CN1462809A - Molecule marker method of wheat Wangshuibai scab resistant major gene locus - Google Patents
Molecule marker method of wheat Wangshuibai scab resistant major gene locus Download PDFInfo
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Abstract
A major gene site resisting bakanae disease extension for wheat "Wanshuibai" and its molecular marker are disclosed. After the gene type, invalid ear rate and affected section length of each parentage for the recombinant selfing line obtained by hybridization between wheat "Wanshuibai" and "Nanda 2419" are analyzed in genetic linkage mode, said major gene sites QFhs.nau-6b, QFhs.nau-7b1 and QFhs.nau-7d are found out. Detecting if the Wanshuibai and its derivative variety contain said major gene sites can determine their level resisting bakanae disease.
Description
One, technical field
The invention provides wheat Wangshuibai anti gibberellic disease expansion major gene loci and molecule marker thereof, belong to the molecular genetics field, be exclusively used in the seed selection of wheat scab disease-resistant variety and the utilization of germ plasm resource.
Two, technical background
Wheat is the important food crop of China.By the wheat scab that gibberella causes, not only cause the serious underproduction of wheat, reduce grain quality, and the toxin deoxynivalenol that this germ produces also influences people, animal health, caused very big loss to Wheat Production.In China, the harm that suffers head blight above 1/4th wheat belt is arranged, and just once be very popular every 3 ~ 5 years, only head blight generation area in 2002 promptly reaches 30,000,000 mu times.Therefore, research prevents and treats wheat scab and has also just become a urgent task on the current wheat breeding.Cultivating disease-resistant variety is to prevent and treat the safest, effective of wheat scab harm and save one of measure of cost.But traditional breeding way is wasted time and energy, and because the head blight phenotypic evaluation is very difficult, makes the wheat anti gibberellic disease breeding process slow unusually.Identify that by the location disease-resistant major gene loci comes assistant breeding effectively to address this problem.
Genetic research shows that wheat scab is controlled by a few major gene loci mainly, and its disease-resistant major gene loci position on karyomit(e) of different resistant materials is inconsistent, and be divided into anti-infect and resist expand two types.But the polymerization by the disease-resistant major gene loci of difference or introduce new anti-source and can select the good kind of resistance.
At present focusing mostly in Soviet Union wheat No. 3 and derive in the utilization of the research of scab resistance hereditary basis and breeding for disease resistance is, and to the utilization research in other anti-source seldom.Wangshuibai is the distinctive local variety in China Jiangsu, has high and stable scab resistance.Up to the present find the wheat breed stronger as yet, and it carries the resistance main effect gene locus (Liao Yu ability 1985) that is different from Soviet Union wheat No. 3 than its resistance.
Three, disclosure of Invention Technical Problem the objective of the invention is: wheat Wangshuibai anti gibberellic disease expansion major gene loci and molecule marker thereof are provided, locate these anti-expansion major gene locis, by detecting and the chain molecule marker of these anti-expansion major gene locis, can predict the scab resistance of wheat plant, accelerate the selection progress of wheat-resistance to scab.Technical scheme wheat Wangshuibai anti gibberellic disease expansion major gene loci is characterized in that: major gene loci QFhs.nau-6b, and by mark Xbarc024 location,
Left end primer sequence CGCCTCTTATGGACCAGCCTAT
Right-hand member primer sequence GCGGTGAGCCATCGGGTTACAAAG
Amplified fragments 191bp, this major gene loci are positioned at wheat Wangshuibai 6B karyomit(e), utilize Datadesk v.5.0 software record with the incoherent probability P value of scab resistance be 0.0003, to the contribution rate 16.6% of scab resistance; Major gene loci QFhs.nau-7b1, by mark Xgwm533-170 location,
Left end primer sequence AAGGCGAATCAAACGGAATA
Right-hand member primer sequence GTTGCTTTAGGGGAAAAGCC
Amplified fragments 170bp, this major gene loci are positioned at wheat Wangshuibai 7B karyomit(e), utilize Datadesk v.5.0 software record with the incoherent probability P value of scab resistance be 0.0016, to the contribution rate 8.6% of scab resistance; Major gene loci QFhs.nau-7d, by mark Xbarc076 location,
Left end primer sequence ATTCGTTGCTGCCACTTGCTG
Right-hand member primer sequence GCGCGACACGGAGTAAGGACACC
Amplified fragments 191bp, this major gene loci are positioned at wheat Wangshuibai 7D karyomit(e), utilize Datadesk v.5.0 software record with the incoherent probability P value of scab resistance be 0.0026, to the contribution rate 12.2% of scab resistance.
Be positioned at anti gibberellic disease expansion major gene loci QFhs.nau-6b, QFhs.nau-7b1 on 6B, 7B and the 7D and the molecule marker of QFhs.nau-7d in the above-mentioned Wangshuibai and be respectively Xbarc024, Xgwm533-170 and Xbarc076.
Above-mentioned wheat Wangshuibai anti gibberellic disease expansion major gene loci, be that to obtain mark by the following method localized: (1) wheat breed Wangshuibai (♀) is hybridized with Nanjing University 2419 (♂) and is obtained hybrid F1, the F1 selfing produces F2, adopts simple grain transmission method (SSD) to obtain the F6 RIL in generation then; (2) extract the DNA that each strain of recombinant inbred lines is with the SDS method, adopt simple repeated sequence mark SSR that two parents are carried out the polymorphism screening, PCR carries out on PE9600 amplification instrument, amplified production carries out electrophoretic analysis on 8% (g/ml) polyacrylamide gel, have polymorphic primer to analyze in recombinant inbred lines between the parent, the pcr amplification program is the same; (3) according to chain exchange rule, utilize colony's genotype data to make up the genetic map of wheat, used software is Mapmaker 2.0, minimum LOD value is made as 3, obtains linkage map; (4) blooming stage carries out the scab resistance evaluation to each family of RIL, and leading indicator is disease spikelet number and sick joint length; (6) utilize Data desk v.5.0 software sick spikelet number and sick joint length that the colony's genotype data of each molecule marker and the scab resistance of its corresponding each family are identified carry out linkage analysis, One-way ANOVA records and the incoherent probability P value of scab resistance, the molecule marker of P＜0.05 promptly shows with a major gene loci chain: the mark Xbarc024 that finds P=0.0003 in the Wangshuibai, the mark Xgwm533-170 of P=0.0016 and the mark Xbarc076 of P=0.0026 and anti-expansion major gene loci close linkage promptly obtain the anti-expansion of wheat Wangshuibai major gene loci QFhs.nau-6b, the mark location of QFhs.nau-7b1 and QFhs.nau-7d; Perhaps the linkage group that major gene loci is arranged is carried out interval mapping analysis with Map Manager QTX software, the peak of curve position is a major gene loci, the anti-expansion of Wangshuibai major gene loci QFhs.nau6b can be obtained by mark Xbarc024 location, the anti-expansion of Wangshuibai major gene loci QFhs.nau-7b1 can be obtained by mark Xgwm533-170 location.Beneficial effect (1) the present invention obtains to have located the major gene loci of 3 anti gibberellic disease expansions in the wheat breed Wangshuibai, common soluble 28.3% scab resistance in the world first.The wheat cdna group is huge, is 40 times of rice genome size, and the accurate location work of wheat anti gibberellic disease major gene loci is occupy the same domain prostatitis; (2) major gene loci locality specific, it is convenient to identify.By detecting and the chain molecule markers of these anti-expansion major gene locis, can define the scab resistance that no anti gibberellic disease is expanded major gene loci and predicted wheat plant, and then rapid screening disease-resistant variety or strain are used for wheat breeding.Major gene loci easy to detect fast, not affected by environment; (3) the assistant breeding select target is clear and definite, saves cost.In traditional breeding way, at first to collect parent and Cultivar and carry out a series of hybridization, and will carry out individual plant to scab resistance and select with disease-resistant gene.Wheat scab is carried out phenotypic evaluation will wait until blooming stage, be subjected to bigger environmental influence, the result reliability of phenotypic evaluation is low.Therefore breeding for disease resistance is not only time-consuming, and difficulty is big, the cost height.By detecting the anti gibberellic disease major gene loci, can just identify the individual plant of high anti gibberellic disease in seedling stage, eliminate other plant, not only save production cost but also improve the efficiency of selection of wheat-resistance to scab greatly.
Four, the interval distribution plan of description of drawings .1 Wangshuibai linkage chromosome map and major gene loci
The left side is the chromosomal inheritance linkage map, and the map distance between mark marks with data; The graphic representation on the right is the interval graph of major gene loci, and two straight lines are respectively the expected value and remarkable value that has major gene loci, exceed the second straight line and promptly show major gene loci of existence.
Five, detailed embodiment is of the present invention is described in detail as follows:
Studies show that wheat scab resistance is controlled by the minority major gene loci mainly.U.S. Anderson laboratory finds that the scab resistance of No. 3/Stoa of four major gene locis and Soviet Union wheat colony is significantly relevant, lays respectively at 2Al, 3BS, 4BS and 6BS and goes up (Anderson, 1998; Waldron, 1999; Anderson, 2000,2001).People such as U.S. Bai obtain two the scab resistance sites relevant with the RAPD mark, and Zhou integrated use SSR, AFLP in 2002 and aneuploid technology are positioned at this major gene loci that to lack be that 3BS-8 does not hold a last length to be about the chromosomal region of 8cM.Japan Ban infers that the resistance main effect gene locus of Soviet Union wheat 3 may be positioned on the 5AL.Austrian Buerstmayr (2000,2001) finds that three genome areas and the anti-extendability significant correlation of head blight are arranged, and is positioned on 3BS, 5A, the 1B.The present invention finds, has 3 anti-expansion major gene locis respectively on 6B, the 7B of Wangshuibai and 7D karyomit(e).These major gene locis can be used to instruct the seed selection work of anti gibberellic disease kind, but screen with chain with it molecule marker enantiopathy kind, make different disease-resistant major gene loci rapid polymerizations in same plant, thereby accelerate breeding efficiency greatly.Materials and methods: the structure and the phenotypic evaluation of (one) Wangshuibai RIL: (1) is that acquisition F1 is hybridized in Nanjing University 2419 (♂) to the choosing of China's Jiangsu local variety Wangshuibai (♀) and Italian wheat breed Mentana, the F1 selfing produces F2,136 optional plantations of F2 individual plant, the method that adopts simple grain to pass is bred F6 generation, obtain RIL, comprise 136 familys; (2) RIL is planted in Agricultural University Of Nanjing in experimental plot, the greenhouse and academy of agricultural sciences, Jiangsu Province in the school.Blooming stage carries out scab resistance to each family to be identified.Adopt single flower instillation, promptly choose the small ear of just having bloomed in wheat head middle part, instiling contains mixed bacteria liquid 20 microlitres of F4, F15, several strong gibberella spores that cause a disease of F17, F34, and atomizing was preserved moisture 3 days then.Inoculate after 21 days, measure state of an illness indexs such as disease spikelet number and sick joint length.(2) molecular marker analysis of recombinant inbred lines
(1) each strain of SDS method extraction recombinant inbred lines is DNA;
(2) at first Wangshuibai and 2419 parents' of Nanjing University dna polymorphism is carried out initial analysis with 976 pairs of SSR primers.The PCR reaction volume is 25 microlitres, 10 * buffer, 2.5 microlitres wherein, and 25m,MMg,Cl2 1.5 microlitres, 2.5mM dNTPs 2 microlitres, Taq enzyme (5 units/microlitre) 0.2 microlitre, template DNA 20 nanograms add water to 25 microlitres.After the SSR reaction system is 94 ℃ of pre-sex change 3min of DNA, 94 ℃ of sex change 1min, 60 ℃ of annealing 1min, 72 ℃ are extended exhibition 2min, circulate 35 times, and last 72 ℃ are extended 10min.In the enterprising performing PCR amplification of PE 9600 amplification instrument, amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel (containing 7.6 gram acrylamides and 0.4 gram methylene diacrylamide in the 100ml polyacrylamide solution), on ultraviolet transilluminator, take a picture then, the record result, there is polymorphic primer in recombinant inbred lines, to analyze between the parent, obtains colony's genotype data;
(3) according to chain exchange rule, utilize colony's genotype data to make up the genetic map of wheat, used software is Mapmaker 2.0, minimum LOD value is made as 3, obtains linkage map;
(4) v.5.0 software and Map Manager QTX software carry out linkage analysis, the location major gene loci to colony's genotype data of each molecule marker with sick spikelet number and sick joint length that the scab resistance of its corresponding each family is identified to utilize Data desk.(3) result and analysis:
Molecular marker screening is the result show, has 383 pairs of SSR primers variant between parents.Utilize Datadesk v.5.0 software carry out linkage analysis to colony's genotype data of each molecule marker with sick spikelet number and sick joint length that the scab resistance of its corresponding each family is identified, One-way ANOVA records and the incoherent probability P value of scab resistance and the site contribution rate R to scab resistance
2(table 2), the molecule marker of P＜0.05 are promptly chain with a major gene loci.The genotype of gained molecule marker in colony is by the genotype classification of parent's Wangshuibai and Nanjing University 2419, if the strain that genotype is identical with Wangshuibai is a phenotype is anti-, then the localized disease-resistant major gene loci of this mark is from Wangshuibai: find the mark Xgwm533-170 of mark Xbarc024, P=0.0016 of P=0.0003 and the mark Xbarc076 and anti-expansion major gene loci close linkage of P=0.0026 in the Wangshuibai, promptly obtain the mark location of the anti-expansion of wheat Wangshuibai major gene loci QFhs.nau-6b, QFhs.nau-7b1 and QFhs.nau-7d; Perhaps the linkage group that major gene loci is arranged is carried out interval mapping analysis with Map Manager QTX software, the peak of curve position is a major gene loci, the anti-expansion of Wangshuibai major gene loci QFhs.nau6b can be obtained by mark Xbarc024 location, the anti-expansion of Wangshuibai major gene loci QFhs.nau-7b1 can be obtained by mark Xgwm533-170 location.
Be tested and appraised above-mentioned major gene loci and predict the wheat plant resistance, expectation can improve the breeding process of China's disease-resistant wheat kind rapidly.
The sequence of table 1. labeled primer and amplified fragments size
Clip size mark left end primer sequence right-hand member primer sequence
(bp)Xbarc024 CGCCTCTTATGGACCAGCCTAT GCGGTGAGCCATCGGGTTACAAAG 191Xgwm533-170AAGGCGAATCAAACGGAATA GTTGCTTTAGGGGAAAAGCC 170Xbarc076 ATTCGTTGCTGCCACTTGCTG GCGCGACACGGAGTAAGGACACC 191
The One-way ANOVA of the anti-expansion of table 2. Wangshuibai major gene loci
The long major gene of the sick joint of sick spikelet number
Mark 2,002 2,003 2,002 2003 sites
P R
2(％) P R
2(％) P R
2(％) P R
2(％)QFhs.nau
Xbarc024 0.0003 16.6-6bQFhs.nau Xgwm533- 0.005 7.5 0.0016 8.6 0.0001 13.4 0.0018 8.4-7b1 170QFhs.nau
Xbarc076 0.072 4.7 0.0026 12.2 0.0621 5.0 0.0444 5.6-7dP: expression and the incoherent probability of scab resistance, P＜0.05 shows this mark and scab resistance close linkage R
2: the site is to the contribution rate of scab resistance, value is big more show relevant more with scab resistance
Claims (3)
1, wheat Wangshuibai anti gibberellic disease expansion major gene loci is characterized in that: major gene loci QFhs.nau-6b, and by mark Xbarc024 location,
Left end primer sequence CGCCTCTTATGGACCAGCCTAT
Right-hand member primer sequence GCGGTGAGCCATCGGGTTACAAAG
Amplified fragments 191bp, this major gene loci are positioned at wheat Wangshuibai 6B karyomit(e), utilize Data desk v.5.0 software record with the incoherent probability P value of scab resistance be 0.0003, to the contribution rate 16.6% of scab resistance; Major gene loci QFhs.nau-7b1, by mark Xgwm533-170 location,
Left end primer sequence AAGGCGAATCAAACGGAATA
Right-hand member primer sequence GTTGCTTTAGGGGAAAAGCC
Amplified fragments 170bp, this major gene loci are positioned at wheat Wangshuibai 7B karyomit(e), utilize Data desk v.5.0 software record with the incoherent probability P value of scab resistance be 0.0016, to the contribution rate 8.6% of scab resistance; Major gene loci QFhs.nau-7d, by mark Xbarc076 location,
Left end primer sequence ATTCGTTGCTGCCACTTGCTG
Right-hand member primer sequence GCGCGACACGGAGTAAGGACACC
Amplified fragments 191bp, this major gene loci are positioned at wheat Wangshuibai 7D karyomit(e), utilize Data desk v.5.0 software record with the incoherent probability P value of scab resistance be 0.0026, to the contribution rate 12.2% of scab resistance.
2, wheat Wangshuibai anti gibberellic disease expansion major gene loci according to claim 1, it is characterized in that: these major gene locis are that to obtain mark by the following method localized: (1) wheat breed Wangshuibai (♀) is hybridized with Nanjing University 2419 (♂) and is obtained hybrid F1, the F1 selfing produces F2, adopts simple grain transmission method (SSD) to obtain the F6 RIL in generation then; (2) extract the DNA that each strain of recombinant inbred lines is with the SDS method, adopt simple repeated sequence mark SSR that two parents are carried out the polymorphism screening, PCR carries out on PE9600 amplification instrument, amplified production carries out the electrophoretic separation analysis on 8% (g/ml) polyacrylamide gel, according to the molecular marker screening result, filtering out has polymorphic primer between the parent, has polymorphic primer to analyze in recombinant inbred lines between the parent, the pcr amplification program is the same, obtains colony's genotype data; (3) according to chain exchange rule, utilize colony's genotype data to make up the genetic map of wheat, used software is Mapmaker 2.0, minimum LOD value is made as 3, obtains linkage map; (4) blooming stage inoculation head blight pathogenic bacteria carries out scab resistance to each family of RIL and identifies, obtains the sick spikelet number and the sick joint length of each family of RIL; (5) utilize Data desk v.5.0 software sick spikelet number and sick joint length that the colony's genotype data of each molecule marker and the scab resistance of its corresponding each family are identified carry out linkage analysis, One-way ANOVA records and the incoherent probability P value of scab resistance, the molecule marker of P＜0.05 is promptly chain with a major gene loci, the position of anti gibberellic disease expansion major gene loci is determined by the chromosome position of molecule marker: the mark Xbarc024 that finds P=0.0003 in the Wangshuibai, the mark Xgwm533-170 of P=0.0016 and the mark Xbarc076 of P=0.0026 and anti-expansion major gene loci close linkage promptly obtain the anti-expansion of wheat Wangshuibai major gene loci QFhs.nau-6b, the mark location of QFhs.nau-7b1 and QFhs.nau-7d; Perhaps the linkage group that major gene loci is arranged is carried out interval mapping analysis with MapManager QTX software, the peak of curve position is a major gene loci, the anti-expansion of Wangshuibai major gene loci QFhs.nau6b can be obtained by mark Xbarc024 location, the anti-expansion of Wangshuibai major gene loci QFhs.nau-7b1 can be obtained by mark Xgwm533-170 location.
3, the molecule marker of claim 1 or 2 described wheat Wangshuibai anti gibberellic diseases expansion major gene locis is characterized in that: be positioned at anti gibberellic disease expansion major gene loci QFhs.nau-6b, QFhs.nau-7b1 on 6B, 7B and the 7D and the molecule marker of QFhs.nau-7d in the Wangshuibai and be respectively Xbarc024, Xgwm533-170 and Xbarc076.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100336905C (en) * | 2004-05-08 | 2007-09-12 | 南京农业大学 | Wheat fertility recovery gene molecular mark and its obtaining method |
| CN102586291A (en) * | 2011-12-20 | 2012-07-18 | 南京农业大学 | Receptor protein kinase gene and expression vector and application thereof |
| CN102599047A (en) * | 2012-03-28 | 2012-07-25 | 南京农业大学 | Method by adopting molecular marker-assisted backcross to improve gibberellic disease expansion resistance of wheat |
| CN103954231A (en) * | 2014-03-28 | 2014-07-30 | 电子科技大学 | Non-contact measuring method for deformed plate and strip cross section profile in cold roll forming process |
| CN105543239A (en) * | 2016-01-22 | 2016-05-04 | 南京农业大学 | Wheat gibberellic-disease-resistant gene Tafhb1 and application thereof |
| CN107338310A (en) * | 2017-07-31 | 2017-11-10 | 中国农业科学院作物科学研究所 | A kind of mark and application method for detecting wheat anti gibberellic disease gene PFT |
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- 2003-06-23 CN CN 03131963 patent/CN1246479C/en not_active Expired - Fee Related
Cited By (9)
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| CN100336905C (en) * | 2004-05-08 | 2007-09-12 | 南京农业大学 | Wheat fertility recovery gene molecular mark and its obtaining method |
| CN102586291A (en) * | 2011-12-20 | 2012-07-18 | 南京农业大学 | Receptor protein kinase gene and expression vector and application thereof |
| CN102599047A (en) * | 2012-03-28 | 2012-07-25 | 南京农业大学 | Method by adopting molecular marker-assisted backcross to improve gibberellic disease expansion resistance of wheat |
| CN103954231A (en) * | 2014-03-28 | 2014-07-30 | 电子科技大学 | Non-contact measuring method for deformed plate and strip cross section profile in cold roll forming process |
| CN103954231B (en) * | 2014-03-28 | 2016-08-17 | 电子科技大学 | The contactless measurement of Deformation in Cold-Bend Forming strip cross-sectional profiles |
| CN105543239A (en) * | 2016-01-22 | 2016-05-04 | 南京农业大学 | Wheat gibberellic-disease-resistant gene Tafhb1 and application thereof |
| CN105543239B (en) * | 2016-01-22 | 2018-12-28 | 南京农业大学 | A kind of wheat anti gibberellic disease gene Tafhb1 and its application |
| CN107338310A (en) * | 2017-07-31 | 2017-11-10 | 中国农业科学院作物科学研究所 | A kind of mark and application method for detecting wheat anti gibberellic disease gene PFT |
| CN114600767A (en) * | 2022-04-07 | 2022-06-10 | 四川省农业科学院作物研究所 | Method for rapidly breeding wheat durable stripe rust resistant variety |
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