CN102433327B - Molecular marker closely linked with powdery-mildew-resistant gene of wheat Tabasco - Google Patents
Molecular marker closely linked with powdery-mildew-resistant gene of wheat Tabasco Download PDFInfo
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Abstract
The invention discloses a molecular marker closely linked with a powdery-mildew-resistant gene of wheat Tabasco, belonging to the technical field of agrobiology. Disease-resistant wheat Tabasco and disease-infected wheat Ningru No.1 are hybridized to structure a F1, F2 and F2:3 family colony, a resistance gene is located onto a chromosome 5DS by means of a disease resistance evaluation result and a molecular marker technology, and in order to find out a new molecular marker more approximate to an unknown Tabasco powdery-mildew-resistant gene, an EST (Expressed Sequence Tag) sequence which is located on the 5DS is utilized to develop an STS (Sequence Tagged Site)-marked Xmp510, which is 1.3cm away from a target gene. Compared with related markers obtained through screening in the past, the molecular marker which is used as the new molecular marker for the Tabasco powdery-mildew-resistant gene has better advantage when applied to auxiliary selective breeding of wheat powdery-mildew-resistant varieties, so that the defects of conventional breeding are overcome, a selection method is simplified, the breeding efficiency is increased, and the breeding progress of the disease-resistant varieties is accelerated.
Description
One, technical field
The present invention relates to and the closely linked molecule marker of wheat Tabasco mildew-resistance gene, can be applicable to molecular mark, belong to agricultural biological technical field.
Two, background technology
Wheat powdery mildew is a kind of serious leaf diseases that is caused by obligatory parasitism fungi wheat powdery mildew (Erysiphe graminis f.sp.tritici).All harm be can produce in Wheat Seedling to the strain phase, leaf sheath, stem stalk and fringe section also can be endangered when serious.Powdery Mildew is except the nutrient of predation wheat plant, and subiculum covering wheat strain surface is increased wheat strain respiration, and transpiration is strengthened, and photosynthesis usefulness reduces, and Carbohydrate Accumulation and conveying reduce.Wheat powdery mildew is in the past only popular in the wheat planting district with the maritime of abundant rainfall and half continent sex climate environment and cause serious production loss (Bennett, 1984, Resistance to powdery mildew in wheat:a review of its use in agriculture and breeding programmes, Plant Pathology33,279～300).Since the sixties in 20th century, the popularization of, Semi-dwarf cultivar of short stem along with wheat, the increase of amount of application of nitrogen fertilizer, the occurrence and harm of Powdery Mildew is on the rise, and main Mai Qu rises to Major Diseases by Minor diseases in the world, becomes a large obstacle (Wang Xinyu etc. of impact and restriction wheat stable yields and high yield, 2000, the RAPD mark of powdery mildew resistance gene in wheat Pm6, Acta Genetica Sinica 27,1072～1079).According to statistics, the production loss that wheat powdery mildew causes is generally 5～10%, can be up to 50% when seriously occuring, even total crop failure.Nineteen ninety and 1991 are very popular in China whole nation, and the generation area in 2 years is all above 1,200 ten thousand hm
2(National Agricultural technology popularization service centre 2008 annual datas).Nineteen ninety Powdery Mildew cause wheat yield lose 14.39 hundred million kg (Jin Shanbao, 1996, Wheat in China is learned, Beijing: Chinese agriculture press).Cause that this twice pandemic major cause is the 1B/1R translocation line wheat breed forfeiture resistance (Shi Ainong etc., 1998, the seed selection of powdery-mildew-resistance wheat new lines and Resistance gene analysis thereof, Plant Pathology 25,218～222) of carrying Pm8.Therefore, still very important to the control of wheat powdery mildew.
Cultivate and promote disease-resistant variety and be acknowledged as most economical, effective, the safe approach (Qiu etc. of control wheat powdery mildew, 2005, Microsatellite mapping of a Triticum urartu Tum.derived powdery mildew resistance gene transferred to common wheat (Triticum aestivum L), Theoretical and Applied Genetics 111,1524～1531).It can reduce the use of agricultural chemicals on the one hand, avoids contaminate environment, can reduce production costs on the other hand.The key of breeding resistant variety is excavation, the research to powdery mildew resistance gene in wheat and rationally utilizes (Yang Zuomin etc., 1994, the strategic issue of wheat breeding for disease resistance---wheat is to foundation and the application in rust, the anti-source of Powdery Mildew the second line, Acta Agronomica Sinica 20,385～394).At present, to excavate the powdery mildew resistance gene in wheat of identifying more, wherein the quality resistance accounts for major part.But the characteristics such as colony is large because wheat powdery mildew has, wide accommodation, physiological strain is many and the virulence variation is fast, the single disease-resistant variety of spread is lost resistance so that most of resistant gene has been overcome by new physiological strain in addition.For this reason, seek disease-resistant new germ plasm, excavate new gene, cultivate the new variety that contain new disease-resistant gene, then can tackle and produce the upper stronger and generally popular microspecies of toxicity.In addition, rational deployment disease-resistant gene and disease-resistant variety are aggregated to a plurality of disease-resistant genes in the kind and go, and widen anti-spectrum and improve resistance to persistence, also be to delay the varietal resistance forfeiture, prevent and treat the popular important method of disease big area.
The wheat powdery mildew resistance shows as quality resistance (qualitative resistance) and two kinds of form (Friebe etc. of quantity resistance (quantitative resistance), 1994, Cytogenetically monitored transfer of powdery mildew resistance from rye into wheat, Crop Science 34,621～625).The quality resistance is by single-gene or oligogene control, the mutual work of plant and pathogenic bacteria meet Flor's " gene pairs gene hypothesis ", the microspecies specialization is stronger, generally express (Bennett in seedling stage, 1984, Resistance to powdery mildew in wheat a review ofits use inagricuture and breeding programmes, Plant Pathology 33,279～300).The research of wheat powdery mildew quality resistant gene is comparatively deep with utilization, and technology is also comparatively ripe.At present, great majority are the quality resistance in the powdery mildew resistance gene in wheat of having reported, namely most of resistances are the race specific resistances by Dominant gene.The quantity resistance without the microspecies specialization, shows as the continuous variability by controlled by multiple genes.The quantity resistance can be present in seedling stage, strain phase or whole breeding time.This resistance mainly delays infecting of pathogenic bacteria, development and fecundity, be also referred to as partial resistance (Hautea etc., 1987, Inheritance of partial resistance to powdery mildew in spring wheat, Theoretical and Applied Genetics 73,609～615), strain resistance (Griffey etc., 1993, Effectiveness of adult-plant resistance in reducing grain yield loss to powdery mildew in winter wheat, Plant Disease 77,618～622) or slow disease resistance (Roberts and Caldwell, 1970, General resistance (slow mildewing) to Erysiphe graminis f.sp.tritici in Knox wheat, Phytopathology 60,1310).At present, have 43 that are positioned different loci of definite designation are respectively Pm1～Pm43 (McIntosh etc., 2008 in more than 60 the wheat main effect mildew-resistance gene of having reported, Catalogue of gene symbols for wheat, In Appels R, Eastwood R, Lagudah E et al (eds), Proceedings of l lth international wheat genet symposium, Sydney University Press, Sydney, Australia; Luo etc., 2009, Characterization and chromosomal location of Pm40 in common wheat:a new gene for resistance to powdery mildew derived from Elytrigia intermedium, Theoretical and Applied Genetics 118,1059～1064; Li etc., 2008, Molecular characterization of a new powdery mildew resistance gene Pm41 on chromosome 3BL derived from wild emmer (Triticum turgidum var.dicoccoides), Theoretical and Applied Genetics 119,531～539; Hua etc., 2009, Identification and genetic mapping of pm42, a new recessive wheat powdery mildew resistance gene derived from wild emmer (Triticum turgidum var.dicoccoides), Theoretical and Applied Genetics 119,223～230; He etc., 2009, Inheritance and mapping of powdery mildew resistance gene Pm43 introgressed from Thinopyrum intermedium into wheat, Theoretical and Applied Genetics 118,1173～1180).In these disease-resistant genes, except Pm5, Pm9, Pm26, pm42, mlRD30, pmY212, PmLK906 and pm2026 etc. are the recessive gene, major part is dominant inheritance.Although the mildew-resistance major gene of having named is many, because the wheat powdery mildew physiological strain is many, the virulence variation is fast, causes the kind that contains single resistance generally to use 3～5 years in production and just loses resistance.In addition, since some disease-resistant gene and some the bad linkage of characters and difficulty in breeding, utilize.The limited amount that therefore, really can be applied to produce.
In China, the mildew-resistance gene that is applied in the production has Pm2, Pm4a, Pm4b, Pm5, Pm6, Pm8, Pm21, Pm23 and Pm24 etc.(the Xiang Qijun etc. such as Xiang Qijun, 1996, the effective anti-genetic analysis of wheat powdery mildew anti-source material, Acta Agronomica Sinica, 22,741～744) by measuring resistant gene the resistance level of white powder flora body is found that the disease-resistant usefulness of Pm1, Pm3a, Pm3b, Pm3c, Pm3f, Pm5, Pm7, Pm8 is very poor in China, on producing without practical value; Pm2, Pm4a, Pm4b are lower in the southwest resistance, but still effective in other area of China; The resistance of Pm2+Mld, Pm2+Pm6, Pm21 and China farm variety Xiao Bai winter wheat (XBD gene) is done well.Pm21 is the strongest gene of present resistance, and its carrier kind is without obvious bad proterties, and disease resistance all shows stablely under different wheat genetic backgrounds, and now transformation and has obtained application producing in the production kind.Pm8 is a comparatively typical example in China and even the breeding of world wheat mildew-resistance.Breeding units adopts the anti-source from the rye blood lineage mostly since 20 century 70s, such as the kind of deriving of " Niu Zhute ", " Caucasia ", " A Fuleer " and 1B/1R transpositions such as " Luo Fulin " or substitution line, its contained mildew-resistance gene mostly is Pm8.Because the unicity in the anti-source of commercial variety, 20th century, the mid-80 began, the anti-white powder gene of Pm8 has worldwide been lost disease resistance in succession, has caused being very popular of wheat powdery mildew over nearly 10 years, thereby causes finally that Powdery Mildew repeatedly is very popular in 20 end of the century China's Wheat Production.
So, because neoantigen must be excavated and utilize to the single resistant lose phenomenon that is similar to Pm8 that again causes of antigen energetically, cultivate the kind that contains new disease-resistant gene in order to prevent Pm21, effectively control the popular of Powdery Mildew.Simultaneously, rational deployment disease-resistant gene and disease-resistant variety are aggregated to a plurality of disease-resistant genes in the kind and go, and widen anti-spectrum and improve resistance to persistence, also be to delay the varietal resistance forfeiture, prevent and treat the popular important method of disease big area.Traditional selection of disease-resistant variety is by selecting disease-resistant plant behind the inoculated identification, make to affect sometimes in this way efficiency of selection because inoculation is insufficient or onset condition is not suitable for.Along with generation and the development of molecular marking technique are perfect, the evaluation that can utilize molecule marker to carry out disease-resistant gene is selected, thereby has developed molecular marker assisted selection (Marker-assisted selection, MAS) method.Carry out the detection of disease-resistant gene with molecule marker, just can select in generation early, improve the accuracy of selecting, dwindle breeding population, and be not subjected to environment, the restriction of the season of growth, in conjunction with increasing generation technique greatly shortening the breeding cycle, raising breeding efficiency.Therefore molecular marking technique has become the important means of assistant breeding.
Mildew-resistance new germ plasm Tabasco is the red grain of the hard winter wheat that introduce from Germany in this laboratory (Jiangsu Province Agriculture Science Institute), belong to hexaploid, this germplasm all shows high resist powdery mildew of wheat mixed strains in Germany and China's In Nanjing, is a desirable Powdery Mildew new resistance source material.The peaceful glutinous wheat of sense Powdery Mildew material is for No. 1 first Waxy wheat kind of the whole nation that cultivate in this laboratory, has good cultivation characteristic and mouthfeel, this kind sense Powdery Mildew, and pedigree is derived and is not contained any effective mildew-resistance gene.As the parent, be conducive to the excavation of the new gene of mildew-resistance with these two materials, can directly create the disease-resistant new germ plasm that is applied to breeding simultaneously.
Three, technical scheme
Technical problem
The present invention is directed to above-mentioned research background, take strong powdery-mildew-resistance wheat kind Tabasco as disease-resistant material screening with seek new and stable existence and the closely linked molecule marker of powdery mildew resistance gene and method thereof, be used for the wheat powdery mildew assisted selection, can overcome the shortcomings such as conventional genetic breeding cycle length.Because therefore the material of research material therefor for the local popular mixed strains in Nanjing is had resistance of wide spectrum be expected to excavate new powdery mildew resistance gene.
Technical scheme
With the closely linked molecule marker primer of wheat Tabasco mildew-resistance gene, it is characterized in that, this primer is the primer with the closely linked STS mark of wheat Tabasco powdery mildew resistance gene Xmp510, chain and the genetic distance of mark Xmp510 and target gene is 1.3cM, and primer sequence is as follows:
Left primer: 5 '-CATCCAAACATCCAATGC-3 ';
Right primer: 5 '-ACACTACCACCATCACCGC-3 '.
The application of described molecule marker primer, it is characterized in that, be object with Tabasco or its derived varieties and product, with primer PCR amplification wheat plant DNA described and the closely linked STS mark of wheat Tabasco powdery mildew resistance gene Xmp510, if there is general 570bp and 600bp two strip-types of Xmp510 mark, represent that then the resistant gene of wheat powdery mildew in this plant exists.
Include template 10～20ng, each 2pmol of left and right sides primer, MgCl
215nmol, 0.1U Taq archaeal dna polymerase and 1 * PCR damping fluid;
The reaction cycle program is as follows: 94 ℃ of denaturation 3min, then press " 94 ℃, 30s; 58 ℃, 30s; 72 ℃, 1min " carry out 34 cyclic amplifications, last 72 ℃ are extended 10min;
The PCR product carries out electrophoretic separation with the non-denaturing polyacrylamide gel of mass ratio 8%, and reads the separation band with silver staining color.
Beneficial effect
Powdery Mildew is a kind of serious plant disease that affects Wheat Production, and the present invention utilizes molecule marking method to develop new and the closely linked STS mark of powdery mildew resistance gene, in wheat breeding practice and disease-resistant theoretical investigation very important value is arranged.Its advantage specifically is summarized as following 3 points:
(1) among the present invention with the closely linked STS mark of powdery mildew resistance gene in wheat, it is the new mark that in the stable filial generation individual plant of the wheat breed Tabasco that Germany is introduced and resistance thereof, obtains, 15 different Powdery Mildew physiological strains all there is resistance, and stable existence, can be for wheat powdery mildew cultivar identification and disease-resistant wheat offspring's assisted selection.
(2) this research material therefor Tabasco is new Powdery Mildew anti-source material, to 15 popular all immunity of mixing Powdery Mildew of In Nanjing, has the Powdery Mildew resistance of wide spectrum.Therefore, be expected to excavate the powdery mildew resistance gene that makes new advances according to this molecule marker.
(3) this is labeled as stable STS mark, and with the mildew-resistance gene close linkage, genetic distance is 1.3cM.Can be directly used in the assisted selection of wheat anti-powdery mildew molecule marker, accelerate breeding process.Have laid a good foundation for cloning the new gene of wheat anti-powdery mildew and functional study thereof simultaneously.
Four, description of drawings
By following detailed description also by reference to the accompanying drawings, with clearer understanding above and other purpose of the present invention, feature and other advantages, wherein:
Fig. 1 is that the upper Xgwm205 of 5DS is marked at parent and F
2Separation in the colony, swimming lane M is molecular weight Marker, and swimming lane 1,2,3 and 4 is respectively Tabasco, peaceful glutinous wheat No. 1, anti-pond and sense pond, and the arrow place is shown the polymorphic banding pattern of this mark amplification.
Fig. 2 is that the upper Xcfd81 of 5DS is marked at the amplification banding pattern that China spring and nullisomic thereof, limbs are fastened.
Fig. 3 is the separation of STS mark Xmp510 in parent and anti-sense pond.Swimming lane M is molecular weight Marker, and swimming lane 1,2,3 and 4 is respectively Tabasco, peaceful glutinous wheat No. 1, anti-pond and sense pond.
Fig. 4 is the genetic linkage map of Tabasco mildew-resistance gene (PmTa), and wherein Xmp510 and PmTa genetic distance are 1.3cM.
Fig. 5 is the performance of STS mark Xmp510 in the cross combination offspring take Tabasco as the parent.Swimming lane M is molecular weight Marker, and swimming lane 1,2,3 and 4 is respectively Tabasco, peaceful glutinous wheat No. 1, anti-pond and sense pond.Swimming lane 5-22 is the filial generation F take Tabasco as one of parent
3The amplification banding pattern, in full accord with resistant phenotype.
Five, embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The powder mildew resistance of embodiment 1, Tabasco is identified and genetic analysis
1, for the examination material
Vegetable material: with disease-resistant variety Tabasco (Beschreibende Sortenliste 2009-Getreide, Mais,
Leguminosen und Hackfr ü chte au β er Kartoffeln, P.124, ISSN 0948-4167) and No. 1 (Soviet Union examines wheat 200803) hybridization of the peaceful glutinous wheat of susceptible variety, make up F
2Target group, results F when ripe
2:3The family seed is used for seedling resistance to be identified.China spring and the (CS of corresponding nulli-tetrasomes system, N5A-T5D, N5D-T5B, N7B-T7D, N4D-T4B) (Sears, 1954, The aneuploids of common wheat, Missouri Agricultural Experiment Station Research Bulletin 572,1～58) is used for chromosomal localization; Ulka/8*cc (Crops In China germplasm Information Network
Http:// icgr.caas.net.cn/Unified number MY008575) for containing the near isogenic line of Pm2, is used for anti-spectrum analysis; Sumai 3 (Crops In China germplasm Information Network
Http:// icgr.caas.net.cn/Unified number ZM010242) as susceptible contrast.
Bacterial classification material: wheat powdery mildew bacterial classification Bgt (Blumeria graminis f.sp.tritici) separates and obtains (Red Star perhaps from the local popular mixed strains in Nanjing, 2008, the doctorate paper, the evaluation of the new gene of one grained wheat mildew-resistance and mark secondary transfer), identify seedling stage and adopt Bgt19 (Ma etc., 2011, Identification and mapping of a new powdery mildew resistance gene on chromosome 6D of common wheat, Theoretical and Applied Genetics, DOI 10.1007/s00122-011-1651-3) carries out inoculated identification.
2, Resistance Identification
(1) standard of perfection
Pathography is pressed 6 grade standards of Sheng Baoqin (Sheng Baoqin, 1988, with response type record Wheat Seedling Powdery Mildew, plant protection Isosorbide-5-Nitrae 9): 0 grade: immunity, and plant is without scab; 0; Level: necrotic reaction, blade have withered spot; 1 grade: high resistance, scab little (general diameter is less than 1mm) the thin visible green of subiculum blade face, accidental larger scab, but still reveal the green, produce the amount of embracing seldom; 2 grades: in anti-, the leaf spot lesion diameter is less than 1mm, but subiculum is thicker, does not reveal the green, and can produce a certain amount of spore; 3 grades: middle sense, leaf spot lesion is many, and general diameter is greater than 1mm, the mycelia bed thickness, sporulation quantity is large, but scab is not in flakes; 4 grades: high sense, leaf spot lesion diameter be generally greater than 1mm, the mycelia bed thickness, and sporulation quantity is many, and scab is in flakes.
(2) colony identifies
The F that No. 1 hybridization of Tabasco and peaceful glutinous wheat obtains
1All be seeded in the wheat powdery mildew evaluation garden of setting up in experimental plot, academy of agricultural sciences, Jiangsu Province with anti-sense parents and carry out Resistance Identification.The long 1.35m of cell row, line-spacing 0.25m, both sides are vertically planted susceptible variety Sumai 3 (producing with planting) and are done and bring out row.Identify that used bacterial classification Bgt is that the hybrid bacterial strain that local field, Nanjing gathers comes through getting over summer preservation breeding.Before the wheat jointing, transplant a large amount of diseased plants in the ranks bringing out, make its continuous infection-induced capable, guarantee that early stage bacterium source, disease garden is sufficient.
F
2In growth cabinet, use physiological strain Bgt19 inoculated identification in seedling stage.Material to be identified is planted in 4 * 4cm
2In the dish of * 72 rectangle cave, spread conidial mode and inoculate by trembling when in the greenhouse that pollutes without powdery mildew, treating that wheat growth to first leaf launches fully.Plant at random Sumai 3 in the dish of cave as the contrast of inoculation homogeneity, as susceptible contrast, disease-resistant parent is as disease-resistant contrast with Susceptible parent.Keep indoor relative humidity more than 80% after the inoculation, illumination every day 14 hours, diurnal temperature is respectively 22 ℃ and 18 ℃.After 7～14 days, when susceptible synopsis reveals obvious illness, carry out the resistance investigation, check after 2 days.
To the F of all results to seed
2:3Family is got 25 and is sent out the seedling evaluation, and inoculation and evaluation mode are the same, and result disease-resistant to isozygoty (RR), resistance are separated (Rr) and susceptible (rr) family record that isozygotys.
3, genetic analysis
F
1Colony and anti-sense parent identify through the land for growing field crops natural occurrence and show: (IT 0～0 for equal high resistance wheat powdery mildew of Tabasco seedling stage and strain phase; ); Peaceful glutinous wheat No. 1, seedling stage to the strain phase is high sense wheat powdery mildew (IT4) all; F
1(IT 0～0 for equal high resistance wheat powdery mildew of seedling stage to strain phase; ).With wheat powdery mildew physiological strain Bgt19 to Tabasco, peaceful glutinous wheat No. 1, F
2And F
2:3Family is carried out evaluation in seedling stage in the greenhouse.The result shows: (IT 0～0 for the high mildew-resistance of Tabasco; ), No. 1 high sense Powdery Mildew of peaceful glutinous wheat (IT 4), F
2For 359 strains disease-resistant (IT 0～2) in the 472 strain individualities, 113 strains susceptible (IT 3～4).Through x
2Check meets 3: 1 segregation ratio (x
2 3: 1=0.23, P=0.63) (table 1).To all F that obtains
2:3The every system of family chooses 25 seeds and carries out powder mildew resistance evaluation in seedling stage, and the result shows: 101 F are arranged
2:3The family performance is isozygotied disease-resistant; 238 F
2:3Family anti-sense occurs and separates; 97 F
2:3For family all show isozygoty susceptible.Its resistance segregation ratio meets 1: 2: 1 segregation ratio (x
2 1: 2: 1=3.58, P=0.17) (table 1).In conjunction with F
1, F
2And F
2:3The qualification result in generation, deducibility Tabasco is controlled by 1 pair of dominant major gene to the resistance of white powder bacterial classification Bgt19.
The F that table 1 Tabasco and peaceful glutinous wheat are No. 1
2And F
2:3Segregating population is to the reaction of wheat powdery mildew Bgt19 microspecies
RR: the disease-resistant family of isozygotying; Rr: the disease-resistant family of heterozygosis; Rr: the susceptible family of isozygotying.x
2 0.05，1＝3.84，x
2 0.05，2＝5.99
1, DNA extraction and anti-sense pond are set up
Extracting genome DNA is with reference to (Ma etc., 1994, RFLP markers linked to powdery mildew resistance genes Pm1 such as Ma, Pm2, Pm3, and Pm4 in wheat, Genome 37,871～875) the SDS method of describing is carried out.Concrete steps are as follows: get wheat seedling and be organized in an amount of DNA extraction liquid (0.1M Tris pH8.0,0.05MEDTApH8.0, the 1.25%SDS of the rear adding of grinding in the liquid nitrogen, 0.5MNaCl, 3.8g/L NaBisufite), process 30min, put upside down mixing therebetween 4～5 times for 65 ℃.Chloroform/primary isoamyl alcohol (24: 1) extracting, the centrifuging and taking supernatant liquor.With the raw spirit of-20 ℃ of precoolings precipitation DNA, and place 1h or spend the night down at-20 ℃.Wash with 70% alcohol, add at last an amount of TE dissolving, stand-by in 4 ℃ or-20 ℃ of storages.
Adopt chorista grouping hybrid analysis method (Bulk segregant analysis, BSA) carry out the screening (Michelmore etc. of gene linkage mark, 1991, Identification of markers linked to disease-resistance genes by bulked segregant analysis:a rapid method to detect markers in specific genomic regions by using segregating populations, Proceedings of the National Academy of Sciences 88,9828～9832).Choose F
2Generation 0～0; The DNA balanced mix of disease-resistant individual plant 6 strains become disease-resistant pond (BR), the DNA balanced mix of 4 grades of susceptible individual plant 6 strains becomes susceptible pond (BS).
2, ssr analysis
From the full genome of wheat, select barc take the genetic distance of 10～20cM as unit, gwm, wmc, and 44 pairs of the complete genomic SSR primer 2s of covering of the series such as cfp carry out full genome polymorphism and detect.DNA take anti-sense parent, disease-resistant pond and susceptible pond carries out pcr amplification as template, seeks the primer that consistent polymorphism is arranged between parent and the anti-sense pond, and to F
2Segregating population carries out analysis verification, detects the linkage degree of SSR mark and disease-resistant gene.
Found that at the SSR mark Xgwm205 on the 5DS has consistent polymorphism (Fig. 1) between anti-sense parent and anti-sense pond, with 96 F
2The microcommunity of individual plant DNA is verified this mark, finds that Xgwm205 and this disease-resistant gene are chain, and further screening is positioned at other SSR marks of 5DS and 5DL kinetochore near zone, find Xcfd81, Xgpw302, Xcfd67 and Xwmc608 be equal tool polymorphism between the parent, through F
2Microcommunity detects that they are all chain with target gene.This powdery mildew resistance gene tentatively is decided to be on 5DS karyomit(e).
3, the physical positioning of Tabasco mildew-resistance gene
Use China spring and corresponding nulli-tetrasomes system, to carrying out physical positioning with the chain mark of target gene.Because the mark Xcfd81 nearest apart from goal gene also has amplification site (http://wheat.pw.usda.gov/GG2/index.shtml) on 4D and 7B, this selects China spring (CS) and corresponding the 4th, the 5th and the 7th homology group's nulli-tetrasomes system that goal gene is carried out physical positioning.Above-mentioned 5 are marked at CS and CS N5A-T5D all can amplify corresponding band, and can not increase out on CS N5D-T5B.Xcfd81 can both expand at CS N4D-T4B and CS N7B-T7D and corresponding band in addition, but on CS N5D-T5B, can not increase out (Fig. 2).Prove that thus this disease-resistant gene is positioned on the chromosomal galianconism of wheat 5D.
4、EST-S
The exploitation of TS mark and the structure of linkage map
In order to seek and the more closely linked mark of this disease-resistant gene, in the hope of providing more reliable detection means for molecular mark (MAS), database (http://wheat.pw.usda.gov/wEST/binmaps/wheat5_rice.html) according to GrainGenes, est sequence on the wheat 5DS is tentatively chosen 2～3 take each Deletion bin as unit, carry out the exploitation of corresponding STS mark.Design of primers adopts software MACVECTOR V10.0 (Accelrys, UK).STS mark pleiomorphism detecting method is similar to the SSR labeled analysis.Do not detect between the parent when polymorphic when target STS is marked at, PCR selectivity of product ground is digested with restriction enzyme.Endonuclease reaction carries out according to the respective description book, and product separates with 8% native polyacrylamide gel electrophoresis.
Developed altogether 14 pairs of STS marks, wherein Xmp510 primer obvious difference (Fig. 3) between anti-sense parent is used F
2Crowd surveillance is found chain with target gene and genetic distance is 1.3cM (Fig. 4).STS mark Xmp510 is designed according to the sequence of EST BE498794, and primer sequence is as follows:
Left primer: 5 '-CATCCAAACATCCAATGC-3 ';
Right primer: 5 '-ACACTACCACCATCACCGC-3 '.
To Tabasco and No. 1 F of peaceful glutinous wheat
3The family individual plant increases, the Xmp510 amplification as shown in Figure 5, the anti-emotion condition of labeled analysis prediction of result and the actual disease detected result fit like a glove (table 2) that connects, proof mark Xmp510 can be applied to the resistance screening of Powdery Mildew, and the selection breeding that contains the Tabasco mildew-resistance gene.
F among table 2 Fig. 5
3The corresponding powder mildew resistance phenotypic evaluation of strain result
Above-mentioned enforcement does not limit the present invention in any form.
SEQUENCE LISTING
＜110〉Jiangsu Province Agriculture Science Institute
＜120〉with the closely linked molecule marker of wheat Tabasco mildew-resistance gene
<130> 000
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 18
<212> DNA
＜213〉artificial
<220>
＜221〉left primer
<222> (1)..(18)
<223>
<400> 1
<210> 2
<211> 19
<212> DNA
＜213〉artificial
<220>
＜221〉right primer
<222> (1)..(19)
<223>
<400> 2
Claims (2)
1. with the closely linked molecule marker primer of wheat Tabasco mildew-resistance gene, it is characterized in that,
This primer is and the closely linked STS mark of wheat Tabasco powdery mildew resistance gene
Xmp510Primer, mark
Xmp510Chain and genetic distance is 1.3cM with target gene, and primer sequence is as follows:
Left primer: 5 '-CATCCAAACATCCAATGC-3 ';
Right primer: 5 '-ACACTACCACCATCACCGC-3 '.
2. the application of the described molecule marker primer of claim 1 is characterized in that, take Tabasco or its derived varieties as object, with claim 1 described with the closely linked STS mark of wheat Tabasco powdery mildew resistance gene
Xmp510Primer PCR amplification wheat plant DNA, if there is
Xmp510The 570bp of mark and 600bp two strip-types represent that then the resistant gene of wheat powdery mildew in this plant exists.
3
.The application of molecule marker primer as claimed in claim 2 is characterized in that,
PCR reaction system 10 μ l:
Include template 10 ~ 20ng, each 2pmol of left and right sides primer, MgCl
215nmol, 0.1U Taq archaeal dna polymerase and 1 * PCR damping fluid;
The reaction cycle program is as follows: 94 ℃ of denaturation 3min, then press " 94 ℃, 30s; 58 ℃, 30s; 72 ℃, 1min " carry out 34 cyclic amplifications, last 72 ℃ are extended 10min;
PCR product mass ratio is that 8% non-denaturing polyacrylamide gel carries out electrophoretic separation, and reads the separation band with silver staining color.
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| CN105112416B (en) * | 2015-09-28 | 2018-03-30 | 江苏省农业科学院 | Molecular labeling primer and application with red common house centipede wheat mildew-resistance gene close linkage |
| CO2018009511A2 (en) * | 2016-02-22 | 2018-09-20 | Bejo Zaden Bv | Carrot powdery mildew resistance genes |
| CN106222285B (en) * | 2016-08-11 | 2019-06-18 | 江苏省农业科学院 | The molecular labeling isolated with powdery mildew resistance gene in wheat Pm48 |
| CN107354232A (en) * | 2017-09-18 | 2017-11-17 | 江苏省农业科学院 | A kind of method for developing chromosome segment linkage molecule mark specific with wheat |
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