CN102653759A - Molecular marker of wheat scab resistance expansion gene Fhbl and application thereof - Google Patents
Molecular marker of wheat scab resistance expansion gene Fhbl and application thereof Download PDFInfo
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- CN102653759A CN102653759A CN2012100851955A CN201210085195A CN102653759A CN 102653759 A CN102653759 A CN 102653759A CN 2012100851955 A CN2012100851955 A CN 2012100851955A CN 201210085195 A CN201210085195 A CN 201210085195A CN 102653759 A CN102653759 A CN 102653759A
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Abstract
The invention belongs to the field of crop breeding, and relates to a molecular marker of wheat scab resistance expansion gene Fhbl and application thereof. The molecular marker is one of MAG6067 and MAG6660 with a genetic distance to an Fhbl gene being respectively 0.05cM and 0cM. A molecular marker which is closest linked to the Fhbl for the first time internationally by utilizing the invention. The Fhbl gene can be detected by marking the MAG6660 to determine whether the Fhbl exists or not and the existing state of the Fhbl, and the scab resistance of wheat can be predicted, so that a plant carrying the Fhbl can be quickly screened; and the molecular marker can be used for breeding a scab-resistance variety. Furthermore, laboratory detection can be carried out by utilizing the molecular marker to avoid the influence of the environment to the phenotype. According to the molecular marker closely linked with the Fhbl, time and labor for screening scab resistance materials can be greatly saved, the prediction accuracy can be enhanced, and clone of the Fhbl gene can be accelerated.
Description
Technical field
The invention belongs to the breeding of plants field, relate to molecule marker and the application thereof of wheat anti gibberellic disease extension gene Fhb1.
Technical background
Wheat is one of most important food crop in the world, and human about 20% energy is provided.Many biologies and abiotic stress directly influence the yield and quality of wheat in Wheat Production.Head blight is exactly wherein most important a kind of biological adverse circumstance, has a strong impact on quality of wheat and output.The seed selection of disease-resistant variety and application are the most economical effective meanss of control head blight, and the excavation of disease-resistant gene is the prerequisite and the basis of breeding for disease resistance, and exploitation and the closely linked molecule marker of disease-resistant gene are used the key of disease-resistant gene especially.
Wheat mainly contains five types (Mesterhazy 1995) to the resistance of head blight, and wherein anti-expansion (Type I) and anti-expansion (Type II) (Schroeder and Christensen 1963) are topmost two types.Type I resistance mainly reflects the first expansion of wheat opposing pathogenic bacteria, is the first road barrier that wheat is resisted gibberella harm, in the resistance reaction, plays crucial effects.
Many researchs show that wheat is typical quantitative character to the resistance of head blight, receives controlled by multiple genes.About more than 200 anti gibberellic disease QTL have been located up to now; The anti gibberellic disease expansion QTL that wherein is arranged on the 3B karyomit(e) is present in a plurality of resistance germplasms; Expression is stable and effect is the strongest, and has good breeding for disease resistance potentiality (Liu and Anderson 2003).Liu et al. (2006) is interval to the XSTS3B-189-XSTS3B-206 at a distance of 1.2cM with this QTL Fine Mapping, and with its called after Fhb1.Lin et al (2006) utilizes Wangshuibai * Nanjing University's 2419 recombinant inbred lines on the 3B of Wangshuibai karyomit(e), also to detect this QTL, is positioned the Xbarc147-Xgwm493 interval, explains about 30% phenotypic variation.
Because wheat scab resistance has typical quantitative character characteristic, easy affected by environment, early generation is difficult to it is selected.In addition, the economical character of many anti gibberellic disease germplasms is relatively poor, and regional adaptability is stronger, utilizes traditional breeding way cultivation resistant variety to waste time and energy and possibly also can not get expected result.The molecular marker assisted selection breeding is compared with traditional breeding method; Can confirm the target gene type in early days from generation to generation; And select not receive the influence of genetic expression and envrionment conditions, can improve the accuracy of selection, thereby accelerate breeding process (Collard and Mackill 2008).Therefore with its more the exploitation of compact linkage molecule mark help improving application efficiency to Fhb1.In addition, the corresponding physical interval does not also know to have much in Wangshuibai between the present location of Fhb1, is necessary to develop clone's research that more closely linked molecule marker is accelerated this gene.
Summary of the invention
The objective of the invention is above-mentioned deficiency, the more closely linked molecule marker with wheat anti gibberellic disease extension gene Fhb1 is provided to prior art.
Another object of the present invention provides the application of the molecule marker of this wheat anti gibberellic disease extension gene Fhb1.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is following:
The molecule marker of wheat anti gibberellic disease extension gene Fhb1; With labeled primer MAG6067-F:SEQ ID NO.1, MAG6067-R:SEQ ID NO.2 amplification wheat breed DNA, the amplified fragments of acquisition is 280bp; Be the chain molecule marker MAG6067 of wheat anti gibberellic disease extension gene Fhb1; This is labeled as the dominance molecule marker, and with the Fhb1 close linkage, the genetic distance that utilizes Mapmaker Macintosh V 3.0 to record this mark and Fhb1 gene is 0.05cM;
Or with labeled primer MAG6660-F:SEQ ID NO.3; MAG6660-R:SEQ ID NO.4 amplification wheat breed DNA; The amplified fragments that obtains is 510bp, is the chain molecule marker MAG6660 of wheat anti gibberellic disease extension gene Fhb1, and this is labeled as the codominance molecule marker; With the Fhb1 close linkage, utilize Mapmaker Macintosh V 3.0 to record this mark and Fhb1 gene and be divided into and leave.
The application in identification of described molecule marker anti gibberellic disease extension gene Fhb1 in the wheat germplasm resource.
The molecule marking method of wheat anti gibberellic disease extension gene Fhb1; With the above-mentioned any a pair of molecule marker primer PCR wheat cdna group DNA to be checked that increases; And detection amplified production; If the amplified fragments that can amplify 280bp with primer MAG6067-F and the MAG6067-R of molecule marker MAG6067, the amplified fragments that perhaps can amplify 510bp with primer MAG6660-F and the MAG6660-R of molecule marker MAG6660 indicates that then there is anti gibberellic disease extension gene Fhb1 in wheat to be checked.
The application of molecule marker of the present invention in the screening wheat-resistance to scab.
Utilize the method for above-mentioned molecular marker screening wheat-resistance to scab to be: with the above-mentioned any a pair of molecule marker primer PCR wheat cdna group DNA to be checked that increases; And detection amplified production; If the amplified fragments that can amplify 280bp with primer MAG6067-F and the MAG6067-R of molecule marker MAG6067; The amplified fragments that perhaps can amplify 510bp with primer MAG6660-F and the MAG6660-R of molecule marker MAG6660 indicates that then wheat to be checked is the wheat-resistance to scab that has anti gibberellic disease extension gene Fhb1.
The application of the primer of described molecule marker in clone's anti gibberellic disease extension gene Fhb1.
The molecule marker of above-mentioned wheat anti gibberellic disease extension gene Fhb1 obtains through following method:
(1) Wangshuibai Fhb1 near isogenic line NMAS016 and its recurrent parent Mianyang 99-323F
2:3The screening of the establishment of colony and Fhb1 section recombinant chou:
(1) NMAS016 (♀) and breed of wheat Mianyang 99-323
Hybridize and obtain hybrid F
1, F
1Selfing produces F
2Colony;
(2) utilize the boundary marker of Fhb1 to screen F
2At the heterozygosis individual plant of this section generation reorganization, utilize same tag in the colony at its F
3The individual plant that isozygotys of reorganization takes place in the generation screening at this section.
(2) evaluation of the disease-resistant phenotype of recombinant chou
Adopt the inoculation of single flower instillation when (3) recombinant chou is bloomed.Inoculate and divide individual plant to investigate disease spikelet number and its anti-extendability of sick axial length evaluation after 15 days;
(3) molecular marker analysis
(4) extract disease-resistant parent NMAS016, susceptible parent Mianyang 99-323 and F with the SDS method
2The DNA of each individual plant of colony; With 69 SSR marks of China spring 3B sequence exploitation to NMAS016, Mianyang 99-323, China spring and China spring disappearance are that 3BS8-0.78-0.87 (Endo and Gill 1996) carries out polymorphism analysis and specificity analyses.
(5) be chosen in amplify polymorphic between the parent and can deletion mapping to the molecule marker of 3BS8-0.78-0.87bin at F
2Obtain the genotype data of each individual plant of colony for amplification in colony's individual plant, and utilize these marker detection heterozygosis recombinant chous offspring F
3The genotype of each individual plant of family;
The PCR reaction system is 12.5 μ l, 10 * buffer, 1.25 μ l wherein, 25mM MgCl
20.75 μ l, 2.5mMdNTPs 1 μ l, each 0.2 μ M of left and right sides primer, Taq enzyme (5u/ μ l) 0.1 μ l, template DNA 10ng adds water to 12.5 μ l;
After the pcr amplification program is 94 ℃ of preparatory sex change 3min, 94 ℃ of sex change 30sec, 60 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate 35 times, and last 72 ℃ are extended 8min; In the enterprising performing PCR amplification of PE9600 amplification appearance, amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel, on ultraviolet transilluminator, takes a picture the record result then.
(4) molecule marker obtains
(6) according to chain exchange rule, in conjunction with F
2For each individual plant genotype data of colony and heterozygosis recombinant chou F
3The disease-resistant phenotype in the field of family utilizes software Mapmaker Macintosh V3.0 to make up the genetic linkage map of Wangshuibai Fhb1, has obtained and the most closely linked molecule marker MAG6067 of Fhb1 and MAG6660.
Beneficial effect
The present invention has obtained and closely linked molecule marker MAG6067 of Fhb1 and MAG6660 in the world first.The application of disease-resistant gene Fhb1 in wheat breeding for disease resistance can be quickened, and the clone of Fhb1 gene can be used for.
1, obtained and closely linked molecule marker MAG6067 of Fhb1 and MAG6660.Chain with Fhb1 in known molecule marker is MAG6660 the most closely, is divided into the Fhb1 gene and leaves, can help this gene in commercial variety, to shift and with the polymerization of other disease-resistant gene.
2, identify conveniently.These two molecule markers are the codominant marker all, have easy to detect, stable amplification, advantage such as easy.Detect the Fhb1 gene with mark MAG6660, the existence that can confirm Fhb1 whether and existence, and the scab resistance of prediction wheat, and then rapid screening carries the plant of Fhb1 and is used for the seed selection of disease-resistant variety.Utilize the molecule marker chamber of experimentizing to detect simultaneously and can avoid the influence of environment kind.
3, efficient is identified in the selection that improves disease-resistant variety, practices thrift cost.In traditional anti gibberellic disease breeding process, the parent and the Cultivar that need to carry disease-resistant gene carry out a series of hybridization, the progeny population individual plant is carried out disease resistance identify, and need the phenotypic evaluation of multiple years; And disease-resistant phenotypic evaluation is subject to the influence of environment, and phenotypic evaluation result has certain error.Therefore the seed selection of disease-resistant variety is not only time-consuming, and difficulty is big, and cost is high.Through detecting and the closely linked molecule marker of anti gibberellic disease extension gene Fhb1, can the work of phenotypic evaluation be significantly reduced, and just can identify the individual plant that carries disease-resistant gene Fhb1, thereby eliminate non-target plant in seedling stage.Therefore, be total to isolating molecule marker MAG6660 through detection with anti gibberellic disease extension gene Fhb1 and select, not only practice thrift the efficiency of selection that the breeding cost improves disease-resistant variety simultaneously greatly.
4, can be used for cloning anti gibberellic disease extension gene Fhb1 research.The prerequisite of map based cloning anti gibberellic disease extension gene Fhb1 is to obtain and the closely linked molecule marker of Fhb1.MAG6067 and MAG6660 are chain the tightst with Fhb1 in all known molecular marks.
Description of drawings
The genetic linkage map of Fig. 1 MAG6067 and MAG6660 and wheat anti gibberellic disease extension gene Fhb1, the right are the mark of genetic linkage map, and left data is the genetic distance between mark.
Fig. 2 MAG6067 banding pattern that increases.1 is NMAS016, and 2 is Mianyang 99-323, and 3,4,5,6,12,18,19,20,21 is disease-resistant gene type individual plant, and all the other are susceptible or heterozygous genes type individual plant.The arrow indication is the specific amplified band.
Fig. 3 MAG6660 banding pattern that increases.M is PUM, and the left side is molecular weight marker stripe size (bp).1 is NMAS016, and 2 is Mianyang 99-323, and 3,8,15,16,17 is disease-resistant gene type individual plant, and 2,5,9,13,19,20 is susceptible genotype individual plant, and 4,6,7,10,11,12,14,18,21 is heterozygous genes type individual plant.The arrow indication is the specific amplified band.
Embodiment
The molecule marker of above-mentioned wheat anti gibberellic disease extension gene Fhb1 obtains through following method:
(1) Wangshuibai Fhb1 near isogenic line NMAS016 and its recurrent parent Mianyang 99-323F
2:3The screening of the establishment of colony and Fhb1 section recombinant chou:
(1) NMAS016 (♀) and breed of wheat Mianyang 99-323
Hybridize and obtain hybrid F
1, F
1Selfing produces the F that includes 2875 individual plants
2Colony;
(2) the boundary marker Xbarc147 and the Xgwm493 (http://wheat.pw.usda.gov/GG2/) that utilize Fhb1 are at F
2Be sieved to 71 heterozygosis individual plants that take place to recombinate at this section in the colony, utilize same tag at its F
3The generation screening obtains 99 individual plants that isozygoty that reorganization takes place at this section.
(2) evaluation of the disease-resistant phenotype of recombinant chou
When blooming, adopts recombinant chou the inoculation of single flower instillation.Inoculate and divide individual plant to investigate disease spikelet number and its anti-extendability of sick shaft length evaluation after 15 days.Qualification result shows: these recombinant chou resistances are separated obviously, and the strain system that carries the Fhb1 gene compares with susceptible parent, resistance be significantly increased (table 1).
(3) screening of polymorphic molecular marker and recombinant gene type analysis
(1) at first utilize 69 SSR flag F hb1 near isogenic line NMAS016 of China spring 3B sequence exploitation to carry out polymorphum with Mianyang 99-323 and screen, finally obtaining 2 has polymorphic SSR mark MAG6067 and MAG6660 between the parent.
(3) be utilized in polymorphic molecule marker BARC147 is arranged between the parent, GWM493, MAG6067 and MAG6660 analyze all recombinant chous that isozygoty, these recombinant chous only can be divided into 6 kinds of recombinant types (table 1).
(4) acquisition of molecule marker
According to chain exchange rule, in conjunction with F
2For each individual plant genotype data of colony and heterozygosis recombinant chou F
3The disease-resistant phenotype in the field of family (table 1); Utilize software Mapmaker Macintosh V3.0 to make up the genetic linkage map of Wangshuibai Fhb1; Obtained and closely linked molecule marker MAG6067 of Fhb1 and MAG6660, they are respectively apart from Fhb1 gene 0.05cM, 0cM.MAG6067 and MAG6660 molecule marker primer amplification banding pattern are seen Fig. 2 and Fig. 3.
Table 1NMAS016-Mianyang 99-323F
2:3Isozygoty in the colony genotype and the phenotype of recombinant chou
W represents the Wangshuibai genotype, and M represents Mianyang 99-323 genotype, and C1 represents susceptible contrast Mianyang 99-323, and C2 represents disease-resistant contrast NMAS016.
*Representative is remarkable with the phenotypic difference of disease-resistant contrast NMAS016 on the P=0.01 level.
Claims (6)
1. the molecule marker of wheat anti gibberellic disease extension gene Fhb1 is characterized in that:
With labeled primer MAG6067-F:SEQ ID NO.1; MAG6067-R:SEQ ID NO.2 amplification wheat breed DNA; The amplified fragments that obtains is 280bp, is the chain molecule marker MAG6067 of wheat anti gibberellic disease extension gene Fhb1, and this is labeled as the dominance molecule marker; With the Fhb1 close linkage, the genetic distance that utilizes Mapmaker Macintosh V 3.0 to record this mark and Fhb1 gene is 0.05cM;
Or with labeled primer MAG6660-F:SEQ ID NO.3; MAG6660-R:SEQ ID NO.4 amplification wheat breed DNA; The amplified fragments that obtains is 510bp, is the chain molecule marker MAG6660 of wheat anti gibberellic disease extension gene Fhb1, and this is labeled as the codominance molecule marker; With the Fhb1 close linkage, utilize Mapmaker Macintosh V 3.0 to record this mark and Fhb1 gene and be divided into and leave.
2. the application in identification of the described molecule marker of claim 1 anti gibberellic disease extension gene Fhb1 in the wheat germplasm resource.
3. the molecule marking method of wheat anti gibberellic disease extension gene Fhb1; It is characterized in that with the described any a pair of molecule marker primer PCR of the claim 1 wheat cdna group DNA to be checked that increases; And detection amplified production; If the amplified fragments that can amplify 280bp with primer MAG6067-F and the MAG6067-R of molecule marker MAG6067; The amplified fragments that perhaps can amplify 510bp with primer MAG6660-F and the MAG6660-R of molecule marker MAG6660 indicates that then there is anti gibberellic disease extension gene Fhb1 in wheat to be checked.
4. the application of the described molecule marker of claim 1 in the screening wheat-resistance to scab.
5. application according to claim 4; It is characterized in that utilizing the described molecule marker of claim 1 to be: with the described any a pair of molecule marker primer PCR of the claim 1 wheat cdna group DNA to be checked that increases in the method for screening wheat-resistance to scab; And detection amplified production; If the amplified fragments that amplifies 280bp with primer MAG6067-F and the MAG6067-R of molecule marker MAG6067; The amplified fragments that perhaps can amplify 510bp with primer MAG6660-F and the MAG6660-R of molecule marker MAG6660 indicates that then wheat to be checked is the wheat-resistance to scab that has anti gibberellic disease extension gene Fhb1.
6. the application of the primer of the described molecule marker of claim 1 in clone's anti gibberellic disease extension gene Fhb1.
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Cited By (5)
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| 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 |
| CN108359739A (en) * | 2018-04-24 | 2018-08-03 | 南京农业大学 | The SNP marker primer of one anti gibberellic disease related gene TaRLK-B and its application |
| CN108486278A (en) * | 2018-06-26 | 2018-09-04 | 江苏省农业科学院 | Primer pair and application for identifying peaceful wheat No. 9 and its derived varieties scab resistance |
| CN109402292A (en) * | 2018-12-12 | 2019-03-01 | 江苏省农业科学院 | A pair is for detecting primer and its application of wheat scab resistance |
| CN115927698A (en) * | 2022-07-15 | 2023-04-07 | 江苏省农业科学院 | Multiple PCR (polymerase chain reaction) marker primer group for simultaneously detecting wheat scab resistant genes Fhb1 and Fhb7 and application thereof |
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| CN101892307A (en) * | 2010-05-12 | 2010-11-24 | 江苏省农业科学院 | SSCP marker closely linked with major wheat scab resistance QTL and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108359739A (en) * | 2018-04-24 | 2018-08-03 | 南京农业大学 | The SNP marker primer of one anti gibberellic disease related gene TaRLK-B and its application |
| CN108359739B (en) * | 2018-04-24 | 2021-03-30 | 南京农业大学 | SNP (Single nucleotide polymorphism) marker primer of gibberellic disease resistant related gene TaRLK-B and application of SNP marker primer |
| CN108486278A (en) * | 2018-06-26 | 2018-09-04 | 江苏省农业科学院 | Primer pair and application for identifying peaceful wheat No. 9 and its derived varieties scab resistance |
| CN108486278B (en) * | 2018-06-26 | 2020-12-01 | 江苏省农业科学院 | Primer pair for identifying gibberellic disease resistance of Ningmai No. 9 and derivative variety thereof and application |
| CN109402292A (en) * | 2018-12-12 | 2019-03-01 | 江苏省农业科学院 | A pair is for detecting primer and its application of wheat scab resistance |
| CN109402292B (en) * | 2018-12-12 | 2020-12-11 | 江苏省农业科学院 | Primer pair for detecting wheat scab resistance and application thereof |
| CN115927698A (en) * | 2022-07-15 | 2023-04-07 | 江苏省农业科学院 | Multiple PCR (polymerase chain reaction) marker primer group for simultaneously detecting wheat scab resistant genes Fhb1 and Fhb7 and application thereof |
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Application publication date: 20120905 |