CN102653758A - Molecular marker of genes Mlm2033 for resisting powdery mildew of einkorn wheat and application of molecular marker - Google Patents
Molecular marker of genes Mlm2033 for resisting powdery mildew of einkorn wheat and application of molecular marker Download PDFInfo
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- CN102653758A CN102653758A CN2012100851584A CN201210085158A CN102653758A CN 102653758 A CN102653758 A CN 102653758A CN 2012100851584 A CN2012100851584 A CN 2012100851584A CN 201210085158 A CN201210085158 A CN 201210085158A CN 102653758 A CN102653758 A CN 102653758A
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Abstract
The invention belongs to the field of molecular genetics and relates to a molecular marker of genes Mlm2033 for resisting powdery mildew of wheat. The genetic distance between the molecular marker MAG5827 and the gene Mlm2033 is 0.4cM. Single plants containing the Mlm2033 are selected by virtue of the molecular marker MAG5827, and the genes Mlm2033 for resisting the powdery mildew of the einkorn wheat can be transplanted to the common wheat by virtue of a bridge parent method. By virtue of the molecular marker which is in close interlink with the Mlm2033, the plants containing the Mlm2033 can be screened conveniently and rapidly; and by virtue of the application of the molecular marker MAG5827, the time and labor for screening disease-resistant materials are saved greatly, and the prediction accuracy is enhanced.
Description
Technical field
The invention belongs to the molecular genetics field, relate to molecule marker and the application thereof of powdery mildew resistance gene in wheat Mlm2033.
Background technology
Wheat is global topmost food crop, also is second largest food crop of time paddy rice only in China.At present, the annual output of world wheat is about 6.2 hundred million tons, and 1/5 energy of necessary for human is provided, and the production of wheat is concerning human survival.Common wheat is allohexaploid species, contains A, B, three different dyeing bodies of D group, has with the wheat cdna group and has contained abundant genetic resources in homology or homeologous wild species and the original species.Utilizing allelic variation excellent in the wheat germplasm resource is the key factor that realizes wheat stable yields, raising the output.
Wheat powdery mildew is that the cereal powdery mildew wheat specialized form Blumeria graminis f.sp.tritici by Ascomycetes camber obligatory parasitism infects and a kind of gas of causing passes sick.The wheat powdery mildew that it causes is by a kind of serious wheat leaf diseases.After the infection process wheat, absorb plant nutrition, the photosynthetic capacity of wheat plant is descended even completely lose, finally cause mature spike number, grain number per spike and thousand seed weight to descend, can cause wheat yield loss 5%～34%, can reach 45% even total crop failure when serious.
In numerous wheat powdery mildew prophylactico-therapeutic measuress, cultivate and promote disease-resistant variety economical and effective and environmental protection the most.The mildew-resistance utilization mainly is the quality resistant gene in the current production.But; Because Powdery Mildew quality resistance has the microspecies specialization; Nontoxic gene of pathogenic bacteria and resistant gene carry out coevolution, and big area is promoted the use of single disease-resistant variety and is easy to cause producing new toxicity microspecies on producing, thereby makes entrained its resistance of disease-resistant gene forfeiture.From the wheat germplasm resource, excavate with to utilize new anti-white powder gene be the wheat genetic scholar with breeding man long-term objective and challenge.
Summary of the invention
The objective of the invention is above-mentioned deficiency, the molecule marker of powdery mildew resistance gene in wheat Mlm2033 is provided to prior art.
Another object of the present invention provides the molecule marker of this powdery mildew resistance gene in wheat Mlm2033 and uses.
The object of the invention can be realized through following technical scheme:
The molecule marker of powdery mildew resistance gene in wheat Mlm2033, described molecule marker MAG5827;
Described molecule marker MAG5827:
The left end primer sequence is SEQ ID NO.1,
The right-hand member primer sequence is SEQ ID NO.2,
Amplified production is 282bp, and this molecule marker is the codominance molecule marker from one grained wheat TA20337AL karyomit(e), and the genetic distance that utilizes Mapmaker Macintosh V 2.0 to record with the Mlm2033 gene is 0.4cM.
Application in the evaluation of described molecule marker mildew-resistance gene in the wheat germplasm resource and the transfer.
The molecule marking method of one grained wheat mildew-resistance gene Mlm2033, with the following any a pair of molecule marker primer PCR wheat cdna group DNA to be checked that increases, and detect amplified production:
Molecule marker MAG5827:
The left end primer sequence is SEQ ID NO.1,
The right-hand member primer sequence is SEQ ID NO.2;
If the amplified fragments with primer MAG5827 can amplify 282bp indicates that then there is mildew-resistance gene Mlm2033 in wheat to be checked.
A kind of method of screening powdery-mildew-resistance wheat; It is characterized in that with the described primer of claim 1 to one of the amplification wheat cdna group DNA to be checked; If the amplified fragments with primer MAG5827 can amplify 282bp indicates that then this wheat to be checked is the powdery-mildew-resistance wheat that has mildew-resistance Mlm2033.
The molecule marker of above-mentioned powdery mildew resistance gene in wheat Mlm2033 can obtain through following method:
(1) TA2033 and M389F
2The establishment in generation and phenotypic evaluation:
(1) einkorn wheat TA2033
Hybridize with einkorn wheat M389 (♀) and to obtain hybrid F
1, F
1Selfing produces F
2Colony.
(2) F
2Coil in the cave for colony's single-strain planting, place the greenhouse, daytimes 22, degree was 14 hours, and evenings 18, degree was 10 hours.One leaf, one heart stage inoculation Powdery Mildew is inoculated and is carried out the resistance investigation after 7 days.Qualification result is seen table 1.
(3) F
2For after the individual plant investigation with the disease plant land for growing field crops of transplanting seedlings, selfing gained seed is F
3Family.Each F
3Select 18 to carry out the filial generation test in the family.
(4) pass through F
2Carry out genetic analysis, infer the number of the disease-resistant gene that carries in the disease-resistant germplasm.
(2) polymorphic molecular marker screening
(5) with the disease-resistant F of 6 strains
2The blade balanced mix Cheng Kangchi of individual plant, the susceptible F of 6 strains
2The blade balanced mix of individual plant becomes the sense pond.Extract the DNA in disease-resistant parent TA2033, susceptible parent M389, anti-pond and sense pond with SDS method (Ma and Sorrells, 1995), use simple repeated sequence mark SSR and BSA (Bulk segregant analysis) bonded method and carry out the polymorphum screening.
(6) at first utilize 37 pairs of SSR primers that the polymorphum screening is carried out in TA2033, M389, anti-pond and sense pond.
The PCR reaction volume is 25 microlitres, 10 * buffer, 2.5 microlitres wherein, and 25mM MgCl21.5 microlitre, 2.5mM dNTPs 2 microlitres, Taq enzyme (5 units/microlitre) 0.2 microlitre, each 0.5 microlitre of 10 μ M primers (F/R), template DNA 20 nanograms add water to 25 microlitres;
After the SSR reaction system is 94 ℃ of preparatory sex change 3min of DNA, 94 ℃ of sex change 1min, 50-65 ℃ (look primer and decide) annealing 1min, 72 ℃ are extended 1min, circulates 35 times, last 72 ℃ of extension 10min;
In the enterprising performing PCR amplification of pcr amplification appearance, amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel, on ultraviolet transilluminator, takes a picture the record result then:
Be chosen between the parent and F
2For amplifying the identical primer that multiformity is arranged between the R of colony, S pond, with 1 pair of the chain candidate molecular marker of Mlm2033;
(3) molecule marker obtains
(7) exploitation of molecule marker; Above-mentioned mark ZMG132 is located in wheat 7AL; Because the 7th group of wheat with false bromegrass first group has macroscopical collinearity; Utilize the corresponding section search wheat EST of false bromegrass and develop mark, the mark that wherein transforms according to EST CJ579028, the PCR product of MAG5827 has the polymorphic of unanimity in resisting sense parent and anti-sense pond.
(8) according to chain exchange rule; Utilize the genotype data and the resistance of pure and mild susceptible each individual plant of family of amplification acquisition to identify that the resistance data that obtains makes up the linkage map of 1 pair of molecule marker and Mlm2033 gene; Used software is Mapmaker Macintosh V 2.0; Obtained the closely linked molecule marker MAG5827 with Mlm2033, the genetic distance that utilizes Mapmaker Macintosh V 2.0 to record with the Mlm2033 gene is respectively 0.4cM;
(4) acquisition of anti-spectrum
(9) utilize 15 whole Powdery Mildew physiological strains of this laboratory to inoculate disease-resistant material TA2033 respectively, write down the disease-resistant performance of disease-resistant material TA2033 different physiological strains.
(5) to the transfer of common wheat
(10) utilize the molecule marker that obtains,, import to the common wheat kind (abbreviating bridge parent method as) that common wheat obtains to carry Mlm2033 again through earlier disease-resistant gene Mlm2033 being imported in the tetraploid.
Beneficial effect:
The present invention has found a mildew-resistance gene Mlm2033 in one grained wheat, the disease-resistant performance that Mlm2033 is excellent makes it in the mildew-resistance breeding, significant values arranged.Obtained molecule marker MAG5827 closely linked with it.The molecule marker that utilize to obtain is transferred to commercial variety through bridge parent method with Mlm2033 and is raised in the wheat 158, and what successfully obtained to carry Mlm2033 disease-resistantly raises wheat 158.
1, in one grained wheat, identified a mildew-resistance gene Mlm2033.
2, the anti-spectrum of Mlm2033 is measured, the result shows whole 15 the Powdery Mildew physiological strains in anti-this laboratory of Mlm2033, has excellent resistance, is disease-resistant gene good in the Powdery Mildew breeding.
3, obtained closely linked molecule marker MAG5827 with powdery mildew resistance gene in wheat Mlm2033.The genetic distance of MAG5827 and gene is 0.4cM, also is the codominance molecule marker simultaneously, can help this gene in commercial variety, to shift and with other disease-resistant gene polymerization.
4, identify conveniently.That molecule marker has is easy to detect, stable amplification, advantage such as easy.Detect powdery mildew resistance gene in wheat Mlm2033 with mark MAG5827, the existence that can confirm Mlm2033 whether and existence, and prediction powder mildew resistance, and then accelerate to shift the utilization of Mlm2033.
5, the present invention proposes the method for utilizing mildew-resistance gene in the one grained wheat germ plasm resource: utilize molecular marker screening, Mlm2033 is imported in the hexaploid wheat through bridge parent method; That utilizes that this method successfully obtained mildew-resistance raises wheat 158.
Description of drawings
Fig. 1 molecule marker amplification banding pattern
The MAG5827 banding pattern that increases wherein 1,2,3,4 is respectively disease-resistant variety TA2033, susceptible variety M389, anti-pond and sense pond, and M:pUC19/MspI, arrow indication are 282bp specific amplified band;
The genetic linkage map of Fig. 2 MAG5827 and powdery mildew resistance gene in wheat Mlm2033, the left side are the map distance between mark, and the arrow direction is the telomere direction.
Embodiment
(1) TA2033 and M389F
2The establishment in generation and phenotypic evaluation:
(1) TA2033 and M389F
2The establishment in generation and phenotypic evaluation:
(1) einkorn wheat TA2033
Hybridize with einkorn wheat M389 (♀) and to obtain hybrid F
1, F
1Selfing produces F
2Colony.
(2) F
2Coil in the cave for colony's single-strain planting, place the greenhouse, daytimes 22, degree was 14 hours, and evenings 18, degree was 10 hours.One leaf, one heart stage inoculation Powdery Mildew is inoculated and is carried out the resistance investigation after 7 days.Qualification result is seen table 1.
Table 1Bgt19 inoculation back M389 * TA2033F
2Colony
R, S represent disease-resistant individual plant, susceptible individual plant respectively.χ
2 0.05，1＝3.84，χ
2 0.05，2＝5.99
(3) F
2For after the individual plant investigation with the disease plant land for growing field crops of transplanting seedlings, selfing gained seed is F
3Family.Each F
3Select 18 to carry out the filial generation test in the family.Verify whether susceptible individual plant is pure and mild susceptible family.
(4) pass through F
2Carry out genetic analysis, find to carry among the disease-resistant germplasm TA2033 dominance disease-resistant gene.
(2) polymorphic molecular marker screening
(1) with the disease-resistant F of 6 strains
2The blade balanced mix Cheng Kangchi of individual plant, the susceptible F of 6 strains
2The blade balanced mix of individual plant becomes the sense pond.Extract the DNA in disease-resistant parent TA2033, susceptible parent M389, anti-pond and sense pond with SDS method (Ma and Sorrells, 1995), use simple repeated sequence mark SSR and BSA (Bulk segregant analysis) bonded method and carry out the polymorphum screening.
(2) at first utilize 37 pairs of SSR primers (barc:Song QJ, Shi JR, Singh S; Fickus EW; Costa JM, Lewis J, Gill BS; Ward R, Cregan PB (2005) Development and mapping of microsatellite (SSR) markers in wheat.Theor Appl Genet 110:550-560; Gwm:
MS; Korzun V; Wendehake K, Plaschke J, Tixer MH; Leroy P, Ganal MW (1998) A microsatellite map of wheat.Genetics 149:2007-2023; Wmc:Wheat Microsatellite Consortium (http://wheat.pw.usda.gov); Cfa, gpw:Sourdille ' s lab (http://wheat.pw.usda.gov); Cfd:Guyomarc ' h H; Sourdille P; Charmet G; Edwards KJ, Bernard M (2002) Characterization of polymorphic microsatellite markers from Aegilops tauschii and transferability to the D-genome of bread wheat.Theor Appl Genet 104:1164-1172; Gdm:Pestsova E; Ganal MW,
MS (2000) Isolation and mapping of microsatellite markers speciWc for the D genome of bread wheat.Genome 43:689-697) the polymorphum screening is carried out in TA2033, M389, anti-pond and sense pond.
The PCR reaction volume is 25 microlitres, 10 * buffer, 2.5 microlitres wherein, and 25mM MgCl21.5 microlitre, 2.5mM dNTPs 2 microlitres, Taq enzyme (5 units/microlitre) 0.2 microlitre, each 0.5 microlitre of 10 μ M primers (F/R), template DNA 20 nanograms add water to 25 microlitres;
After the SSR reaction system is 94 ℃ of preparatory sex change 3min of DNA, 94 ℃ of sex change 1min, 50-65 ℃ (look primer and decide) annealing 1min, 72 ℃ are extended exhibition 1min, circulates 35 times, last 72 ℃ of extension 10min;
Wherein SSR mark ZMG132 anti-sense parent with anti-feel detected between the pond consistent polymorphic.
(3) exploitation of molecule marker
(3) exploitation of molecule marker; Positioning result (Singr ü n C according to Singr ü n; Hsam SLK, Zeller FJ, Wenzel G; Mohler V (2004) Localization of a novel recessive powdery mildew resistance gene from common wheat line RD30in the terminal region of chromosome 7AL.Theor Appl Genet 109:210-214), ZMG132 is located in 7AL.Because the 7th group of wheat with false bromegrass first group has macroscopical collinearity; Utilize the corresponding section search wheat EST of false bromegrass and develop mark; The mark that wherein transforms according to EST CJ579028, the PCR product of MAG5827 has consistent polymorphic (Fig. 1) in anti-sense parent and anti-sense pond.
(4) according to chain exchange rule; Utilize the genotype data and the resistance of pure and mild susceptible each individual plant of family of amplification acquisition to identify that the resistance data that obtains makes up the linkage map of 1 pair of molecule marker and Mlm2033 gene; Used software is Mapmaker Macintosh V 2.0; Obtained the closely linked molecule marker MAG5827 with Mlm2033, the genetic distance that utilizes Mapmaker Macintosh V 2.0 to record with the Mlm2033 gene is respectively 0.4cM (Fig. 2);
(4) acquisition of anti-spectrum
(5) utilize 15 whole Powdery Mildew physiological strains of this laboratory to inoculate disease-resistant material TA2033 respectively, the result finds that TA2033 is disease-resistant to 15 physiological strain performances, is good disease-resistant gene.
(5) to the transfer of common wheat
(6) utilize the molecule marker that obtains,, import to the common wheat kind (abbreviating bridge parent method as) that common wheat obtains to carry Mlm2033 again through earlier disease-resistant gene Mlm2033 being imported in the tetraploid.Utilize above-mentioned strategy, what successfully obtained to carry Mlm2033 disease-resistantly raises wheat 158.
Contain abundant genetic resources in the wheat germplasm resource, utilize these genetic resourceses to have vital role for wheat.The present invention is through hybridizing disease-resistant germplasm TA2033 and susceptible variety; Confirm to carry in the disease-resistant material disease-resistant gene of a dominance through genetic analysis; Pass through molecular marker screening; And the structure segregating population, find that disease-resistant gene is positioned on the wheat 7AL karyomit(e), utilize molecular marker method to make up the genetic linkage map of disease-resistant gene Mlm2033 simultaneously through making up segregating population.Anti-spectrum test finds that Mlm2033 has excellent powder mildew resistance, is the good anti-source of wheat anti-powdery mildew breeding.Utilize molecule marker MAG5827, successfully mildew-resistance gene Mlm2033 is imported to commercial variety through bridge parent method and raise in the wheat 158.In the method for traditional breeding method, owing to do not know the heredity and the chain molecule marker thereof of disease-resistant material, per generation shifts all needs resistance to identify; Waste time and energy, therefore, the known and closely linked molecule marker of Mlm2033; The plant that can simple and efficient screening contains Mlm2033; The application of molecule marker MAG5827, the screening time and the labour that can save disease-resistant material greatly, and strengthen prediction accuracy.
Claims (4)
1. the molecule marker of one grained wheat mildew-resistance gene Mlm2033 is characterized in that described molecule marker MAG5827;
Described molecule marker MAG5827:
The left end primer sequence is SEQ ID NO.1,
The right-hand member primer sequence is SEQ ID NO.2,
Amplified production is 282bp, and this molecule marker is the codominance molecule marker from one grained wheat TA20337AL karyomit(e), and the genetic distance that utilizes Mapmaker Macintosh V 2.0 to record with the Mlm2033 gene is 0.4cM.
2. the application in the evaluation of the described molecule marker of claim 1 mildew-resistance gene in wheat wild germ plasm resource and the transfer.
3. the molecule marking method of one grained wheat mildew-resistance gene Mlm2033 is characterized in that: with the following a pair of molecule marker primer PCR wheat cdna group DNA to be checked that increases, and detect amplified production:
Molecule marker MAG5827:
The left end primer sequence is SEQ ID NO.1,
The right-hand member primer sequence is SEQ ID NO.2;
If the amplified fragments with primer MAG5827 can amplify 282bp indicates that then there is mildew-resistance gene Mlm2033 in wheat to be checked.
4. method of screening powdery-mildew-resistance wheat; It is characterized in that with the described primer of claim 1 to one of the amplification wheat cdna group DNA to be checked; If the amplified fragments with primer MAG5827 can amplify 282bp indicates that then this wheat to be checked is the powdery-mildew-resistance wheat that has mildew-resistance Mlm2033.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114262746A (en) * | 2021-12-30 | 2022-04-01 | 四川农业大学 | Diploid wild one-grain wheat 4AbChromosome molecular marker primer pair and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769448A (en) * | 2004-05-08 | 2006-05-10 | 南京农业大学 | Wheat fertility recovery gene molecular mark and its obtaining method |
| CN101736076A (en) * | 2008-11-19 | 2010-06-16 | 朱玉丽 | Research progress in molecular marker positioning of wheat powdery mildew resistance gene |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769448A (en) * | 2004-05-08 | 2006-05-10 | 南京农业大学 | Wheat fertility recovery gene molecular mark and its obtaining method |
| CN101736076A (en) * | 2008-11-19 | 2010-06-16 | 朱玉丽 | Research progress in molecular marker positioning of wheat powdery mildew resistance gene |
Non-Patent Citations (1)
| Title |
|---|
| YAO ET AL: "Genetic mapping of two powdery mildew resistance genes in einkorn (Triticum monococcum L.) accessions", 《THEOR APPL GENET》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114262746A (en) * | 2021-12-30 | 2022-04-01 | 四川农业大学 | Diploid wild one-grain wheat 4AbChromosome molecular marker primer pair and application thereof |
| CN114262746B (en) * | 2021-12-30 | 2023-05-23 | 四川农业大学 | Diploid wild one-grain wheat 4A b Chromosome molecular marker primer pair and application thereof |
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Application publication date: 20120905 |