosaic virus material initiative in application.
Present invention also offers for the identification of with screening soybean mosaic virus resistant gene R
sC8molecule marker ZL-39 and ZL-52, and for the primer pair of amplifier molecule mark ZL-39 with to assist application in the breeding of Soybean Resistance soybean mosaic virus at molecule marker for the primer pair of amplifier molecule mark ZL-52.
Present invention also offers a kind of containing the test kit for the primer pair of amplifier molecule mark ZL-39 and the primer pair for amplifier molecule mark ZL-52.
The present invention also comprises the application of mentioned reagent box in the initiative of Soybean Resistance soybean mosaic virus material, and mentioned reagent box assists the application in the breeding of Soybean Resistance soybean mosaic virus at molecule marker.
The invention has the beneficial effects as follows:
Soybean Mosaic is a kind of virus disease having a strong impact on Soybean production, and the present invention utilizes the method for molecular biology and information biology to screen and soybean mosaic virus resistant gene R
sC8closely linked molecule marker, the application of this mark can accelerate the seed selection of SMV resistant variety.Its advantage is specifically summarized as follows:
(1) soybean mosaic virus resistant gene R
sC8come from No. 1, the resistance parent Ke Feng of broad-spectrum high efficacy, by R
sC8gene carries out molecular marker assisted selection, can go out soybean line or the kind of a large amount of resistance by quickly breeding.
(2) traditional breeding method utilizes the parent containing resistant gene to carry out a series of hybridization with the upper conventional kind of production, then carries out Single-plant selection to SMV resistance.Because the resistance of this disease is comparatively large by the impact of envrionment conditions, the reliability of phenotypic evaluation result needs repeated authentication, is therefore identified by field resistance or indoor inoculation qualification judges SMV resistant genotype, and not only waste time and energy, and difficulty is large, cost is high.Detected by the banding pattern marked its resistant gene close linkage, its resistant genotype measurable, so not only saves cost of expert testimony, and greatly improves the efficiency of selection of SMV resistance soybean varieties.
(3) the present invention obtain with SMV resistant gene R
sC8closely linked molecule marker, it detects fast, simply, result is stable, reliable, is applicable to the detection a large amount of breeding material being carried out to marker genetype at short notice, to judge whether it has the resistance to soybean Mosaic, row filter of going forward side by side.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 is the soybean mosaic virus resistant gene R of the embodiment of the present invention
sC8position on No. 2 karyomit(e)s and and molecule marker of the present invention between Genetic linkage map information.
Fig. 2 is the electrophoretogram that the F2 plant molecule marker ZL-39 of the section of the embodiment of the present invention rich No. 1 × Nan Nong 1138-2 detects, wherein P1, P2 and F1 are respectively the first-filial generation of rich No. 1 of section, Nan Nong 1138-2 and Ke Feng No. 1 × Nan Nong 1138-2,1,2,4-7,12-14,19-21,23,27,28,30 are SMV resistance homozygous genotype plant, 3,8,9,11,15,16,18,22,24-26,29 is SMV resistance heterozygous genotypes plant, 10,17 is susceptible genotype, and M is Marker.
Fig. 3 is the electrophoretogram that the F2 plant molecule marker ZL-52 of the section of the embodiment of the present invention rich No. 1 × Nan Nong 1138-2 detects, wherein P1, P2 and F1 are respectively the first-filial generation of rich No. 1 of section, Nan Nong 1138-2 and Ke Feng No. 1 × Nan Nong 1138-2,11,20-22,25,26,30 is SMV resistance homozygous genotype plant, 2-4,6,7,9,10,12-19,27 is SMV resistance heterozygous genotypes plant, 1,5,8,23,24,28,29 is susceptible genotype, and M is Marker.
Embodiment
1. the large rich No. 1 SMV resistance gene R of pulse family
sC8location and the acquisition of molecule marker
1) material:
Rich No. 1 of disease-resistant variety section: (Hebei local variety Systematic selection, containing resistant gene R for the local soybean varieties of wide spectrum High resistance to SMV
sC8), most of SMV strain of anti-qualification at present, genotype is R
sC8r
sC8, 94 days average breeding times of this kind, plant height 53.6 centimetres, lanceolata, spends in vain, palm fiber, and limited pod bearing habit, plant type restrains, and stem 15.0 saves, effective branch 3.3.End pod height 14.1 centimetres, the effective pod number of individual plant 106.0,100-grain weight 7.33 grams, seed kidney shape, seed coat black.Gross protein value 43.90%, crude fat content 19.60%.
Susceptible variety south agriculture 1138-2: the kind that last century extensively promotes in In Middle And Lower Reaches of Changjiang River is the backbone parent of multiple soybean varieties that the later stage is bred as.Soybean Mosaic resistant genotype is r
sC8r
sC8, 110 days average breeding times of this kind, plant height 79.9 centimetres, blade oval, pale reddish brown, palm fiber, limited pod bearing habit, plant type half is opened a business, and stem 20.0 saves, effective branch 4.2.End pod height 22.4 centimetres, the effective pod number of individual plant 124.0,100-grain weight 19.43 grams, seed is oval, and seed coat is yellow.Gross protein value 44.10%, crude fat content 20.40%.
Segregating population: utilize rich No. 1 of SMV resistance soybean varieties section to be female parent, to feel SMV soybean varieties south agriculture 1138-2 for paternal hybrid, obtain Hybrid F1, and then selfing obtains 2212 F2 individual plants formation mapping populations.
2) F2 SMV resistance qualification:
By the F2 colony for examination at fly net indoor pot, when first pair of true leaf launches completely, get phosphoric acid buffer (about every the fresh blade of 1g add 5mL damping fluid) that the sick leaf with typical SMV symptom adds 0.1mol/L, pH 7.3-7.4 to add a little silicon carbide (600 order) be again ground into homogenate in mortar, then with hairbrush, homogenate is coated onto first on the true leaf launched, finally use tap water, when first pair of compound leaf launches, repeated inoculation once.Within one week, starting to observe and records incidence, Continuous Observation two months after inoculation, there is the plant of symptom in viewing duration listing mark, in case the impact of hidden disease afterwards.Regular spray medicine, in case the cross infection that aphid transmission causes.Resistant reaction is divided into disease-resistant (do not have symptom after inoculation or only occur local lesion and superior leaf is asymptomatic on inoculation leaf) and susceptible (system floral leaf, shrinkage, curling etc.) two classes.
3) gene type assay
A) extracting genome DNA: adopt CTAB method to extract No. 1, parent Ke Feng, Nan Nong 1138-2 and F1, F2 for colony plant DNA.Detailed step is as follows:
1. get soybean tender leaf 2-3g, grinding powder in liquid nitrogen, pour the filling in the centrifuge tube of 650 μ L CTAB Extraction buffers of 65 DEG C of preheating about 30min into, then add 20 μ L beta-mercaptoethanols, 65 DEG C of water-bath about 60min, period jog 3-5 time;
2. add the chloroform of 624 μ L and the isoamyl alcohol of 26 μ L, and fully shake up, be placed on jog 20min on shaking table;
3. through the centrifugal 15min of 10000rpm, supernatant liquor is proceeded in a new centrifuge tube;
4. add the Virahol that 200 μ L are freezing, shake up up and down;
5. through the centrifugal 5min of 8000rpm, outwell liquid, add 1000 μ L TE damping fluids, until completely dissolved, extracting is once again for the isoamyl alcohol adding the chloroform of 624 μ L and 26 μ L;
6. through the centrifugal 5min of 8000rpm, Aspirate supernatant forwards in a new centrifuge tube;
7. add the freezing dehydrated alcohol of 1000 μ L, at-20 DEG C, leave standstill 20min to precipitate DNA;
8. the centrifugal 5min of 10000rpm, abandoning supernatant;
9. 70% ethanol 800 μ L cleans twice, and Vacuumdrier dries up;
10. be dissolved in TE by DNA, the agarose gel electrophoresis with 1% detects; Be placed in-20 DEG C stand-by.
B) ssr analysis
1. the exploitation of SSR marker: utilize (2010) such as Wang to soybean mosaic virus resistant gene R
sC8the PRELIMINARY RESULTS of location, and in conjunction with the data that soybean gene group database website (http://phytozome.jgi.doe.gov/pz/portal.html) has been announced, utilize the data sequence announced and with SSR search software-SSRHUNTER2.0, the SSR site that may exist in the sequence in target section inquired about, screened, and designing, synthesized 43 pairs of SSR primers.
2. the screening of polymorphism primer: utilize above-mentioned 43 pairs of primers carry out polymorphism screening and utilize F2 colony between No. 1, resistance parent Ke Feng and Susceptible parent south agriculture 1138-2, found that 13 pairs of primers present polymorphism in resistance parent and Susceptible parent combination.
3. the ssr analysis of F2 colony: what 13 couple obtained with above-mentioned steps had a polymorphism is labeled as primer, the DNA of simultaneously increase No. 1, parent Ke Feng, Nan Nong 1138-2 and F1 and F2 colony plant, carries out genotype identification, obtains SSR marker data.The banding pattern that No. 1, parent Ke Feng is designated as a, and the banding pattern of parent Nan Nong 1138-2 is designated as b, and the banding pattern of section rich No. 1 × Nan Nong 1138-2F1 is designated as h.What F2 colony banding pattern derived from rich No. 1 of section is designated as a, and what derive from Nan Nong 1138-2 is designated as b, and what derive from section rich No. 1 × Nan Nong 1138-2F1 is designated as h.
4. linkage map builds: according to SSR marker data, utilizes mapping software JoinMap4.0 to build genetic map, in conjunction with F2 colony inoculation SMV phenotypic data Fine Mapping SMV resistance gene R
sC8.Find SSR marker ZL-39 and ZL-52 and its close linkage, genetic distance is respectively 2.2cM, 1.6cM (table 1, Fig. 1).
Table 1 is for the identification of soybean mosaic virus resistant gene R
sC8sSR marker
5. significance analysis: when SSR label primer ZL-39 and ZL-52 is increased in filial generation, we find that rich with section No. 1 amplifies same strap or containing the equal corresponding SMV resistance genotype of filial generation of rich No. 1 amplified band of section, can not increase to graduate from old-type opera school that the filial generation of rich No. 1 band is all corresponding feels SMV genotype.Disease resistance corresponding for the plant amplifying different banding pattern is carried out t inspection, finds differences and all reach significantly or pole conspicuous level.
2. molecule marker provided by the invention is at selection soybean mosaic virus resistant gene R
sC8on application test
1) utilize rich No. 1 of soybean SMV resistance material supply section to be female parent, SMV soybean varieties south agriculture 1138-2 is male parent, adopts simple grain transmission method to build and obtains a F7 be made up of 184 parts of materials for RIL (RILs).
2) carry out marker detection to obtained F7 filial generation, concrete grammar is: extract the genomic dna of F7 for individual plant in seedling stage; Take genomic dna as substrate, with the primer pair of the SSR marker ZL-39 developed and ZL-52 for primer carries out pcr amplification respectively, described primer is:
ZL-39: the positive/negative TTAACGACGTTAACTTCAAACT/CTTCTCTATCAAACACAATGTAAAC of primer sequence.
ZL-52: the positive/negative GGTGAAGAACATGCACACAATTAG/TGACTTGTTATGTGATGGTGGGT of primer sequence.
3) PCR amplification system:
ZL-39：
Amplification system is 4.0 μ L Taq × Master PCR Mix, and 2.0 μ L concentration are 1.0ppmol/L upstream and downstream primer, 20.0ng/ μ L template DNA 3.0 μ L, ddH
2o 1.0 μ L; Reaction total amount 10.0 μ L;
ZL-52：
Amplification system is 4.0 μ L Taq × Master PCR Mix, and 2.0 μ L concentration are 1.0ppmol/L upstream and downstream primer, 20.0ng/ μ L template DNA 3.0 μ L, ddH
2o 1.0 μ L; Reaction total amount 10.0 μ L;
4) pcr amplification program:
ZL-39：
94 DEG C of denaturation 5min, each circulation 95 DEG C of sex change 60s, 55 DEG C of annealing 50s, 72 DEG C extend 60s, totally 30 circulations, and last 72 DEG C extend 10min, 12 DEG C of preservations;
ZL-52：
94 DEG C of denaturation 5min, each circulation 95 DEG C of sex change 60s, 55 DEG C of annealing 50s, 72 DEG C extend 60s, totally 30 circulations, and last 72 DEG C extend 10min, 12 DEG C of preservations;
5) PCR primer detects:
PCR reaction product is on mass ratio 8% non-denaturing polyacrylamide gel electrophoresis, observes under gel imaging system with cma staining, records.
6) electrophoresis result finds, when increasing with primer pair ZL-39,94 filial generations are had to amplify No. 1 identical band rich in section, 90 filial generations are had not amplify No. 1 identical band rich in section, and when increasing with primer pair ZL-52, there are 87 filial generations to amplify the band identical with southern agriculture 1138-2, have 97 filial generations not amplify the band identical with southern agriculture 1138-2.Prediction is when increasing with ZL-39, and the material amplifying rich No. 1 same strap with section will higher than the resistance that can not expand stripping to the resistance of SMV.And when increasing with ZL-52, can amplify will lower than the resistance that can not expand stripping to the resistance of SMV with the material of southern agriculture 1138-2 same strap.
7) in conjunction with the phenotypic data of greenhouse expert evidence, finding by analyzing, when increasing with ZL-39, the disease resistance of the rich No. 1 same strap material with section can be amplified all higher than the material that can not amplify its band; And when increasing with ZL-52, can amplify with the disease resistance of southern agriculture 1138-2 same strap material all lower than the material that can not amplify its band.Respectively disease resistance corresponding for the material of the different bands gone out at two pairs of primer amplifications is carried out t inspection, find with rich No. 1 same strap of disease-resistant variety section with the material of susceptible variety south agriculture 1138-2 same strap, disease resistance difference all reaches significantly or pole conspicuous level, and actual result is consistent with expected results.Illustrate that the SSR marker ZL-39 that the present invention develops and ZL-52 can jointly for SMV resistant gene R
sC8selection.
Above-described embodiment of the present invention, does not form limiting the scope of the present invention.Any amendment done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within claims of the present invention.
Claims (9)
1. qualification and screening soybean mosaic virus resistant gene R
sC8molecule marker, it is characterized in that: comprise molecule marker ZL-39 and ZL-52; Described molecule marker ZL-39 and ZL-52 and resistant gene R
sC8between genetic distance be respectively 2.2cM and 1.6cM.
2. for the primer pair of the molecule marker ZL-39 as claimed in claim 1 of increasing, it is characterized in that, described primer pair comprises the upstream primer with the nucleotide sequence shown in SEQ ID NO:1 and the downstream primer with the nucleotide sequence shown in SEQ IDNO:2.
3. for the primer pair of the molecule marker ZL-52 as claimed in claim 1 of increasing, it is characterized in that, described primer pair comprises the upstream primer with the nucleotide sequence shown in SEQ ID NO:3 and the downstream primer with the nucleotide sequence shown in SEQ IDNO:4.
4. qualification and screening soybean mosaic virus resistant gene R
sC8the preparation method of molecule marker, comprise the following steps:
(1) using material DNA to be identified as template, the primer pair described in claim 2 or primer pair according to claim 3 is utilized to carry out pcr amplification; The wherein extraction of soybean material genomic dna, with seedling stage fresh blade for material, adopt CTAB method to extract DNA, detailed step is as follows:
1) get soybean tender leaf 2-3g, grinding powder in liquid nitrogen, pour the filling in the centrifuge tube of 650 μ L CTAB Extraction buffers of 65 DEG C of preheating about 30min into, then add 20 μ L beta-mercaptoethanols, 65 DEG C of water-bath about 60min, period jog 3-5 time;
2) add the chloroform of 624 μ L and the isoamyl alcohol of 26 μ L, and fully shake up, be placed on jog 20min on shaking table;
3) through the centrifugal 15min of 10000rpm, supernatant liquor is proceeded in a new centrifuge tube;
4) add the Virahol that 200 μ L are freezing, shake up up and down;
5) through the centrifugal 5min of 8000rpm, outwell liquid, add 1000 μ L TE damping fluids, until completely dissolved, extracting is once again for the isoamyl alcohol adding the chloroform of 624 μ L and 26 μ L;
6) through the centrifugal 5min of 8000rpm, Aspirate supernatant forwards in a new centrifuge tube;
7) add the freezing dehydrated alcohol of 1000 μ L, at-20 DEG C, leave standstill 20min to precipitate DNA;
8) the centrifugal 5min of 10000rpm, abandoning supernatant;
9) 70% ethanol 800 μ L cleans twice, and Vacuumdrier dries up;
10) be dissolved in TE by DNA, the agarose gel electrophoresis with 1% detects; Be placed in-20 DEG C stand-by;
(2) pcr amplification:
Claim 2 or molecular marker primer pair according to claim 3 are added PCR reaction system, and the DNA of soybean breeder colony plant is increased; Amplification system is 4.0 μ L Taq × Master PCRMix, and 2.0 μ L concentration are 1.0ppmol/L upstream and downstream primer, 20.0ng/ μ L template DNA 3.0 μ L, ddH
2o 1.0 μ L; Reaction total amount 10.0 μ L; PCR program: 94 DEG C of denaturation 5min, each circulation 95 DEG C of sex change 60s, 55 DEG C of annealing 50s, 72 DEG C extend 60s, totally 30 circulations, and last 72 DEG C extend 10min, 12 DEG C of preservations;
(3) PCR primer detects:
PCR reaction product is on mass ratio 8% non-denaturing polyacrylamide gel electrophoresis, observes under gel imaging system with cma staining, records.
5. molecule marker according to claim 1 or primer pair according to claim 2 or the application of primer pair according to claim 3 in the initiative of Soybean Resistance soybean mosaic virus material.
6. molecule marker according to claim 1 or primer pair according to claim 2 or primer pair according to claim 3 assist the application in the breeding of Soybean Resistance soybean mosaic virus at molecule marker.
7. contain the test kit of primer pair according to claim 2 or primer pair according to claim 3.
8. the application of test kit according to claim 7 in the initiative of Soybean Resistance soybean mosaic virus material.
9. test kit according to claim 7 assists the application in the breeding of Soybean Resistance soybean mosaic virus at molecule marker.
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| CN110343159A (en) * | 2019-08-16 | 2019-10-18 | 安徽省农业科学院作物研究所 | A kind of Mung Bean Blooming gene VrELF3 and its application |
| CN110387376A (en) * | 2019-08-16 | 2019-10-29 | 安徽省农业科学院作物研究所 | A kind of Mung Bean Blooming gene VrFT5a and its application |
| CN110438133A (en) * | 2019-08-16 | 2019-11-12 | 安徽省农业科学院作物研究所 | A kind of Mung Bean Blooming gene VrFT2a and its application |
| CN111996282A (en) * | 2020-09-18 | 2020-11-27 | 南京农业大学 | SSR marker CH0211 for identifying soybean mosaic virus resistant SC3 strain of soybean as well as detection method and application thereof |
| CN116355913A (en) * | 2023-03-22 | 2023-06-30 | 青岛农业大学 | Soybean vacuole ATPase assembled protein gene Vma12 and application thereof |
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| CN110438133A (en) * | 2019-08-16 | 2019-11-12 | 安徽省农业科学院作物研究所 | A kind of Mung Bean Blooming gene VrFT2a and its application |
| CN110343159B (en) * | 2019-08-16 | 2021-05-14 | 安徽省农业科学院作物研究所 | Application of expression vector of mung bean flowering gene VrELF3 |
| CN110387376B (en) * | 2019-08-16 | 2021-06-04 | 安徽省农业科学院作物研究所 | Application of expression vector containing mung bean flowering gene VrFT5a |
| CN110438133B (en) * | 2019-08-16 | 2021-06-04 | 安徽省农业科学院作物研究所 | Application of expression vector containing mung bean flowering gene VrFT2a |
| CN111996282A (en) * | 2020-09-18 | 2020-11-27 | 南京农业大学 | SSR marker CH0211 for identifying soybean mosaic virus resistant SC3 strain of soybean as well as detection method and application thereof |
| CN111996282B (en) * | 2020-09-18 | 2022-07-15 | 南京农业大学 | SSR marker CH0211 for identifying soybean mosaic virus resistant SC3 strain of soybean as well as detection method and application thereof |
| CN116355913A (en) * | 2023-03-22 | 2023-06-30 | 青岛农业大学 | Soybean vacuole ATPase assembled protein gene Vma12 and application thereof |
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