CN103088016B - Specific marker linked with stripe rust resistance gene of Yilong Tuo wheat, and application of specific marker - Google Patents
Specific marker linked with stripe rust resistance gene of Yilong Tuo wheat, and application of specific marker Download PDFInfo
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- CN103088016B CN103088016B CN201210479255.1A CN201210479255A CN103088016B CN 103088016 B CN103088016 B CN 103088016B CN 201210479255 A CN201210479255 A CN 201210479255A CN 103088016 B CN103088016 B CN 103088016B
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Abstract
The invention relates to the agricultural biotechnology engineering and the field of disease-resistant genetic breeding of wheat, and in particular relates to a specific marker linked with a stripe rust resistance gene of Yilong Tuo wheat, a preparation method and application of the specific marker. The preparation method substantially comprises the following steps: adopting Taichang 29/F2 group of the Yilong Tuo wheat as the mapping population according to the linkage separation rules; and acquiring an SSR (Simple Sequence Repeat) marker Xgpw 7342-350bp linked with the stripe rust resistance gene of the wheat according to the identification result of the stripe rust resistance in field and the linkage analysis of SSR marker data (forward primer: 5'-GCGGTGGCAACAACCTAC-3'), and reverse primer: 5'-GTGCATAAAGAGGGCAGAGC-3'). The marker can be used for assisting the selective breeding of the stripe rust resistance of the wheat, the shortcomings that in the conventional breeding, the stripe rust resistance identification of the wheat is easily influenced by the environment, the stability and the repeatability are poor, the workload is large, the time and manpower are wasted, and the breeding period is long, can be overcome, so that the selection method is simplified, the breeding efficiency is improved, and the breeding progress of the wheat stripe rust resistance variety is improved.
Description
Technical field
The present invention relates to agricultural biotechnology engineering and disease-resistant wheat genetic breeding field, relate to particularly Yilong stick together the mutually chain specific molecular marker of wheat Stripe Rust Resistance Gene and application thereof.
background technology
By stripe shape handle rest fungus (Puccinia striiformis Westend.f.sp.tritici, Pst) stripe rust of wheat causing is one of the heaviest typical air infection diseases of wheat hazard rating, before wheat is as the mankind's staple food crop, just exist, all there is generation in various degree in more than 60, the each continent country in the whole world except the Antarctica.China is maximum and relatively independent Epidemics of Wheat Strip Rust district in the world, and stripe rust of wheat is to threaten one of most important wheat diseases in Winter Wheat Area, ground and spring wheat district, northwest such as NORTHWEST CHINA, southwest, Huang-Huai-Hai always.In recent years due in new toxicity microspecies bar 32 and bar in 33 appearance and the popular stripe rust trend of causing disaster of more having aggravated, China's stripe rust of wheat is since two thousand always in the state of being very popular.Therefore,, in the urgent need to excavating new Stripe Rust Resistance Gene efficient transformation in existing main breed, strengthen the resistance of Wheat cultivar.
The excavation of disease-resistant new gene and utilization are the bases of breeding work, are also the fundamental ways that solves this difficult problem of resistant lose.Although traditional chemical control can alleviate the harm of stripe rust to a certain extent, a large amount of agricultural chemicals that use have not only increased production cost, also can produce very large threat to ecotope and human health.Therefore, breeding resistant variety is to prevent and treat stripe rust economy, safe and effective method.In recent years, China's stripe rust show that high, the scope of outburst frequency is wide, harm increases the weight of year by year, disease time morning etc. feature.Therefore, from wheat wild sibling species genus, rare species and local wheat, excavate new Stripe Rust Resistance Gene, and effectively utilize, the stripe rust resisting kind that Breeding and popularization is new, has become vital task in the sick breeding of China's wheat stripe rust resisting.
Along with molecular biological development, the application of DNA molecular marker technology in Molecular Selection assistant breeding field is more and more extensive.Molecular marking technique can be evaluated crop germplasm resource and identify on gene level, simultaneously for location, clone and the polymerization of disease-resistant gene provide foundation.Have speed just because of molecular marking technique compared with conventional tag means fast, marker site is many, and codominance is difficult for feature affected by environment, so molecular marking technique has obtained developing rapidly in recent years.At present, mainly utilize several DNA molecular marker technology such as RAPD, SSR, AFLP, RGAP, RFLP in anti-rust major gene of the stripe rust resisting major gene of 50 definite designations and tentatively titled, to find and the molecule marker of 48 anti-rust gene linkage.In all labeling techniques, Litt equal 1989 exploitation SSR mark have easy, quick, stability is high and allelic diversity advantages of higher, aspect the researchs such as genetic diversity, germ plasm resource evaluation, genetic mapping, the assignment of genes gene mapping and molecular marker breeding, be widely used.Utilize at present the multiple stripe rust resistance genes of the successful screening and identification of this technology, such as Yr2, Yr5, Yr24, Yr26, Yr30, Yr33, Yr34, Yr36, YrZH84, YrSph etc.But the stripe rust resistance genes linked marker obtaining based on segregating population single or that minority combines, versatility is often subject to the restriction of kind background; And most marks are far away apart from target gene, cause these to be marked at application in breeding few.
From the Wheat landraces Yilong of the Sichuan Province China wheat that sticks together, to identify and show through multiple years field stripe rust resistance, this kind of confrontation China popular physiological strain of current Stripe Rust has good resistance.Genetic analysis shows, the Yilong wheat that sticks together contains a pair of 31,32 and 33 dominant genes that mix physiological strains that can resist in China's bar.Therefore, screen this resistant gene linkage molecule mark, be expected to directly apply to the seed selection of disease-resistant wheat kind, to improve efficiency of selection and to accelerate wheat breeding for disease resistance process.
Summary of the invention
In order to address the above problem, present inventor proposes and has completed the present invention.
One of object of the present invention is to provide the molecule marker mutually chain with wheat stripe rust resisting ospc gene.
Two of object of the present invention is to provide the method that obtains the mutually chain molecule marker of above-mentioned and wheat stripe rust resisting ospc gene.
Three of object of the present invention is to provide the application of the mutually chain molecule marker of above-mentioned and wheat stripe rust resisting ospc gene.
The present invention is directed to above-mentioned research background, with stick together wheat/director 29 F of Yilong
2colony is mapping population, by the linkage analysis between Disease Resistance Identification result and SSR flag data, has obtained the mark Xgpw7342 chain with wheat stripe rust resistance genes
-350bp, this mark can be applicable to the assisted selection of stripe rust of wheat disease-resistant variety.
According to the molecule marker Xgpw7342 mutually chain with wheat stripe rust resisting ospc gene of the present invention
-350bp, by using SSR primer: forward primer: 5 '-GCGGTGGCAACAACCTAC-3 '; Reverse primer: 5 '-GTGCATAAAGAGGGCAGAGC-3 ' increases and obtains, and the size of described molecule marker is 350bp, and it is positioned on wheat 7B galianconism, and genetic distance is 1.8cM.
The present invention also provides the application of above-mentioned molecule marker aspect evaluation wheat stripe rust resisting disease.Mode according to a particular embodiment of the invention, wheat and the susceptible variety director 29 height generation (F as parent's cross combination sticks together take disease-resistant variety Yilong
3) single wheat strain (L1-L4) object, by detecting its specific molecular marker Xgpw7342
-350bpbanding pattern data, in conjunction with the disease-resistant evaluation phenotypic data in field, can predict the resistance of this strain to stripe rust of wheat.
Comprise and use SSR primer according to the method for evaluation wheat stripe rust resisting disease of the present invention: forward primer: 5 '-GCGGTGGCAACAACCTAC-3 '; The step that reverse primer: 5 '-GTGCATAAAGAGGGCAGAGC-3 ' increases.
The method of the mutually chain molecule marker of acquisition according to the present invention and wheat stripe rust resisting ospc gene, said method comprising the steps of:
(1) take susceptible wheat breed director 29 as maternal, stick together wheat as male parent take Wheat landraces Yilong, Sichuan Province, build F
2and F
2:3genetic mapping colony, the F of structure
2colony is Stripe Rust Resistance Gene genetic mapping colony;
(2) extract wheat parent seedling and F thereof by CTAB method
2the individual plant DNA of colony;
(3) adopt SSR molecular marking technique to carry out the screening of wheat stripe rust resistance molecule marker, wherein, use SSR primer Xgpw7342
-350bp(forward primer: 5 '-GCGGTGGCAACAACCTAC-3 '; Reverse primer: 5 '-GTGCATAAAGAGGGCAGAGC-3 ') the disease-resistant parent that increases, Susceptible parent, disease-resistant gene pond, susceptible gene pool and their F
2genetic mapping colony, through linkage analysis, finds that this mark is mutually chain with wheat stripe rust resistance genes.
The method of the mutually chain molecule marker of acquisition according to the present invention and wheat stripe rust resisting ospc gene, wherein, adopts SSR molecule marker to screen with the method for the mutually chain molecule marker of wheat stripe rust resisting ospc gene to be:
(1) use SSR primer at disease-resistant parent, Susceptible parent, disease-resistant gene pond, susceptible gene pool and their F
2in genetic mapping colony, carry out DNA polymorphism analysis;
(2) to the Yilong F that wheat and Susceptible parent director 29 hybridization obtain that sticks together
2individual plant resistance is carried out field test, investigation F
2the disease resistance of individual plant, extracts F
2the individual plant DNA of colony, carry out pcr amplification by reaction system, primer and the program same with step (1), statistics resists, feels the frequency of occurrences and the switch type of strain indicia band and calculate crossover value, with the genetic distance between MAPMARKER/EXP 3.0b calculating mark and Stripe Rust Resistance Gene.
According to the specific embodiment of the present invention, the SSR mark Xgpw7342 chain with wheat stripe rust resistance genes provided by the present invention
-350bp, obtain by the following method:
1), take susceptible wheat breed director 29 as maternal, the disease-resistant variety Yilong wheat that sticks together is male parent, builds F
2genetic mapping colony, to the F obtaining
2individual plant resistance is carried out Resistance Identification.
2) extract parent, disease-resistant gene pond, susceptible gene pool and F with CTAB method
2the individual plant DNA of colony.
3) according to the upper SSR primer sequence synthetic primer of announcing of GrainGenes (http://www.wheat.pw.usda.gov), after PCR, adopt 6% native polyacrylamide gel electrophoresis analysis, obtain molecular marker data;
4) utilize in bar 31,32 and bar in 33 stripe rust physiology mix microspecies Yilong sticked together to wheat with Susceptible parent director 29 is hybridized, the F of selfing acquisition
2individual plant resistance is carried out field test, and stripe rust is seeded in two leaves stem and leaf of Wheat wholeheartedly, by Stripe Rust artificial inoculation to disease identify garden to bring out row upper, inoculate by natural propagation, in the time bringing out row and fall ill completely, measure F
2the disease resistance of individual plant, investigation rank be divided into high resistance (HR), anti-(R), in anti-(MR), sense (S) and highly feel (HS) Pyatyi.Wherein, measure sick level for high resistance (HR), anti-(R) or in anti-individual plant be defined as disease-resistantly, be defined as susceptible and measure sick level for the individual plant of middle sense or high sense.
5) utilize in bar 31,32 and bar in 33 stripe rust physiology mix microspecies Yilong sticked together to wheat with Susceptible parent director 29 is hybridized, the F of selfing acquisition
2:3strain resistance is carried out field test, and stripe rust is seeded in the stem and leaf of Wheat in tillering phase, by Stripe Rust artificial inoculation to disease identify garden to bring out row upper, inoculate by natural propagation, in the time bringing out row and fall ill completely, measure F
2:3the each individual plant disease resistance of strain, investigation rank be divided into high resistance (HR), anti-(R), in anti-(MR), sense (S) and highly feel (HS) Pyatyi.Wherein, measure sick level for high resistance (HR), anti-(R) or in the individual plant of anti-(MR) be defined as disease-resistantly, be defined as susceptible and measure sick level for the individual plant of sense (S) or high sense (HS).
6) in conjunction with 4) and 5) result, determine F
2individual plant disease resistance and carry disease-resistant gene heterozygosity.
7) extract wheat parent seedling and F thereof by CTAB method
2the individual plant DNA of colony; And according to 6) result, screen respectively 15 disease-resistant genes F that isozygotys
2individual plant and 15 susceptible gene pure F
2individual plant equivalent DNA mixes, and builds disease-resistant gene pond and susceptible gene pool.
8) adopt SSR molecule marking method to carry out the screening of wheat stripe rust resistance genes molecule marker, its concrete method that obtains the molecule marker mutually chain with wheat stripe rust resisting ospc gene is:
(1) SSR primer is at disease-resistant parent, Susceptible parent, disease-resistant gene pond, susceptible gene pool and their F thereof
2dNA polymorphism analysis between individual plant:
Screen by SSR labeling technique, according to the upper SSR primer sequence synthetic primer of announcing of GrainGenes (http://www.wheat.pw.usda.gov), in the enterprising performing PCR amplification of ABI-9700 PCR instrument, pcr amplification reaction system: 50ng/ μ l wheat cdna group DNA 1.0 μ l, 10 × PCR Buffer (with Mg
2+) 2.0 μ l, 2.5mM dNTP1.6 μ l, 100 μ M primer 1.0 μ l, 2.5U/ μ l Taq archaeal dna polymerase 0.3 μ l, ddH
2o 13.1 μ l, total system 20 μ l.Amplified reaction program: 94 ℃ of sex change 5 minutes, 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ are extended 1 minute, 40 circulations, last 72 ℃ are extended 10 minutes; Product detects: 6% native polyacrylamide gel electrophoresis analysis, click damping fluid is 1 × TBE, 80 watts of firm powers.Gel-colored employing cma staining; Take a picture and record experimental result on white light lamp box.
(2) linkage analysis of molecule marker:
According to linkage inheritance law, according to MapmakerEXP3.0b software requirement, disease-resistant parent Yilong is sticked together to the wheat banding pattern assignment identical with disease-resistant gene pond for " A ", the banding pattern assignment identical with susceptible gene pool with Susceptible parent director 29 is " B ", the assignment that simultaneously amplifies disease-resistant parent, disease-resistant gene pond and Susceptible parent, two kinds of banding patterns of susceptible gene pool is " H ", banding pattern assignment unclear or disappearance is "-", in conjunction with 4) field resistance qualification result, calculate genetic distance with Kosambi function.
9) filter out a SSR molecule marker Xgpw7342
-350bp(forward primer: 5 '-GCGGTGGCAACAACCTAC-3 '; Reverse primer: 5 '-GTGCATAAAGAGGGCAGAGC-3 '), the disease-resistant parent that increases, Susceptible parent, disease-resistant gene pond, susceptible gene pool and their F
2genetic mapping colony, finds to have a mark at 350bp place, through linkage analysis, finds that this mark and wheat stripe rust resistance genes are chain.
SR molecule marker Xgpw7342
-350bpin the application of resistant gene assisted Selection, its concrete method is:
(1) utilize in bar 31,32 and bar in 33 stripe rust physiology mix microspecies enantiopathy kind Yilongs and stick together the height of the cross combination that wheat and susceptible variety river agriculture 16 be parent for (F
5) 4 wheat strains (L1-L4), 4 (comprising: the Palestine and China wheat that sticks together from the disease-resistant variety Yilong different Sichuan Wheat landraces of wheat that sticks together, the white wheat of Kaijiang, Yibin pig wheat, wheat is reprinted by Hejiang), 10 (comprising: interior wheat No. 8 with the stick together Bred Wheat Varieties of wheat affinity-less relation of disease-resistant variety Yilong, interior wheat No. 9, river wheat 47, section becomes wheat No. 2, river wheat 42, river agriculture 16, No. 5, river spoke, No. 3, continuous agriculture, Mianyang 26, river wheat 22) resistance carries out field test, stripe rust is seeded in two leaves stem and leaf of Wheat wholeheartedly, Stripe Rust artificial inoculation to disease is identified to bringing out on row of garden, inoculate by natural propagation, when bringing out row while falling ill completely, measure disease resistance, investigation rank is divided into high resistance (HR), anti-(R), in anti-(MR), sense (S) and high sense (HS) Pyatyi.Wherein, measure sick level for high resistance (HR), anti-(R) or in anti-material decision be disease-resistant, and measure sick level for the material decision of middle sense or high sense be susceptible.
(2) extract (1) by SDS method and mention wheat lines DNA;
(3) molecule marker Xgpw7342
-350bpin (2) DNA polymorphism analysis:
SSR molecule marker Xgpw7342
-350bpin the enterprising performing PCR amplification of ABI-9700PCR instrument, pcr amplification reaction system: 50ng/ μ l wheat cdna group DNA 1.0 μ l, 10 × PCR Buffer (with Mg
2+) 2.0 μ l, 2.5mM dNTP1.6 μ l, 100 μ M primer 1.0 μ l, 2.5U/ μ l Taq archaeal dna polymerase 0.3 μ l, ddH
2o 13.1 μ l, total system 20 μ l.Amplified reaction program: 94 ℃ of sex change 5 minutes, 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ are extended 1 minute, 40 circulations, last 72 ℃ are extended 10 minutes; Product detects: 6% native polyacrylamide gel electrophoresis analysis, click damping fluid is 1 × TBE, 80 watts of firm powers.Gel-colored employing cma staining; Take a picture and record experimental result on white light lamp box.
(4) utilize molecule marker Xgpw7342
-350bpthe evaluation of antagonism gene:
Height generation (the F of the cross combination that wheat and the agriculture of susceptible variety river 16 that stick together in disease-resistant parent, disease-resistant gene pond and disease-resistant variety Yilong is parent
5) 4 wheat strain (L1-L4) storerooms have amplified the polymorphism band line of a 350bp size, and at Susceptible parent, susceptible gene pool, 4 (comprising: the Palestine and China wheat that sticks together from the disease-resistant variety Yilong different Sichuan Wheat landraces of wheat that sticks together, the white wheat of Kaijiang, Yibin pig wheat, wheat is reprinted by Hejiang) and 10 (comprising: interior wheat No. 8 with the stick together Bred Wheat Varieties of wheat affinity-less relation of disease-resistant variety Yilong, interior wheat No. 9, river wheat 47, section becomes wheat No. 2, river wheat 42, river agriculture 16, No. 5, river spoke, No. 3, continuous agriculture, Mianyang 26, river wheat 22) in all can not amplify the special banding pattern of this 350bp.Therefore, in conjunction with field phenotype Resistance Identification result, thereby confirm this resistant gene specific molecular marker Xgpw7342
-350bpcan identify the existence of this resistant gene in wheat lines.
Enthusiasm effect of the present invention and application are as follows:
1, the PCR system that the present invention uses, does not need special PCR instrument and special reaction reagent, and the product that common commercial company produces all can reach requirement.
2, the present invention obtain with the closely linked molecule marker of stripe rust resistance genes, it is the new mark obtaining in the Wheat landraces Yilong, Sichuan to China's stripe rust high resistance sticks together wheat and genetic mapping colony thereof, this mark and resistant gene genetic distance are near, pcr amplification is reproducible, stability is strong, can be for stripe rust of wheat cultivar identification and disease-resistant wheat offspring's molecule assisted Selection, and then accelerate the breeding process of disease-resistant variety.
3, have laid a good foundation for cloning wheat stripe rust resisting ospc gene, gene sequencing and turning the research of Stripe Rust Resistance Gene wheat.
Accompanying drawing explanation
Fig. 1 shows SSR mark Xgpw7342
-350bpat disease-resistant parent, Susceptible parent, disease-resistant gene pond, susceptible gene pool and F thereof
2amplification in colony.(RP is the disease-resistant parent Yilong wheat that sticks together; SP is Susceptible parent director 29; RB is disease-resistant gene pond; SB is susceptible gene pool; R is disease-resistant phenotype; H is heterozygous; S is susceptible phenotype; "+" is for having specific mark; "-" is without specific mark band; Arrow represents SSR mark Xgpw7342
-350bp).
Fig. 2 shows SSR mark Xgpw7342
-350bpheight generation (the F of the cross combination that wheat and the agriculture of susceptible variety river 16 that stick together in disease-resistant parent, Susceptible parent, disease-resistant gene pond, susceptible gene pool and disease-resistant variety Yilong is parent
5) 4 wheat strains (L1-L4), 4 and disease-resistant variety Yilong the stick together pcr amplification of Bred Wheat Varieties (comprising: interior wheat No. 8, interior wheat No. 9, river wheat 47, section become wheat No. 2, river wheat 42, river agriculture 16, No. 5, river spoke, No. 3, continuous agriculture, Mianyang 26, river wheat 22) of wheat affinity-less relation of the different Sichuan Wheat landraces of wheat (comprising: stick together wheat, the white wheat of Kaijiang, Yibin pig wheat, Hejiang of Palestine and China reprinted wheat), 10 and disease-resistant variety Yilong that sticks together.(RP is the disease-resistant parent Yilong wheat that sticks together; SP is Susceptible parent director 29; RB is disease-resistant gene pond; SB is susceptible gene pool; Solid black arrow represents SSR mark Xgpw7342
-350bp).
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but limitation of the present invention not, all any this areas of doing according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Embodiment 1: hold in the palm the chain SSR molecule marker Xgpw7342 of wheat yellow rust resistant gene that sticks together with Wheat landraces Yilong, Sichuan
-350bpacquisition
1, for examination material
Vegetable material: take susceptible variety director 29 as maternal, local wheat Yilong, the disease-resistant variety Sichuan wheat that sticks together is male parent, builds F
2genetic mapping colony.Positive season in 2010 is by F
2152 single-strain plantings of colony are identified garden in test base, Wenjiang, school district, Sichuan Agricultural University Chengdu disease, for Resistance Identification.Get the partial blade of each individual plant seedling stage, for ssr analysis.When ripe, results F
2:3the seed of family, dries preservation, and 2011 for Resistance Identification.
Stripe Rust mixes physiological strain: for F
2colony and F
2:3the Stripe Rust of family Resistance Identification mixes physiological strain and is made up of 31,32 and 33 3 microspecies balanced mix in bar.
2, Resistance Identification
Field test design: 2010 and positive season in 2011, test base, Wenjiang, school district, Sichuan Agricultural University Chengdu disease is identified garden respectively susceptible variety director 29, wheat Yilong, place, disease-resistant variety Sichuan are sticked together wheat and F thereof
2colony and F
2:3family monoseeding, the long 2.0m of row, line-spacing 20cm, 10 of every row sowings, arrange the susceptible check variety SY95-71 of a line at interval of 20 row, plant respectively 1 row and bring out susceptible variety SY95-71 and director 29 for stripe rust around evaluation garden.
Resistance Identification: adopt artificial inoculation mode to carry out field resistance evaluation.When wheat seedling grows to, two leaves bring out susceptible variety SY95-71 with streak method to stripe rust wholeheartedly time and Susceptible parent director 29 carries out individual plant artificial inoculation.After Susceptible parent director 29 morbidities fully, record disease resistance.Carry out stripe rust resistance evaluation by following standard:
High resistance (HR): without infecting as seen;
Anti-(R): only produce withered spot or chlorosis reaction, without uredinium;
In anti-(MR): uredinium is less, has withered or chlorosis reaction around;
Sense (S): uredinium is medium, around without withered or chlorosis reaction;
High sense (HS): uredinium is large, around without withered or chlorosis reaction.
Qualification result is found F
2in colony, show as susceptible 35 strains that have, its offspring F
2:3strain performance is susceptible; F
2show as disease-resistant 117 strains that have, the F that it is corresponding
2:3in all plant show as 39 strains that have of resistance, have 78 and show as the strain that anti-sense separates.F
2the anti-sense of colony separates than meeting 3:1, F
2:3the anti-sense of strain separates than meeting 1:2:1, illustrates that local wheat Yilong, the disease-resistant variety Sichuan wheat that sticks together carried a dominant main effect Stripe Rust Resistance Gene.
3, extracting genome DNA
Adopt CTAB method to extract genomic dna.Get the fresh young leaflet tablet of 2g, liquid nitrogen grinding adds the 2 × CTAB extracting solution (2% CTAB, 1.4M NaCl, 0.1MTris-HCl(pH8.0) that is preheated to 65 ℃, 0.1M EDTA(pH8.0 after becoming fine powder), add before use 2% beta-mercaptoethanol 15ml, mix.
65 ℃ of water-bath 30-45min, jog mixes therebetween.
Be cooled to and add isopyknic chloroform after room temperature: primary isoamyl alcohol (24:1), mixes gently to supernatant liquor and be milk shape, the centrifugal 10min of 4000rpm.
Get supernatant liquor, add equal-volume Virahol, be placed in ice bath precipitation DNA.
Tick DNA, wash 2 times with 70% alcohol, dehydrated alcohol is washed once, and gas is done DNA, is dissolved in the TE solution of appropriate pH8.0.
0.8% agarose gel electrophoresis detects DNA quality.The each sample DNA concentration of determined by ultraviolet spectrophotometry, is diluted to 50ng/ μ l by each sample DNA for subsequent use.
4, ssr analysis
According to the upper SSR primer sequence synthetic primer of announcing of GrainGenes (http://www.wheat.pw.usda.gov) (synthetic by the handsome Bioisystech Co., Ltd in Shanghai), in the enterprising performing PCR amplification of ABI-9700 PCR instrument, specific as follows:
1) (20 μ l) for PCR reaction system
2) pcr amplification system
3) electrophoresis detection
With 6% native polyacrylamide gel electrophoresis analysis, develop the color by the silver method of dying, take a picture and also on white light lamp box, record experimental result (Fig. 1).
5, molecule marker linkage analysis
Utilize SSR mark that parent, gene pool polymorphic detection filter out to 152 F
2individual plant is analyzed, according to MAPMAKER software (Lander et al., 1987) requirement, disease-resistant parent Yilong is sticked together to the wheat banding pattern assignment identical with disease-resistant gene pond for " A ", the banding pattern assignment identical with susceptible gene pool with Susceptible parent director 29 is " B ", the assignment that simultaneously amplifies disease-resistant parent, disease-resistant gene pond and Susceptible parent, two kinds of banding patterns of susceptible gene pool is " H ", and banding pattern assignment unclear or disappearance is "-".Calculate genetic distance with Kosambi function.As calculated, obtain a SSR mark Xgpw7342
-350bp, by this SSR molecule marker Xgpw7342
-350bpdisease-resistant parent, Susceptible parent, disease-resistant gene pond, susceptible gene pool and their F increase
2genetic mapping colony, finds to have a mark at 350bp place, and chain with wheat stripe rust resistance genes, genetic distance is 1.8cM.
Embodiment 2: wheat stripe rust resistance genes Molecular Selection
1, for examination material
Vegetable material: the local wheat of susceptible variety director 29, the disease-resistant variety Sichuan Yilong wheat that sticks together, disease-resistant variety Yilong sticks together the height of the cross combination that wheat and susceptible variety river agriculture 16 be parent for (F
5) 4 wheat strains (L1-L4), 4 and the disease-resistant variety Yilong different Sichuan Wheat landraces of wheat that sticks together, comprise: stick together wheat, the white wheat of Kaijiang, Yibin pig wheat, Hejiang of Palestine and China reprinted the stick together Bred Wheat Varieties of wheat affinity-less relation of wheat, 10 and disease-resistant variety Yilong, comprising: interior wheat No. 8, interior wheat No. 9, river wheat 47, section one-tenth wheat No. 2, river wheat 42, river agriculture 16, No. 5, river spoke, No. 3, continuous agriculture, Mianyang 26, river wheat 22.Positive season in 2011 plants above-mentioned materials in test base, Wenjiang, school district, Sichuan Agricultural University Chengdu disease and identifies garden, for Resistance Identification.Get the partial blade of each individual plant seedling stage, for ssr analysis.
Stripe Rust mixes physiological strain: mix physiological strain for the Stripe Rust of Resistance Identification and be made up of 31,32 and 33 3 microspecies balanced mix of bar.
2, Resistance Identification
With embodiment 1.Qualification result finds, susceptible variety director 29 shows as high sense, and the disease-resistant variety Yilong wheat that sticks together shows as high resistance; The stick together height generation (F of the cross combination that wheat and the agriculture of susceptible variety river 16 be parent of disease-resistant variety Yilong
5) 4 wheat strains (L1-L4) show as high resistance; 4 show as susceptiblely from disease-resistant variety Yilong the stick together wheat, the white wheat of Kaijiang of Palestine and China that stick together in the different Sichuan Wheat landraces of wheat, and Yibin pig wheat, Hejiang are reprinted wheat and show as disease-resistant; 10 stick together in the Bred Wheat Varieties of wheat affinity-less relation with disease-resistant variety Yilong in become wheat No. 2, river wheat 42 of wheat No. 8, interior wheat No. 9, river wheat 47, section show as disease-resistantly, and river agriculture 16, No. 5, river spoke, No. 3, continuous agriculture, Mianyang 26, river wheat 22 show as susceptible.
3, extracting genome DNA
With embodiment 1.
4, specific molecular marker Xgpw7342
-350bpssr analysis
Utilize this SSR primer (forward primer: 5 '-GCGGTGGCAACAACCTAC-3 ', reverse primer: 5 '-GTGCATAAAGAGGGCAGAGC-3 '), to local wheat Yilong, the above-mentioned disease-resistant variety Sichuan Province wheat that sticks together, susceptible variety director 29, 4 from the disease-resistant variety Yilong different Sichuan Wheat landraces of wheat that sticks together, comprise: the Palestine and China wheat that sticks together, the white wheat of Kaijiang, Yibin pig wheat, wheat is reprinted by Hejiang, 10 with the stick together Bred Wheat Varieties of wheat affinity-less relation of disease-resistant variety Yilong, comprise: interior wheat No. 8, interior wheat No. 9, river wheat 47, section becomes wheat No. 2, river wheat 42, river agriculture 16, No. 5, river spoke, No. 3, continuous agriculture, Mianyang 26, river wheat 22 carries out ssr analysis, ssr analysis is with embodiment 1.
5, Stripe Rust Resistance Gene specific molecular marker Xgpw7342
-350bpassisted Selection
With this SSR molecule marker Xgpw7342
-350bpabove-mentioned materials is increased, result: the height generation (F of the cross combination that stick together in disease-resistant parent, disease-resistant gene pond and disease-resistant variety Yilong wheat and susceptible variety river agriculture 16 are parent
5) to find the polymorphism band line of a 350bp size in 4 DNA bands of a spectrum that wheat strain (L1-L4) storeroom amplifies (be of the present invention and the Sichuan landraces Yilong chain molecule marker Xgpw7342 of wheat Stripe Rust Resistance Gene that sticks together
-350bp, ), and at Susceptible parent, 4 of susceptible gene pools and the disease-resistant variety Yilong different Sichuan Wheat landraces of wheat that sticks together (comprising: the Palestine and China wheat that sticks together, the white wheat of Kaijiang, Yibin pig wheat, wheat is reprinted by Hejiang), 10 (comprising: interior wheat No. 8 with the stick together Bred Wheat Varieties of wheat affinity-less relation of disease-resistant variety Yilong, interior wheat No. 9, river wheat 47, section becomes wheat No. 2, river wheat 42, river agriculture 16, No. 5, river spoke, No. 3, continuous agriculture, Mianyang 26, river wheat 22) in all can not amplify the special banding pattern of this 350bp, in conjunction with field phenotype Resistance Identification result, thereby confirm this resistant gene specific molecular marker Xgpw7342
-350bpcan identify the existence of resistant gene in wheat lines.(Fig. 2).
Claims (4)
1. the molecule marker mutually chain with wheat stripe rust resisting ospc gene, is characterized in that, described molecule marker is by using with following SSR primer Xgpw7342
-350bpamplification Yilong sticks together wheat and obtains, forward primer: 5 '-GCGGTGGCAACAACCTAC-3 '; Reverse primer: 5 '-GTGCATAAAGAGGGCAGAGC-3 '.
2. the mutually chain molecule marker of claimed in claim 1 and wheat antibody rust gene is in the application of identifying aspect wheat breed stripe rust resisting.
3. a method for the mutually chain molecule marker of acquisition and wheat stripe rust resisting ospc gene, is characterized in that, said method comprising the steps of:
(1) take susceptible wheat breed director 29 as maternal, stick together wheat as male parent take Wheat landraces Yilong, Sichuan, build F
2and F
2:3genetic mapping colony, the F of structure
2colony is Stripe Rust Resistance Gene genetic mapping colony;
(2) extract wheat parent seedling and F thereof by CTAB method
2the individual plant DNA of colony;
(3) adopt SSR molecular marking technique to carry out the screening of wheat stripe rust resistance molecule marker, wherein, use SSR primer Xgpw7342
-350bpdisease-resistant parent, Susceptible parent, disease-resistant gene pond, susceptible gene pool and their F increase
2genetic mapping colony, through linkage analysis, finds that this mark is mutually chain with wheat stripe rust resistance genes, described SSR primer Xgpw7342
-350bpcomprise forward primer: 5 '-GCGGTGGCAACAACCTAC-3 ' and reverse primer: 5 '-GTGCATAAAGAGGGCAGAGC-3 '.
4. method according to claim 3, is characterized in that, adopts SSR molecule marker to screen with the method for the mutually chain molecule marker of wheat stripe rust resisting ospc gene to be:
(1) use SSR primer at disease-resistant parent, Susceptible parent, disease-resistant gene pond, susceptible gene pool and their F
2in genetic mapping colony, carry out DNA polymorphism analysis, described SSR primer Xgpw7342
-350bpcomprise forward primer: 5 '-GCGGTGGCAACAACCTAC-3 ' and reverse primer: 5 '-GTGCATAAAGAGGGCAGAGC-3 ';
(2) the linksystem analysis of molecule marker: to the Yilong F that wheat and Susceptible parent director 29 hybridization obtain that sticks together
2individual plant resistance is carried out field test, investigation F
2the disease resistance of individual plant, extracts F
2the individual plant DNA of colony, uses with same above reaction system, primer and program and carries out pcr amplification, and statistics resists, feels the frequency of occurrences and the switch type of strain indicia band and calculate crossover value, calculates the genetic distance between mark and Stripe Rust Resistance Gene.
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| 两系杂交小麦恢复系MR168抗条锈病基因遗传分析及分子标记定位;任勇等;《遗传》;20111130;第33卷(第11期);1263-1270 * |
| 任勇等.两系杂交小麦恢复系MR168抗条锈病基因遗传分析及分子标记定位.《遗传》.2011,第33卷(第11期),1263-1270. |
| 小麦材料PI31抗条锈性鉴定及其抗性基因SSR标记;蒲宗君等;《植物病理学报》;20060825;第36卷(第04期);342-346 * |
| 蒲宗君等.小麦材料PI31抗条锈性鉴定及其抗性基因SSR标记.《植物病理学报》.2006,第36卷(第04期),342-346. |
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