CN1775953A - Method for screening powdery-mildew-resistance wheat and its special primer - Google Patents
Method for screening powdery-mildew-resistance wheat and its special primer Download PDFInfo
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Abstract
The invention discloses a method to filter anti-powdery mildew wheat and the special primer that is made up from the nucleotide sequence of sequence 1 in sequence table and the nucleotide sequence of sequence 2. The method of filtering anti-powdery mildew wheat is that it makes the testing wheat gene group DNA as template, uses the sequence 1 and 2 of nucleotide sequence as primer, takes PCR extending to detect whether the extended product has banding that the size is 197bp and 193bp. The invention has great effect in filtering wheat anti-powdery mildew gene Pm16.
Description
Technical field
The present invention relates to a kind of method and primer special thereof that screens powdery-mildew-resistance wheat.
Background technology
Wheat powdery mildew is the fungal disease that is caused by wheat powdery mildew Blumeria graminis f.sp.tritici, and having become influences the important disease that world wheat is produced.Before the seventies in 20th century, China's wheat powdery mildew is only popular in moistening areas of heavy rainfull such as cloud, expensive, river and Shandong coastal waters, but nearly 30 years, because the improvement of water and fertilizer condition and the application of short-stalked variety, its occurrence scope and area constantly enlarge, and hazard rating obviously increases the weight of, become the main disease that influences China's Wheat Production, all there is generation nearly all wheat belt, and all cause greater loss to production every year, and popular time loss is heavier.Be very popular in the whole nation as Powdery Mildew in 1990 and 1991, area took place all above 1.8 hundred million mu in 2 years, 3,200,000,000 kilograms of year loss wheats, occurrence scope spreads all over each main Mai Qu of the whole nation.
Utilizing disease-resistant variety is the approach that the control wheat powdery mildew is most economical, effective, safety is easily gone, and seed selection is the first-selected measure of control disease popular with promoting disease-resistant variety.Doing a lot of work aspect the wheat powdery mildew breeding for disease resistance both at home and abroad, the disease-resistant variety of breeding has been brought into play certain effect to alleviating the disease loss.But because the selection of overemphasizing immunity or high anti-type in the quick variation of pathogenic bacteria microspecies and the breeding for disease resistance process, varietal resistance is frequently lost, and breeding for disease resistance is in the passive state of dealing with all the time.In Europe, after the mildew-resistance kind is promoted aborning, 2-3 then soon, slowly then 4-5 just lost resistance against diseases (Duan Xiayu, the toxicity monitoring of European wheat powdery mildew and the utilization of disease-resistant gene, plant protection, 20 (3): 36-38,1994).In China, the disease-resistant variety that cost is cultivated is for many years used soon, have in addition also use, with regard to owing to resistance is lost in the variation of germ colony.The enforcement period of the seventh five-year plan, the kind that contains Pm8 is lost resistance in the whole nation; " eight or five " and the enforcement period of the ninth five-year plan, Pm4a in Beijing, the toxicity frequency (33.3%-100%) in rising trend of Guizhou, Hebei, Henan, Shandong, Hubei and Jiangsu 7 provinces and cities, Pm6 is in the approaching forfeiture of the resistance of above-mentioned 7 provinces and cities, and its toxicity frequency is 91.3%-100%; Also found the own transformation success of China and the virose bacterial strain of disease-resistant gene Pm21 (Duan Xiayu, Zhou Yilin, the Sheng Baoxin that name, China's main wheat district wheat powdery mildew toxicity present situation, plant protection 21 century prospect, Beijing: China Science Tech Publishing House, 246-249,1998); The kind that contained the Pm2 gene in 2003 is experimental field also lost resistance at us.High mostly sense of the kind of present domestic popularization or middle sense Powdery Mildew press for better resistance and persistent kind in the production.
Practice shows that a plurality of effective disease-resistant genes that add up are to improve the disease-resistant broad spectrum of kind and prolong one of the effective means in varietal resistance life-span.But, be difficult to identify a plurality of genes material together by conventional means in the practices of breeding.(maker-assisted selection MAS), can identify concrete resistant gene quickly and accurately, thereby realizes the polymerization of disease-resistant gene to utilize molecular marker assisted selection.
Since the nineties, found the molecule marker of many disease-resistant genes of diseases such as wheat rust, head blight, Powdery Mildew.SSR (simple sequence repeat, being simple repeated sequence) mark is called little satellite (Microsatellite) mark again, because have the polymorphism height, the karyomit(e) specialization is strong, good reproducibility, simple operation and other advantages, and be widely used in the research such as molecule marker, genetic mapping of wheat, paddy rice, corn, Soybean and Other Crops.
The wheat master who has found is in the world at present imitated mildew-resistance gene 42, lays respectively at 32 gene locuss, in the middle of these genes, Pm2 only, Pm4b, Pm12, Pm13, Pm16, Pm20, Pm21, Pm30, Pm31 is effectively in China's most of wheat district resistance, and particularly the Pm16 gene is at wide, the strong resistance of the anti-spectrum of China.Yet, up to the present also do not find the molecule marker of Pm16 gene.Therefore, excavate the molecule marker of Pm16 gene, for cultivating multiple gene polymerization body material, cultivation durable resistance kind and further gene clone are significant.
Summary of the invention
The purpose of this invention is to provide a kind of primer that can be used for screening powdery-mildew-resistance wheat.
The primer that is used to screen powdery-mildew-resistance wheat provided by the present invention, name is called xgwm159, a pair of primer of being made up of the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 1 and sequence 2.
Second purpose of the present invention provides a kind of method of screening powdery-mildew-resistance wheat.
The method of screening powdery-mildew-resistance wheat provided by the present invention, be to be template with wheat cdna group DNA to be measured, classify primer as with the nucleotide sequence of sequence in the sequence table 1 and the nucleotides sequence of sequence 2, carry out pcr amplification, detect the band whether 197bp and 193bp size are arranged in the amplified production.
As the band of 197bp and 193bp size is arranged in the amplified production, wheat to be measured is a powdery-mildew-resistance wheat.
Wherein, be example with 25 μ l cumulative volumes, the reaction system of pcr amplification comprises: template DNA (20ng/ μ l) 5 μ l, Taq enzyme (5U/ μ l) 0.2 μ l, primer is to (2 μ M) 3 μ l, dNTPs (2.5mM) 0.5 μ l, 10 * PCR damping fluid, 2.5 μ l, sterile distilled water 13.8 μ l.The composition of 10 * PCR damping fluid is Tris-HCl (pH8.3) 100mM, KCl 500mM, MgCl
215mM.Response procedures is 94 ℃ of pre-sex change 5min, carries out 35 circulations subsequently: 94 ℃ of 40s, 60 ℃ of 30s, 72 ℃ of 40s; Last 72 ℃ are extended 10min, 4 ℃ of preservations.
The method of described detection amplified production can be carries out 6% denaturing polyacrylamide gel electrophoresis to amplified production, silver dyes colour developing then.Specifically can be: add the load sample indicator (98% methane amide, 10mM EDTA pH8.0,0.25% tetrabromophenol sulfonphthalein and 0.25% dimethylbenzene green grass or young crops) of 8 μ l sex change in every part of amplified production, 95 ℃ of sex change 5-10mins.The amplification sample 7 μ l of every part of sex change are electrophoretic separation in 6% the denaturing polyacrylamide gel in concentration, and silver dyes colour developing.Detect the band whether 197bp and 193bp size are arranged in the amplified production.
Because the Pm16 gene had been positioned in (Reader on the wheat 4A karyomit(e) in the past, S.M., and T.E.Miller, 1991:The introduction into bread wheat of a major gene for resistanceto powdery mildew from wild emmer wheat.Euphytica 53,57-60.), the contriver selects 16 pairs of SSR primers that are positioned on the 4A karyomit(e) for use, and 15 pairs of SSR primers on paddy rice the 3rd karyomit(e) of portion homologous relation is arranged to wheat lines 70281 (containing disease-resistant gene Pm16) with wheat 4A karyomit(e), Chanceller (susceptible), disease-resistant pond, increase in susceptible pond.The result shows that the amplified production of all primers between disease-resistant pond and susceptible pond all do not have polymorphism, only has 4 pairs of primers that polymorphism is arranged between the parent, is not inconsistent with expected result.Because (Naranjo such as Naranjo, T., A.Roca, P.G.Goicoechea, and R.Giraldez, 1987:Arm homoeology of wheat and rye chromosomes.Genome 29 873-882) finds in the wheat evolutionary process the 4th, exchange has taken place in 5 and 7 homologous chromosomes groups, has obtained people (Nelson such as Nelson afterwards, J.C., M.E.Sorrells, A.E.V.Deynze, Y.H.Lu, M.Atkinson, M.Bernard, P.Leroy, J.D.Faris and J.A.Anderson, 1995:Molecular mapping of wheat:Major genes and rearrangments in homoeologous groups 4,5, and 7.Genetics141, confirmation 721-731.).So further select for use 34 pairs of SSR primers on wheat 4B, 5A, 5B, 7A and the 7B karyomit(e) to susceptible variety Chanceller, contain disease-resistant strain 70281 (PM16/ Beijing 837 of Pm16
3) and with the F of these two parent's cross combinations
2Carry out molecular marker screening and genetic linkage analysis for segregating population, the result shows that all there is tangible polymorphism anti-, sense parent and anti-, sense in primer xgwm159 between the pond, disease-resistant parent and disease-resistant pond all have 197bp and 193bp two bands, susceptible parent and susceptible pond all have 193bp and 191bp two bands, wherein the characteristic strip of 197bp is peculiar by disease-resistant sick parent and disease-resistant pond, and with mildew-resistance gene Pm16 close linkage, its genetic distance is 5.3cM.Through China spring nullisomic-limbs analysis, proved that the Pm16 gene is positioned at wheat 5B the short arm of a chromosome (5BS), be not 4A karyomit(e).And verify that with 7 susceptible wheat breeds and 16 disease-resistant varieties (being) that contain known disease-resistant gene the result shows that SSR molecule marker xgwm159 is the fine mark of powdery mildew resistance gene in wheat Pm16.
The invention provides the SSR molecule marker xgwm159 of powdery mildew resistance gene in wheat Pm16, this mark and Pm16 gene close linkage (5.3cM), the Pm16 gene is at wide, the strong resistance of the anti-spectrum of China, utilize this mark to carry out molecular marker assisted selection by enantiopathy gene Pm16, thereby realization adds up with other effective disease-resistant gene, prolongs the disease resistance of kind.Method of the present invention and primer special thereof will play a significant role in the screening of powdery mildew resistance gene in wheat Pm16 and wheat breeding for disease resistance.
Description of drawings
Fig. 1 a is micro-satellite primers xgwm159 antagonism, sense parent, resists, feels between the pond and F
2The amplification of colony's individual plant
Fig. 1 b is micro-satellite primers Borc004 antagonism, sense parent, resists, feels between the pond and F
2The amplification of colony's individual plant
Fig. 1 c is micro-satellite primers Borc109 antagonism, sense parent, resists, feels between the pond and F
2The amplification of colony's individual plant
Fig. 2 is the linkage map of SSR mark and disease-resistant gene Pm16
Fig. 3 is the pcr amplification product electrophorogram of primer xgwm159 to part nullisomic-limbs material of the 4th, 5 homology groups of China spring
Fig. 4 a-Fig. 4 b is that primer xgwm159 is to 7 susceptible variety and 16 pcr amplification product electrophorograms that contain the disease-resistant variety of known disease-resistant gene
Embodiment
Unreceipted wheat lines source person is all available from national germplasm resource bank among the embodiment.
The acquisition of the SSR molecule marker xgwm159 of embodiment 1, powdery mildew resistance gene in wheat Pm16
1, with No. 15 physiological strains of North China's popular powdery mildew to " (cross combination is PM16/ Beijing 837 in Chanceller * 70281
3) F
2Carry out resistance evaluation in seedling stage for colony, identify F altogether
2Colony's 156 strains, wherein disease-resistant 116 strains, susceptible 40 strains meet 3: 1 anti-sense segregation ratio (x
2=0.286＜x
2 0.01=6.63), show that 70281 contain a dominance disease-resistant gene.Analyze from anti-source, this disease-resistant gene is exactly Pm16.Select for use 10 strains disease-resistant strain of typical case and the susceptible strain of 10 strains to form disease-resistant pond and susceptible pond respectively.
Select 16 pairs of little satellite (SSR) primers on the wheat 4A karyomit(e) for use, 4B, 5A, 5B, 7A, little satellite (SSR) primer 34 on the 7B karyomit(e) is to (primer title and sequence are seen R der M.S., Korzum V., WendehakeK., Plaschke J., Tixier M.H., the Leroy P.and Ganal M.W.1998.A microsatellitemap of wheat.Genetics 149:2002-2023 and the U.S. are big, wheat scab coorporative network http://www.scabusa.org) and the micro-satellite primers 15 on paddy rice the 3rd karyomit(e) to (seeing Temnykh S., Park W.D., Ayres N., Cartinhour S., Hauck N., Lipovich L., Cho Y.G.and IshiiT.2000.Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.) .Theor Appl Genet 100:697-712.), to 70281, Chanceller, disease-resistant pond, increase in susceptible pond.Wherein, the PCR reaction system is: the cumulative volume of each reaction is 25 μ l, comprising: template DNA (20ng/ μ l) 5 μ l, Taq enzyme (5U/ μ l) 0.2 μ l, primer is to (2 μ M) 3 μ l, dNTPs (2.5mM) 0.5 μ l, 10 * PCR damping fluid, 2.5 μ l, sterile distilled water 13.8 μ l.The composition of 10 * PCR damping fluid is Tris-HCl (pH8.3) 100mM, KCl 500mM, MgCl
215mM.Response procedures is 94 ℃ of pre-sex change 5min, carries out 35 circulations subsequently: 94 ℃ of 40s, 60 ℃ of 30s, 72 ℃ of 40s; Last 72 ℃ are extended 10min, 4 ℃ of preservations.The load sample indicator (98% methane amide, 10mM EDTA pH8.0,0.25% tetrabromophenol sulfonphthalein and 0.25% dimethylbenzene green grass or young crops) that adds 8 μ l sex change in every part of amplified production, 95 ℃ of sex change 5-10mins.The amplification sample 7 μ l of every part of sex change are electrophoretic separation in 6% the denaturing polyacrylamide gel in concentration, and silver dyes colour developing.The result is shown in Fig. 1 a-Fig. 1 c, show that xgwm159, the Borc004 and the Borc109 amplified production between anti-sense parent that are positioned on the karyomit(e) 5BS have polymorphism, xgwm159 all amplifies each two on visibly different microsatellite marker band between anti-sense parent and anti-sense pond, disease-resistant parent and disease-resistant pond are 197bp and 193bp two bands, susceptible parent and susceptible pond are 193bp and 191bp two bands, wherein the characteristic strip of 197bp is peculiar by disease-resistant sick parent and disease-resistant pond, shows that tentatively xgwm159 and Pm16 are chain.Among Fig. 1 a, M is 100bpDNA Ladder (arrow shows 200bp), and CK is two anti-(Pm16) (available from variety source institutes of the Chinese Academy of Agricultural Sciences), 7, and P
RBe 70281, P
SBe Chanceller, B
RBe disease-resistant pond, B
SBe susceptible pond, R is F
2The disease-resistant individual plant of colony, S are the susceptible individual plant of F2 colony, and a is 197bp, and b is 193bp, and c is 191bp.Among Fig. 1 b, M is 100bpDNA Ladder (arrow shows 200bp), and CS is a China spring, and PR is 70281, and PS is Chanceller, and R is F
2The disease-resistant individual plant of colony, S are the susceptible individual plant of F2 colony.Among Fig. 1 c, 1 for 100bpDNA Ladder (arrow shows 200bp), and 2 is CS, and 3 is 70281,4 to be Chanceller, and 5-34 is F
2Colony's part individual plant.
2, with 3 primer xgwm159, Borc004 on the 5B and Borc109 to F
2156 individual plants of colony increase respectively, and (the same step 1) of reaction system and reaction conditions is carried out linkage analysis with MAPMAKER/EXP 3.0 softwares to microsatellite marker and gene locus.The result shows that the gene locus of Pm16 and this 3 marker sites are chain as shown in Figure 2, and it puts in order and is Pm16-Xgwm159-Borc109-Borc004.The genetic distance in three intervals is followed successively by 5.3cM, 23.7cM, 2.2cM.Therefore the genetic distance of Pm16 and Xgwm159 is 5.3cM.
3, with the part nullisomic-limbs material (N4AT4D of specific mark primer xgwm159 to the 4th, 5 homology groups of China spring, N4AT4B, N4DT4B, N5BT5D, N5AT5D and N5AT5B, purchase in Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology) amplification (the same step 1) of reaction system and reaction conditions, the result as shown in Figure 3, show in lacking the chromosomal material of 5B and do not amplify the band consistent with susceptible parent, and in other material, all can amplify this band, confirm further that thus the Pm16 gene is positioned on the 5B karyomit(e), and be not 4A karyomit(e).Among Fig. 3, M is pBR322DNA/MspI Markers, and 1,4 is CS, 2,3 is Opata, and 5,6 is CSN5BT5D, and 7 is CSN5AT5D, 8 is CSN5AT5B, and 9 is CSN4AT4D, and 10 is CSN4AT4B, 11 is CSN4DT4B, 12 is two anti-(Pm16), and 13 is 70281,14 to be Chanceller, 15 is disease-resistant pond, and 16 is susceptible pond; Arrow shows does not have amplified fragments.
In order further to detect the specificity of micro-satellite primers Xgwm159 mark, with it to 6 parts of susceptible materials except Chanceller and 70281 with contain 15 parts of the materials of No. 15 microspecies genes of other mildew-resistance bacterium or multiple gene polymerization, carry out pcr amplification (reaction system and reaction conditions are with embodiment 1), susceptible material is: as if China spring, capital 411 7107, Luo Bulin, CA0015, CA0131.Disease-resistant material is: CA9640 (Pm2, come from cross combination CA8695/C39//capital 411), 40275 (Pm4b, come from cross combination V.P.M./hundred farmings 3217), two anti-(Pm16) (available from variety source institutes of the Chinese Academy of Agricultural Sciences), 70281 (Pm16), 41139 (Pm12, come from wheat 9 among the 015/87-1//Line31/3/2* of cross combination agricultural university), 40375 (Pm12, come from cross combination Line31/4* hundred farmings 3217), 40388 (Pm13 comes from cross combination T3BL.3BS-3S1#1S/2* hundred farmings 3217//96 mirror 63), C262 (Pm21, come from cross combination T6AL.6VS/2* agricultural university 93), C266 (Pm21 comes from 9303/T6AL.6VS/ in the cross combination/short morning/3/93 mirror 64), CA9550 (Pm2+Pm4b, come from cross combination CA8695/C39//capital 411), (Pm13+Pm4b comes from hybridization YW243//T3BL.3BS-3S to WM1
1#1S/2* hundred farmings 3217), WM16 (Pm12+Pm4b, come from cross combination Line31/4* hundred agricultural 3217//YW243), WM30 (Pm4b+Pm21+Pm12, come from cross combination Line31/4* hundred agricultural 3217//YW243/3/PM97033) and WM42 (Pm21+Pm4b, come from cross combination Line31/4* hundred agricultural 3217//YW243/3/PM97033), 3B509 (available from China Agricultural University) and 3B625 (available from China Agricultural University) all have two anti-blood relationships.The result shows all not amplify in all susceptible materials and other disease-resistant material and the disease-resistant parent 70281 of containing the Pm16 gene and two anti-identical characteristic strip: 197bp shown in Fig. 4 a-Fig. 4 b.Therefore xgwm159 can be used as the specific molecular marker of mildew-resistance gene Pm16, and is applied to molecular marker assisted selection, the wheat breed and the strain of screening mildew-resistance.Among Fig. 4 a, 1 is pBR322DNA/Msp IMarkers, and 2 is two anti-(Pm16), and 3 is Chanceller, 4 is CS, and 5 is CA0015, and 6 is CA0131, and 7 is capital 411,8 is 70281, as if 9 be 7107,10 to be Luo Bulin, 11 is CA9640,12 is 40275,13 to be two anti-(Pm16), and 14 is 41139,15 is 40375,16 to be 40388,17 to be 3B509,18 is 3B625, and 19 is C262, and 20 is C266.Among Fig. 4 b, 1 is pBR322DNA/Msp I Markers, and 2 is 70281,3 to be Chanceller, and 4 is CA9550, and 5 is WM16, and 6 is WM1, and 7 is WM42, and 8 is WM30.
Sequence table
<160>2
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>1
gggccaacac?tggaacac 18
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>2
gcagaagctt?gttggtaggc 20
Claims (4)
1, is used to screen the primer of powdery-mildew-resistance wheat, a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 1 and sequence 2.
2, a kind of method of screening powdery-mildew-resistance wheat, be to be template with wheat cdna group DNA to be measured, classify primer as with the nucleotide sequence of sequence in the sequence table 1 and the nucleotides sequence of sequence 2, carry out pcr amplification, detect the band whether 197bp and 193bp size are arranged in the amplified production.
3, method according to claim 2, it is characterized in that: the cumulative volume of the reaction system of described pcr amplification is 25 μ l, comprise: template DNA (20ng/ μ l) 5 μ l, Taq enzyme (5U/ μ l) 0.2 μ l, primer is to (2 μ M) 3 μ l, dNTPs (2.5mM) 0.5 μ l, 10 * PCR damping fluid, 2.5 μ l, sterile distilled water 13.8 μ l; Response procedures is 94 ℃ of pre-sex change 5min, carries out 35 circulations subsequently: 94 ℃ of 40s, 60 ℃ of 30s, 72 ℃ of 40s; Last 72 ℃ are extended 10min.
4, according to claim 2 or 3 described methods, it is characterized in that: described detection amplified production method is for carrying out 6% denaturing polyacrylamide gel electrophoresis to amplified production, and silver dyes colour developing then.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100422203C (en) * | 2006-07-10 | 2008-10-01 | 南京农业大学 | Marker primer linked with wheat powdery mildew resistant gene Pm6 and its usage method |
| CN108690883A (en) * | 2018-08-17 | 2018-10-23 | 华南农业大学 | A kind of molecular labeling RMD7 of the soybean powder mildew resistance of auxiliary identification soybean to be measured |
| CN112301142A (en) * | 2019-07-30 | 2021-02-02 | 山东省农业科学院作物研究所 | Leymus speetanus anti-powdery mildew gene Pm12 molecular marker and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1252284C (en) * | 2002-09-27 | 2006-04-19 | 天津师范大学 | Molecular marker linked with wheat mildew-resistance gene |
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2004
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100422203C (en) * | 2006-07-10 | 2008-10-01 | 南京农业大学 | Marker primer linked with wheat powdery mildew resistant gene Pm6 and its usage method |
| CN108690883A (en) * | 2018-08-17 | 2018-10-23 | 华南农业大学 | A kind of molecular labeling RMD7 of the soybean powder mildew resistance of auxiliary identification soybean to be measured |
| CN108690883B (en) * | 2018-08-17 | 2020-09-15 | 华南农业大学 | Molecular marker RMD7 for assisting in identifying soybean powdery mildew resistance of soybean to be detected |
| CN112301142A (en) * | 2019-07-30 | 2021-02-02 | 山东省农业科学院作物研究所 | Leymus speetanus anti-powdery mildew gene Pm12 molecular marker and application thereof |
| CN112301142B (en) * | 2019-07-30 | 2021-11-02 | 山东省农业科学院作物研究所 | Leymus speetanus anti-powdery mildew gene Pm12 molecular marker and application thereof |
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