CN103146699B - Molecular marker MAG7237 of wheat fusarium head blight infection resistant gene Fhb4 and application of molecular marker MAG7237 - Google Patents
Molecular marker MAG7237 of wheat fusarium head blight infection resistant gene Fhb4 and application of molecular marker MAG7237 Download PDFInfo
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Abstract
The invention belongs to the field of breeding of plants and discloses a molecular marker MAG7237 of a wheat fusarium head blight infection resistant gene Fhb4 and application of the molecular marker MAG7237. The genetic distance between the molecular marker MAG7237 and the Fhb4 gene is 0.34 cm. The molecular marker most closely linked with the Fhb4 is acquired by the invention for the first time in the world. The molecular marker is a co-dominant marker with the advantages of being convenient to detect, and stable and simple to amplify; by adopting the marker MAG7237 to detect the Fhb4 gene, the existence of the Fhb4 or not and the existence state can be determined, and the fusarium head blight resistance of the wheat can be forecasted; and furthermore, the plants with the Fhb4 can be fast screened and used for breeding of disease-resistant varieties.
Description
The application is that the name of application on March 27th, 2012 is called " molecule marker MAG7237 and application thereof that wheat anti gibberellic disease infects gene Fhb4 ", the divisional application that application number is 201210082937.9.
Technical field
The invention belongs to breeding of plants field, the molecule marker and the application thereof that relate to wheat anti gibberellic disease and infect gene Fhb4.
Technical background
Wheat is one of most important food crop in the world, and the mankind about 20% energy is provided.In Wheat Production, many biologies and abiotic stress directly affect the yield and quality of wheat.Head blight is exactly the biological adverse circumstance of most important one wherein, has a strong impact on the quality and yield of wheat.The Breeding and application of disease-resistant variety is to control the most economical effective means of head blight, and the excavation of disease-resistant gene is prerequisite and the basis of breeding for disease resistance, and exploitation and the closely linked molecule marker of disease-resistant gene are applied the key of disease-resistant gene especially.
Wheat mainly contains five types (Mesterhazy1995) to the resistance of head blight, wherein anti-ly infects (Type I) and anti-expansion (Type II) (Schroeder and Christensen1963) is topmost two types.Type I resistance mainly reflects that wheat opposing the first of pathogenic bacteria infect, and is the first barrier that wheat is resisted gibberella harm, in resistance reaction, plays vital effect.
Much research shows that wheat is typical quantitative character to the resistance of head blight, is subject to controlled by multiple genes.Locate up to now more than 200 anti gibberellic disease QTL, be wherein arranged in anti gibberellic disease on 4B karyomit(e) and infect QTL and be present in multiple resistance germplasms, expressed stable and effect is stronger.Lin et al(2006) utilize large 2419 recombinant inbred lines in Wangshuibai × south on the 4B of Wangshuibai karyomit(e), to detect that one anti-is infected main effect QTL, the phenotypic variation of soluble 15% left and right, and in each time is tested LOD value all more than 3.Xue et al.(2010a) utilize near isogenic line to confirm the effect of this QTL.Xue et al.(2010b) utilize recombinant inbred lines and near isogenic line colony, 4B QTL Fine Mapping is arrived the Xhbg226 – Xgwm149 interval at a distance of 1.7cM by method by screening and identification recombinant chou, being positioned at wheat disappearance is 4BL5-0.86 – 1.00, and by its called after Fhb4.And there are some researches show that this gene is dominant gene.
Because wheat scab resistance has typical quantitative character feature, be subject to controlled by multiple genes, easily affected by environment, early generation is difficult to it to select.In addition, the economical character of many anti gibberellic disease germplasms is poor, and regional adaptability is stronger, utilizes traditional breeding way cultivation resistant variety to waste time and energy and may also can not get expected result.Molecular marker assisted selection breeding is compared with traditional breeding method, can determine from generation to generation in early days target gene type, and select not to be subject to the impact of genetic expression and envrionment conditions, can improve the accuracy of selection, thereby accelerate breeding process (Collard and Mackill2008).Although existing research and utilization Molecular Marker Assisted Selection Technology seed selection at present the near isogenic line of Fhb4, the Fhb4 near isogenic line plant height of institute's seed selection is higher, this is disadvantageous selection (Xue et al.2010a) in breeding.Therefore Linkage drag when more the exploitation of compact linkage molecule mark is conducive to reduce selection with it, improves the application efficiency to Fhb4.In addition, although Fhb4 by Fine Mapping, the genetic distance between location is still very large, is necessary that the more closely linked molecule marker of exploitation accelerates the clone research of this gene.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, provide with wheat anti gibberellic disease and infect the more closely linked molecule marker of gene Fhb4.
The application that another object of the present invention is to provide this wheat anti gibberellic disease and infects the molecule marker of gene Fhb4.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
Wheat anti gibberellic disease infects the molecule marker of gene Fhb4, with labeled primer MAG5184-F:SEQ ID NO.1, MAG5184-R:SEQ ID NO.2 amplification wheat breed genomic dna, the amplified fragments obtaining is 241bp, be wheat anti gibberellic disease infect gene Fhb4 chain molecule marker MAG5184, this is labeled as codominant marker, and with Fhb4 close linkage, the genetic distance that utilizes Mapmaker Macintosh V3.0 to record this mark and Fhb4 gene is 0.72cM;
Or with labeled primer MAG7237-F:SEQ ID NO.3, MAG7237-R:SEQ ID NO.4 amplification wheat breed DNA, the amplified fragments obtaining is 688bp, be wheat anti gibberellic disease and infect the chain molecule marker MAG7237 of gene Fhb4, this this be marked in anti gibberellic disease kind Wangshuibai and sense head blight kind Mianyang 99-323 with HinfI enzyme cut digest exist polymorphic, this is labeled as codominant marker, with Fhb4 close linkage, the genetic distance that utilizes Mapmaker Macintosh V3.0 to record this mark and Fhb4 gene is 0.34cM;
Or with labeled primer MAG2521-F:SEQ ID NO.5, MAG2521-R:SEQ ID NO.6 amplification wheat breed DNA, the amplified fragments obtaining is 161bp, be wheat anti gibberellic disease and infect the chain molecule marker MAG2521 of gene Fhb4, this is labeled as codominant marker, with Fhb4 close linkage, the genetic distance that utilizes Mapmaker Macintosh V3.0 to record this mark and Fhb4 gene is 0.92cM.
Described molecule marker anti gibberellic disease in Wheat Germplasm Resources infects the application in the qualification of gene Fhb4.
Wheat anti gibberellic disease infects the molecule marking method of gene Fhb4, with the above-mentioned any a pair of molecule marker primer PCR wheat cdna group DNA to be checked that increases, and detect amplified production, if can amplify the amplified fragments of 241bp with the primer MAG5184-F of molecule marker MAG5184 and MAG5184-R, or amplify the amplified fragments of 688bp with the primer MAG7237-F of molecule marker MAG7237 and MAG7237-R, or can amplify the amplified fragments of 161bp with the primer MAG2521-F of molecule marker MAG2521 and MAG2521-R, indicate that wheat to be checked exists anti gibberellic disease to infect gene Fhb4.
The application of molecule marker of the present invention in 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 detect amplified production, if can amplify the amplified fragments of 241bp with the primer MAG5184-F of molecule marker MAG5184 and MAG5184-R, or amplify the amplified fragments of 688bp with the primer MAG7237-F of molecule marker MAG7237 and MAG7237-R, or can amplify the amplified fragments of 161bp with the primer MAG2521-F of molecule marker MAG2521 and MAG2521-R, indicate that wheat to be checked is to exist anti gibberellic disease to infect the wheat-resistance to scab of gene Fhb4.
The primer of described molecule marker infects the application in gene Fhb4 at clone's anti gibberellic disease.
The molecule marker that above-mentioned wheat anti gibberellic disease infects gene Fhb4 obtains by the following method:
(1) Wangshuibai Fhb4 near isogenic line NMAS007 and its recurrent parent Mianyang 99-323F
2:3the screening of the establishment of colony and Fhb4 section recombinant chou:
(1) NMAS007(♀) and breed of wheat Mianyang 99-323
hybridize and obtain hybrid F
1, F
1selfing produces F
2colony;
(2) utilize the boundary marker of Fhb4 to screen F
2the heterozygosis individual plant that restructuring occurs at this section in colony, utilizes same tag at its F
3there is the individual plant that isozygotys of restructuring at this section in generation screening.
(2) qualification of the disease-resistant phenotype of recombinant chou
(3) recombinant chou is bloomed and is broadcasted sowing susceptible wheat inoculation in field about front ten days, after one week, repeats once.Inoculate 15 days sick small ear rates of " Invest, Then Investigate " and evaluate its anti-infectivity;
(3) molecular marker analysis
(4) extract disease-resistant parent NMAS007, Susceptible parent Mianyang 99-323 and F by SDS method
2the DNA of the each individual plant of colony; With 27 pairs of SSR primers (http://wheat.pw.usda.gov/GG2/index.shtml) with come from 59 couples of molecule marker primer pair NMAS007 of Wangshuibai BAC clone, Mianyang 99-323, China spring and China spring disappearance is 4BL-5(Endo and Gill1996) carry out polymorphism analysis and specificity analyses;
(5) be chosen between parent, amplify polymorphic and can deletion mapping to the molecule marker of 4BL5-0.86 – 1.00bin at F
2obtain the genotype data of the 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 the each individual plant of family;
Wherein, PCR reaction system is 12.5 μ l, wherein 10 × buffer1.25 μ l, 25mM MgCl
20.75 μ l, 2.5mMdNTPs1 μ l, the each 0.2 μ M of left and right primer, (5u/ μ is 0.1 μ l l), and template DNA 10ng adds water to 12.5 μ l for Taq enzyme;
After pcr amplification program is 94 DEG C of denaturation 3min, 94 DEG C of sex change 30sec, 60 DEG C of annealing 1min, 72 DEG C are extended 1min, circulate 35 times, and last 72 DEG C are extended 8min; In the enterprising performing PCR amplification of PE9600 amplification instrument, amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel, then on ultraviolet transilluminator, takes a picture, and records result.
(4) molecule marker obtains
(6) according to chain exchange rule, in conjunction with F
2for colony's each individual plant genotype data and heterozygosis recombinant chou F
3the disease-resistant phenotype in field of family, utilizes software Mapmaker Macintosh V3.0 to build the genetic linkage map of Wangshuibai Fhb4, has obtained and the most closely linked molecule marker MAG5184 of Fhb4, MAG7237 and MAG2521.
Beneficial effect:
The present invention has obtained and the closely linked molecule marker MAG5184 of Fhb4, MAG7237 and MAG2521 in the world first.Can accelerate the application of disease-resistant gene Fhb4 in wheat breeding for disease resistance, close and can also be used for the clone of Fhb4 gene.
1, obtained and the closely linked molecule marker MAG5184 of Fhb4, MAG7237 and MAG2521.Chain with Fhb4 in known molecule marker is MAG7237 the most closely, and genetic distance is only 0.34cM, can help this gene to shift in commercial variety and with the polymerization of other disease-resistant gene.
2, qualification is convenient.These three molecule markers are all codominant marker, have the advantages such as easy to detect, amplification is stable, easy.Detect Fhb4 gene with mark MAG7237, whether and existence the existence that can determine Fhb4, and predicts the scab resistance of wheat, and then rapid screening carries the plant of Fhb4 the seed selection for disease-resistant variety.Utilize molecule marker to carry out laboratory detection simultaneously and can avoid the impact of environment on kind.
3, improve the selection determination rates of disease-resistant variety, cost-saving.In traditional anti gibberellic disease breeding process, the parent and the Cultivar that carry disease-resistant gene need to be carried out to a series of hybridization, progeny population individual plant is carried out to Disease Resistance Identification, and need the phenotypic evaluation of multiple years; And disease-resistant phenotypic evaluation is subject to the impact of environment, phenotypic evaluation result has certain error.Therefore the seed selection of disease-resistant variety is not only time-consuming, and difficulty is large, and cost is high.Infect the closely linked molecule marker of gene Fhb4 by detecting with anti gibberellic disease, the work of phenotypic evaluation can be greatly reduced, and just can identify in seedling stage the individual plant that carries disease-resistant gene Fhb4, thereby eliminate non-target plant.Therefore, utilize by detecting with anti gibberellic disease and infect the closely linked molecule marker MAG7237 of gene Fhb4, not only save breeding cost, and greatly improve the efficiency of selection of disease-resistant variety.
4, reduce Linkage drag.Because the scab resistance of wheat often has certain being correlated with some bad economical characters.By the higher plant height of the total performance of the anti gibberellic disease strain that carries Fhb4 of molecular selection, this may be because the interval of QTL Primary Location is larger, causes and select the interval of importing larger, caused some Linkage drags in early days.Utilization and anti gibberellic disease infect the closely linked molecule marker of gene Fhb4 can reduce the Linkage drag while selection, improves efficiency of selection.
5, can be used for cloning anti gibberellic disease and infect gene Fhb4 research.The prerequisite that map based cloning anti gibberellic disease infects gene Fhb4 is to obtain and the closely linked molecule marker of Fhb4.MAG5184 and MAG7237 are chain the tightst with Fhb4 in all known molecular marks.For infecting gene Fhb4, clone's anti gibberellic disease provides basis.
Brief description of the drawings
The isozygoty distribution plan of the sick small ear rate of recombinant chou of 9 of Figure 145.
Fig. 2 MAG5184, MAG7237 and MAG2521 and wheat anti gibberellic disease infect the genetic linkage map of gene Fhb4, the mark that the right is genetic linkage map, and left data is the genetic distance between mark.
Fig. 3 MAG5184 banding pattern that increases.M is PUC19/MspI, and left side is molecular weight marker stripe size (bp).1 is Mianyang 99-323,2 is NMAS007,3,5,8,10,11,16,18,19 is susceptible genotype individual plant, and 6,7,22,23,24,26 is disease-resistant gene type individual plant, and 4,9,12,13,14,15,17,20,21,25 is heterozygous genes type individual plant.Arrow indication is specific amplified band.
Fig. 4 MAG7237 banding pattern that increases.M is D2000, and left side is molecular weight marker stripe size (bp).1 is NMAS007,2 is Mianyang 99-323,3,6,8,9,11,12,16 is susceptible genotype individual plant, and 5,17,18,19,20,22,23 is disease-resistant gene type individual plant, and 4,7,10,13,14,15,21,24,25,26 is heterozygous genes type individual plant.Arrow indication is specific amplified band.
Fig. 5 MAG2521 banding pattern that increases.M is PUC19/MspI, and left side is molecular weight marker stripe size (bp).1 is NMAS007, and 2 is Mianyang 99-323, and 2,3,4,5,10,11,14 is susceptible genotype individual plant, and all the other are heterozygous genes type individual plant.Arrow indication is specific amplified band.
Embodiment
The molecule marker that wheat anti gibberellic disease infects gene Fhb4 obtains by the following method:
(1) Wangshuibai Fhb4 near isogenic line NMAS007 and its recurrent parent Mianyang 99-323F
2:3the screening of the establishment of colony and Fhb4 section recombinant chou:
(1) NMAS007(♀) and breed of wheat Mianyang 99-323
hybridize and obtain hybrid F
1, F
1selfing produces the F that includes 4303 individual plants
2colony;
(2) utilize boundary marker Xhbg226 and the Xgwm149(Torada et al.2006 of Fhb4;
et al.1998) at F
2in colony, be sieved to 187 heterozygosis individual plants that restructuring occurs at this section, utilize same tag at its F
3generation screening obtains 459 individual plants that isozygoty that restructuring occurs at this section.
(2) qualification of the disease-resistant phenotype of recombinant chou
Recombinant chou is bloomed and is broadcasted sowing susceptible wheat in field to these recombinant chou inoculations of isozygotying about front ten days, after one week, repeats once.Inoculate 15 days sick small ear rates of " Invest, Then Investigate ".Qualification result shows: these recombinant chou resistances separate obviously (Fig. 1), carry the strain of Fhb4 gene compared with Susceptible parent, resistance be significantly increased (table 1).
(3) screening of polymorphic molecular marker and recombinant gene type analysis
(1) first utilize 59 molecule markers that are positioned at 27 SSR marks (http://wheat.pw.usda.gov/GG2/index.shtml) of Fhb4 section and the exploitation of Wangshuibai BAC cloned sequence in other collection of illustrative plates to carry out polymorphism screening to Fhb4 near isogenic line and Mianyang 99-323, finally obtaining 2 is having polymorphic SSR mark and 1 between parent, to have polymorphic STS mark, i.e. these three molecule markers of MAG5184, MAG2521 and MAG7237 between parent.
PCR reaction system is 12.5 μ l, wherein 10 × buffer1.25 μ l, 25mM MgCl
20.75 μ l, 2.5mM dNTPs1 μ l, the each 0.2 μ M of left and right primer, (5u/ μ is 0.1 μ l l), and template DNA 10ng adds water to 12.5 μ l for Taq enzyme;
After pcr amplification program is 94 DEG C of denaturation 3min, 94 DEG C of sex change 30sec, 60 DEG C of annealing 1min, 72 DEG C are extended 1min, circulate 35 times, and last 72 DEG C are extended 8min; In the enterprising performing PCR amplification of PE9600 amplification instrument, amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel, then on ultraviolet transilluminator, takes a picture, and records result.
(3) utilize and between parent, have polymorphic molecule marker MAG5184, MAG7237 and MAG2521 to analyze all recombinant chous that isozygoty, these recombinant chous can be divided into 4 kinds of recombinant types (table 1).
(4) acquisition of molecule marker
According to chain exchange rule, in conjunction with F
2for colony's each individual plant genotype data and heterozygosis recombinant chou F
3the disease-resistant phenotype in field (table 1) of family, utilize software Mapmaker Macintosh V3.0 to build the genetic linkage map (Fig. 2) of Wangshuibai Fhb4, obtained and the closely linked molecule marker MAG5184 of Fhb4, MAG7237 and MAG2521, they are respectively apart from Fhb4 gene 0.72cM, 0.34cM, 0.92cM.MAG5184, MAG7237 and MAG2521 molecule marker primer amplification banding pattern are shown in Fig. 3, Fig. 4 and Fig. 5.
Table 1NMAS007 – Mianyang 99-323F
2:3in colony, isozygoty genotype and the phenotype of recombinant chou
W represents Wangshuibai genotype, and M represents Mianyang 99-323 genotype, and C1 represents susceptible contrast Mianyang 99-323, and C2 represents disease-resistant contrast NMAS007.
*representative is remarkable with the disease-resistant phenotypic difference that contrasts NMAS007 in P=0.01 level.
Claims (5)
1. wheat anti gibberellic disease infects gene
fhb4the primer pair MAG7237-F/ MAG7237-R of molecule marker MAG7237, it is characterized in that: MAG7237-F sequence is as shown in SEQ ID NO.3, and MAG7237-R sequence is as shown in SEQ ID NO.4; With labeled primer MAG7237-F:SEQ ID NO.3, MAG7237-R:SEQ ID NO.4 amplification wheat breed DNA, the amplified fragments of acquisition is 688 bp, is wheat anti gibberellic disease and infects gene
fhb4chain molecule marker MAG7237, this is labeled as codominant marker, with
fhb4close linkage, utilize Mapmaker Macintosh V 3.0 record this mark with
fhb4the genetic distance of gene is 0.34 cM.
2. the primer pair of molecule marker MAG7237 claimed in claim 1 anti gibberellic disease in Wheat Germplasm Resources infects gene
fhb4qualification in application.
3. wheat anti gibberellic disease infects gene
fhb4molecule marking method, it is characterized in that the primer pair MAG7237-F/ MAG7237-R pcr amplification wheat cdna group to be checked DNA with molecule marker MAG7237 claimed in claim 1, and detect amplified production, if amplify the amplified fragments of 688 bp, indicate that wheat to be checked exists anti gibberellic disease to infect gene
fhb4.
4. the application of the primer pair MAG7237-F/ MAG7237-R of molecule marker MAG7237 claimed in claim 1 in screening wheat-resistance to scab.
5. application according to claim 4, it is characterized in that utilizing the molecule marker described in claim 1 in the method for screening wheat-resistance to scab to be: to amplify the amplified fragments of 688 bp with the primer pair MAG7237-F/ MAG7237-R of molecule marker MAG7237 claimed in claim 1, indicate that wheat to be checked is for existing anti gibberellic disease to infect gene
fhb4wheat-resistance to scab.
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| 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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| CN1462806A (en) * | 2003-06-23 | 2003-12-24 | 南京农业大学 | Molecule marker method of wheat Nanda 2419 scab resistant major gene locus |
| CN1462808A (en) * | 2003-06-23 | 2003-12-24 | 南京农业大学 | Molecule marker method of wheat Wangshuibai scab resistant major gene locus |
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| CN1462806A (en) * | 2003-06-23 | 2003-12-24 | 南京农业大学 | Molecule marker method of wheat Nanda 2419 scab resistant major gene locus |
| CN1462808A (en) * | 2003-06-23 | 2003-12-24 | 南京农业大学 | Molecule marker method of wheat Wangshuibai scab resistant major gene locus |
Non-Patent Citations (4)
| Title |
|---|
| Fine mapping Fhb4, a major QTL conditioning resistance to Fusarium infection in bread wheat (Triticum aestivum L.);Shulin Xue等;《Theor Appl Genet》;20100303;第121卷(第1期);147-156 * |
| Shulin Xue等.Fine mapping Fhb4, a major QTL conditioning resistance to Fusarium infection in bread wheat (Triticum aestivum L.).《Theor Appl Genet》.2010,第121卷(第1期),147-156. |
| 余桂红.小麦赤霉病抗性遗传分析即分子标记的开发.《中国博士学位论文全文数据库》.2008,(第7期),D047-4. |
| 小麦赤霉病抗性遗传分析即分子标记的开发;余桂红;《中国博士学位论文全文数据库》;20080715(第7期);D047-4 * |
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