CN1314816C - Molecular mark of watermelon linked to gene resistant to field pumpkin yellowwatermelon yellow mosaic virus and uses - Google Patents
Molecular mark of watermelon linked to gene resistant to field pumpkin yellowwatermelon yellow mosaic virus and uses Download PDFInfo
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- CN1314816C CN1314816C CNB200510056426XA CN200510056426A CN1314816C CN 1314816 C CN1314816 C CN 1314816C CN B200510056426X A CNB200510056426X A CN B200510056426XA CN 200510056426 A CN200510056426 A CN 200510056426A CN 1314816 C CN1314816 C CN 1314816C
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Abstract
The present invention discloses a linked RAPD mark AK 13-644 of genes for resisting small pumpkin yellow mosaic virus in watermelons and a successfully converted SCAR mark SCAR 13-64 thereof. The establishment of the marks settles solid basis for a technological system of anti-ZYMV-CH molecular mark auxiliary selection of watermelons and for the breeding of virus disease resistant new breeds and the clone of disease resistant genes.
Description
Technical field
The present invention relates to the linkage molecule mark of the anti-little zucchini yellow mosaic virus gene of a kind of watermelon (Citrullus lanatus (Thunb) Manf.), the chain RAPD mark of the anti-little zucchini yellow mosaic virus gene of particularly a kind of watermelon reaches SCAR (the sequence characterizied amplifiedregion) mark and this SCAR that are obtained by this mark conversion and is marked at the application that detects in the transformation offspring plant.
Background technology
At present, little zucchini yellow mosaic virus (Zucchini yellow mosaic virus, ZYMV) be that the member of Potyvirus is the main virus that infects ground family crop, also be proved to be the main virus of harm China watermelon, in many countries and regions generation is arranged all, have a strong impact on the yield and quality of watermelon, even can cause total crop failure.Xu Yong etc. (2004) find 3 kinds of main diseases viral disease (ZYMV-CH to watermelon first, PRSV-W, WMV) all has the watermelon wild germplasm PI595203 of resistance, confirm that PI595203 is controlled by recessive single-gene zym-CH the resistance of ZYMV-CH, resistance to WMV is controlled by the recessive gene more than 3 at least, and the relatively independent (.Xu of the disease-resistant gene of these two kinds of viruses, Y., D.Kang, Z.Shi, H.Shen, and T.Wehner.Inheritance of resistance to zucchini yellow mosaicvirus and watermelon mosaic virus in watermelon.J.Hered, 2004.95:498-502.).This laboratory finds that also PI595203 also is by recessive single-gene PRSV-W control to the resistance of PRSV-W simultaneously, may there be little effect modifying factor, and watermelon presents linkage relationship to the resistant gene of PRSV-W and ZYMV-CH, but the genetic distance of the two 27cM more greatly.We find to want the disease-resistant gene of transformation PI595203 to these 3 kinds of virus diseases from above two test-results, obtain to hold concurrently in the transformation offspring to resist the plant of these three kinds of virus diseases, just need very big segregating population and more seed selection algebraically.This has proved absolutely arduousness and the complicacy of utilizing traditional breeding technology to carry out watermelon viral diseases transformation work, and the molecular marker assisted selection technology might become the powerful mean of watermelon viral diseases breeding research.
Summary of the invention
We carry out watermelon ZYMV-CH gene linkage Study on Molecular Marker on this basis, and RAPD mark and the SCAR mark chain have successfully been obtained with the ZYMV-CH disease-resistant gene, in the hope of setting up the anti-ZYMV-CH molecular marker assisted selection of watermelon technology, for the seed selection of watermelon viral diseases new variety and the clone of disease-resistant gene lay the foundation.
First purpose of the present invention provides a kind of and the closely linked RAPD mark of the anti-ZYMV-CH gene of watermelon.
Second purpose of the present invention provides a kind of and the closely linked stable SCAR mark of the anti-ZYMV-CH gene of watermelon, so as high-throughput be applied to breeding practice.
In order to reach above-mentioned goal of the invention, the present invention adopts following scheme:
The RAPD mark AK13 of the anti-little zucchini yellow mosaic virus gene linkage of watermelon
-644, its nucleotide sequence is the SEQ ID No:1 in the sequence table.
The SCAR mark SCAK13 of the anti-little zucchini yellow mosaic virus gene linkage of watermelon
-644, its nucleotide sequence is SEQ ID No:2 and the SEQ ID No:3 in the sequence table.
Advantage of the present invention is: RAPD of the present invention and SCAR are marked in the seed selection of watermelon viral diseases new variety has vital role, this test is by selecting hybrid, cultivate a large amount of transformation offsprings, reach plant disease-resistant authenticate technology in seedling stage and RAPD, SCAR mark detection technique and combine.Adopt the anti-ZYMV-CH that holds concurrently, PRSV-W, the watermelon germplasm of three kinds of virus diseases of WMV, and in conjunction with F
8Carry out the mark checking for the transformation plant, the workload that has broken through conventional breeding is big, and the constraint that the seed selection cycle is long has realized that the conventional hybridization breeding is put to the technological line that breeding combined, shortened breeding cycle with molecule marker.
Description of drawings
Fig. 1 be RAPD primer of the present invention to the parent, F
1, anti-sense pond and F
2The RAPD-PCR amplification collection of illustrative plates of individual plant.Wherein, M:Ladder DNA; I.PI595203; 2.98R; 3.F
14. disease-resistant gene pond; 5. susceptible gene pool; 6-34.F
2Individual plant.
Fig. 2 be RAPD primer of the present invention to part anti-ZYMV-CH transformation plant and Crimsonsweet, Calhoun gray, the RAPD amplification of PI296341.Wherein, M:Ladder DNA; 1.PI595203; 2.98R; 3.F
14. disease-resistant gene pond; 5. susceptible gene pool; 6.PI296341; 7.Crimsonsweet; 8.Calhoun gray; 9-32. part transformation plant.
Fig. 3 is mark AK13 among the variety of watermelon PI595203
-644Linkage map with the ZYMV-CH resistant gene.
Fig. 4 be SCAR primer of the present invention to the parent, F
1, anti-sense pond and F
2The SCAR-PCR amplification collection of illustrative plates of individual plant.Wherein, M:Ladder DNA; I.PI595203; 2.98R; 3.F
14. disease-resistant gene pond; 5. susceptible gene pool; 6-34.F
2Individual plant.
Embodiment
Experiment material:
Disease-resistant parent: PI595203; Susceptible parent: 98R; As cross combination, pass 109 F of acquisition with PI595203 * 98R by simple grain
3For strain system, each strain system guarantees that 60 individual plants are as disease-resistant expert evidence.And by PI595203 * 98R filial generation repeatedly being carried out anti-ZYMV-CH screening, 60 disease-resistant F that obtain
8Monosystem is as linked marker checking material.
Disease resistance is identified:
Adopt the frictional inoculation method to carry out disease resistance evaluation in seedling stage, expert evidence comprises the parent, F
1And 109 F
3Each 60 individual plant of strain system, plant 2-3 and begin the performance that observes the symptoms after week, according to the symptom performance and with reference to Boyhan (Boyhan G, Norton JD, Jacobsen BJ, Abrahams BR, Evaluation ofwatermelon and relates germplasm for resistance to zucchini yellow mosaic virus.Plant Dis.1992,76:251-252) give birth to diligent Gu (. Gu Qinsheng, Fan Zaifeng, Li Huaifang. ground family crop virus register, Chinese watermelon and muskmelon .2002 (1): grade scale 45-47) is formulated unified standard of perfection.Carry out once disease-resistant statistics week about, continuous 4 weeks.
With reference to F
3The anti-sense separation case of strain system is inferred F
2Resistance and genotype for individual plant.Testing the known PI595203 of existing result of study according to this is recessive single-gene control to the resistance of ZYMV-CH, so F
3Its F infers in the strain system that representative is now isozygotied disease-resistant
2Genotype for individual plant is aa; F
3Its F infers in the strain system that representative is now isozygotied susceptible
2For the individual plant frequency of genotypes AA; F
3The existing anti-sense of representative separates, and meets the strain system of 1: 1 segregation ratio substantially, can infer its F
2For the individual plant genotype is heterozygous Aa.
The foundation of RAPD mark:
The RAPD-PCR reaction system comprises: ddH
2O 6.85 μ L; 10*buffer (has contained Mg
2+) 1.25 μ L; DNTP (2.5mM) 1.0 μ L; Primer (15ng/ μ L) 2.0 μ L; Template DNA (30ng/ μ L) 2.0 μ L; TaqE (2.5U/ μ L) 0.4 μ L, totally 13.5 μ L.
RAPD-PCR is reflected on the DNA PTC-100 (eastern KingMax true tumor technology company limited) and carries out.Response procedures is as follows: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s then, 37 ℃ of annealing 30s, 72 ℃ are extended 1min, carry out 35 circulations, in 72 ℃ of insulation 7min down, 4 ℃ of preservations, about 2.5 hours of entire reaction programs.
(Bulked segregant analysis is BSA) at F to adopt group's compartment analysis method
2In generation, set up anti-sense gene pool, with the parent, and F
1With anti-sense gene pool is template, 640 RAPD primers are carried out the pcr amplification screening, wherein primer AK13 is the parent, F
1, anti-sense gene pool and 109 F
2Between to amplify a polymorphic bands (644bp) partial results shown in Figure 1: wherein on 21 disease-resistant plant of isozygotying all performance this polymorphism band is arranged; In 21 disease plants of isozygotying, there are 4 performances that band is arranged, recombinate; 5 these bands of disappearance in 67 heterozygotes, the performance reorganization.By the genetic affinity between JoinMap3.0 software analysis gained RAPD mark and the ZYMV-CH resistant gene, proved that the resistant gene of this band and ZYMV-CH presents linkage relationship, the genetic linkage distance is 8cM, names to be AK13
-644(as shown in Figure 2), and in transformation offspring self-mating system, be verified partial results and see Fig. 3: mark AK13
-644All performance has band on the transformation plant, at Crimson sweet, Calhoun gray and PI296341 go up this band of performance disappearance, illustrate that this is marked at the stable linkage relationship that exists on the PI595203 with the ZYMV-CH resistant gene, can be used as the selective marker of disease-resistant transformation.
The foundation of SCAR mark
Adopt the DNA of vast company (BioDev, Beijing) to reclaim purification system " glass milk method " fast the purpose band of RAPD primer amplification is carried out purifying.RAPD (PCR) product and carrier be connected the PGEM-T easy Vector system that adopts Promega company.Carry out the conversion and the screening of recombinant plasmid with reference to the method in " molecular cloning experiment guide ".Utilize alkali formula cracking process to extract recombinant plasmid, adopt enzyme to cut and detect with PCR.Entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to check order synthesizing of (seeing sequence table SEQ ID No:1) and SCAR primer.
The SCAR amplification reaction system is totally 25 μ l, comprising: ddH
2O.6.3 μ l; 10*buffer.2.5 μ l; DNTP (2.5mM) 2.0 μ l; Primer 1 (15ng/ul) .1.0 μ l; Primer 2 (15ng/ μ l) .1.0 μ l; Template DNA (5ng/ μ l) 2.0 μ l TaqE (5U/ μ l) .0.2 μ l.
The response procedures of SCAR amplification is: 94 ℃ of pre-sex change 5min, and 94 ℃ of pre-sex change 1min, different annealing temperature (50 ℃, 55 ℃, 60 ℃, 65 ℃) annealed 1 minute, 72 ℃ are extended 2min, 35 circulations, last 72 ℃ are extended 7min, 4 ℃ of preservations down.
According to sequencing result, the SCA primer of design
5’-TCCCACGAGTCCAGCAGCAAG-3’；
5 '-TCCCACGAGTACTTTTAGAAGT-3 ' carries out SCAR-PCR amplification, the amplification partial results as shown in Figure 4: the amplification band is steady and audible, and corresponding fully with its corresponding RAPD primer amplification result, with its called after SCAK13
-644RAPD mark AK13 is described
-644Successfully transform into the SCAR mark SCAK13 that the performance amplified band has or not
-644(being sequence table SEQ IDNo:2 and SEQ ID No:3).
The application of SCAR mark
The SCAK13-644 that the present invention obtains has passed through F in experimentation
8Checking for anti-ZYMV-CH transformation plant, fully confirmed the stable linkage relationship of this mark and disease-resistant gene, the mark that can directly select as the ZYMV-CH disease-resistant gene, the present invention has set up the anti-ZYMV-CH molecular marker assisted selection of watermelon technological system, for the clone of watermelon viral diseases breeding of new variety and disease-resistant gene establishes solid basis.
Sequence table
＜110〉Beijing City Agriculture and Forestry Institute
＜120〉the linkage molecule mark and the application of the anti-little zucchini yellow mosaic virus gene of watermelon
<160>3
<210>SEQ?ID?No：1
<211>644
<212>DNA
＜213〉watermelon (Citrullus lanatus (Thun) Manf.)
<400>1
tcccacgagt?ccagcagcaa?ggacataagg?caggaggctg?ctagcgaaac?tctatgcagt 60
ctcactcttg?ctctcactgg?acccattggt?taaaaccgac?gaaccgatcg?gaaccgaagc 120
aagttggttc?ggttcttgaa?ccaaaaccaa?tgtgcaccga?ttttcaacga?ttttacattt 180
attgtgaacc?gagaacttca?atttagttca?taagttcagc?ccaaaactgg?accagaccga 240
accgaaccgt?gcaaatccct?aatcttgatt?gataattaca?agtgatcgta?agaactttag 300
aagcaaacca?taggttctta?ataaaactct?taaacatatt?tttgttaaaa?tcatttgttg 360
tcatgtttta?aattatttta?taatataatt?tattcattca?gaataatttt?gaaattgtat 420
gaaaattgca?tctaaagtat?aaaggtaaac?gtttaatcaa?ttttaaatga?aaaaatatat 480
tttaaaatga?ttttaaaaaa?caataaaata?atgttaacca?attcaaaaaa?tactctcata 540
agtctttaat?ccaagtttgg?tgctcattac?tgaaatactt?caaccgtcga?tccagaaaat 600
actggggagg?taaataatta?atacttctaa?aagtactcgt?ggga 644
<210>SEQ?ID?No：2
<211>21
<212>DNA
＜213〉watermelon (Citrullus lanatus (Thunb) Manf.)
<400>2
tcccacgagt?ccagcagcaa?g 21
<210>SEQ?ID?No：3
<211>22
<212>DNA
＜213〉watermelon (Citrullus lanatus (Thunb) Manf.)
<400>3
tcccacgagt?acttttagaa?gt 22
Claims (4)
1. the RAPD mark AK13 of the anti-little zucchini yellow mosaic virus gene linkage of a watermelon
-644, its nucleotide sequence is the SEQ ID No:1 in the sequence table.
2. the SCAR mark SCAK13 of the anti-little zucchini yellow mosaic virus gene linkage of a watermelon
-644, its nucleotide sequence is SEQ ID No:2 and the SEQ ID No:3 in the sequence table.
3. the described RAPD mark of claim 1 AK13
-644Application in detecting transformation offspring plant.
4. the described SCAR mark of claim 1 SCAK13
-644Application in detecting transformation offspring plant.
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|---|---|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146691A (en) * | 2013-02-18 | 2013-06-12 | 北京市农林科学院 | SNP loci linked with blight resistant gene Fon-1 in watermelon, and markers thereof |
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| CN101440366B (en) * | 2007-11-23 | 2010-10-27 | 北京市农林科学院 | Specific fragment tightly linked to musk melon powdery mildew resistance gene Pm-2F and obtaining method thereof |
| CN104560961B (en) * | 2013-10-25 | 2018-03-30 | 北京市农林科学院 | The dominant disease-resistant gene ZYMV 1 of cucurbita pepo ZYMV linkage molecule mark and its application |
| CN104498485A (en) * | 2014-12-01 | 2015-04-08 | 北京市农林科学院 | Linked molecular marker for dominant resistance gene ZYMV-2 of cucurbita pepo L. ZYMV and application of linked molecular marker |
| WO2017134297A1 (en) * | 2016-02-05 | 2017-08-10 | Rijk Zwaan Zaadteelt En Zaadhandel B.V. | Qtls conferring resistance to potyviruses in watermelon |
| CN114381539B (en) * | 2020-10-21 | 2023-11-24 | 北京市农林科学院 | Molecular marker of zucchini ZYMV virus and method for identifying zucchini ZYMV virus resistance |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1428440A (en) * | 2002-09-27 | 2003-07-09 | 天津师范大学 | Molecular marker linked with wheat mildew-resistance gene |
| CN1594588A (en) * | 2003-09-11 | 2005-03-16 | 北京市农林科学院 | Feature sequence amplification mark closely interlocked with Chinese cabbage tangerine core character and obtaining method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1428440A (en) * | 2002-09-27 | 2003-07-09 | 天津师范大学 | Molecular marker linked with wheat mildew-resistance gene |
| CN1594588A (en) * | 2003-09-11 | 2005-03-16 | 北京市农林科学院 | Feature sequence amplification mark closely interlocked with Chinese cabbage tangerine core character and obtaining method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146691A (en) * | 2013-02-18 | 2013-06-12 | 北京市农林科学院 | SNP loci linked with blight resistant gene Fon-1 in watermelon, and markers thereof |
| CN103146691B (en) * | 2013-02-18 | 2014-04-02 | 北京市农林科学院 | SNP loci linked with blight resistant gene Fon-1 in watermelon, and markers thereof |
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