CN101892315B - Aided identification method of wheat with resistance to yellow dwarf and special primer pair thereof - Google Patents
Aided identification method of wheat with resistance to yellow dwarf and special primer pair thereof Download PDFInfo
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- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
Abstract
The invention discloses an aided identification method of wheat with resistance to yellow dwarf, comprising the following steps: taking a genome DNA (or cDNA) of wheat to be tested as a template, and carrying out PCR amplification by a primer pair composed of a DNA shown by sequence 3 of a sequence table and a DNA shown by sequence 4 of the sequence table; if a DNA (or cDNA) fragment of 1016bp (or 512bp) is obtained, judging the tested wheat to be the candidate wheat with resistance to yellow dwarf carrying a TiSTK1 gene; and if a PCR amplified fragment of 1016bp (or 512bp) is not obtained, judging the tested wheat to be the candidate wheat with resistance to yellow dwarf carrying no TiSTK1 gene. The aided identification method has the advantages of high accuracy, and saved time and resources and is capable of realizing early screening, thus having important significance for breeding the wheat with resistance to yellow dwarf.
Description
Technical field
The method and the primer special thereof that the present invention relates to a kind of assistant identification wheat with resistance to yellow dwarf are right.
Background technology
Yellow stunt of wheat is the wheat main diseases viral disease that is caused by the barly yellow dwarf virus (BYDV) that aphid mediates, and promptly pasts medical help in case infect, and causes the serious underproduction of wheat.In recent years, because the change of weather condition, the wheat aphid of propagating barly yellow dwarf virus extensively takes place in China, serious day by day, and master such as yellow stunt of wheat has from Shanxi, Gansu, Shaanxi send out the trend that spreads diffusion to Yangze river and Huai river and southwestern Mai Qu of distinguishing.Therefore, seed selection wheat with resistance to yellow dwarf new variety are most economical effective ways of this disease of control.
Good available disease-resistant gene is the prerequisite of breeding for disease resistance.So far, do not find real effectively resistant gene in the wheat primary gene storehouse as yet.Yet a plurality of strains system of the high anti-or immune barly yellow dwarf virus of wheat kindred plant--middle couchgrass is applied and furthers investigate as important anti-source.Middle couchgrass contains 3 resistance to yellow dwarf genes at least, lay respectively at that the 7th homology crowd karyomit(e) 7Ai#1 is long-armed, 7E is long-armed and the 2nd homologous chromosomes 2Ai-2 galianconism on.Scientist breeds the partial amphidiploid TAF46 of resistance to yellow dwarf through wheat and middle couchgrass distant hybirdization, is the bridge parent with TAF46 again, breeds the disomic addition line L1 (additional 2 7Ai#1 karyomit(e)s) and the 7Ai# 1L both-end body addition line of resistance to yellow dwarf; Continuing it, is anti-source with L1, utilizes the pairing of China spring ph mutation induction homeologous chromosome and two approach of tissue culture, and the resistance to yellow dwarf gene on the 7Ai#1 karyomit(e) is successfully imported wheat; Breed a collection of resistance to yellow dwarf translocation line, comprise YW642 (HW642), YW443, YW243 (Xin, Z., Zhang; Z., Chen, X., Lin; Z., Ma, Y., Xu; H., Banks, P.; And Larkin, P.Development and characterization ofcommon wheat-Thinopyrum intermedium translocation lines with resistance tobarley yellow dwarf virus.Euphytica.2001,119:161-165; Zhang Zengyan, Xin Zhiyong, old filial piety etc., the molecular cytogenetics of wheat with resistance to yellow dwarf new lines YW443 is identified, Acta Genetica Sinica, 2000,27 (7): 614～620; Zhang Zengyan, Xin Zhiyong, wheat with resistance to yellow dwarf biotechnology breeding progress, the crop magazine, 2005,5:4-7), and (Banks such as TC5-TC10, TC14; P., Larkin, P., Bariana, H., Lagudah, E.; Appels, R., Waterhouse, P., Brettell, R., Chen; X., Xu, H., Xin, Z., Qian, Y.; Zhou, M., Cheng, Z., and Zhou, G.The use of cell culture for sub-chromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.Genome.1995,38:395-405).
The fast development of genomic in situ hybridization (GISH), molecule marker provides strong instrument for identifying exogenous chromosome, location foreign gene.Utilize technology such as GISH, RFLP mark, identify the wheat-middle couchgrass translocation line TC5-TC10, TC14 (Banks, P., Larkin, P., the Bariana that provide Bdv2; H., Lagudah, E., Appels, R., Waterhouse; P,, Brettell, R., Chen, X.; Xu, H,, Xin, Z.; Qian, Y., Zhou, M., Cheng; Z., and Zhou, G.The use of cell culture for sub-chromosomal introgressions of barley yellow dwarf virus resi stance from Thinopyrum intermedium to wheat.Genome.1995,38:395-405) and YW642, YW443, YW243 (Zhang Zengyan, Xin Zhiyong; Horse is strong-willed etc., Mapping of a BYDV resistance gene from Thintermedium intermedium in wheat background by molecular markers, Sci ence in China (SeriesC), 1999,42 (6): 663-668; Zhang Zengyan, Xin Zhiyong, old filial piety etc., the molecular cytogenetics of wheat with resistance to yellow dwarf new lines YW443 is identified, Acta Genetica Sinica, 2000,27 (7): 614～620; Xie Hao, old filial piety, Zhang Zengyan, Xin Zhiyong, Lin Zhishan, Du Lipu, horse is strong-willed, Xu Huijun, the seed selection of wheat with resistance to yellow dwarf new lines YW243 and cellular elements biological assay, Acta Agronomica Sinica, 2000,26 (6): 687-691; Xin, Z., Zhang, Z.; Chen, X., Lin, Z.; Ma, Y., Xu, H.; Banks, P., and Larkin; P.Development and characterization of common wheat-Thinopyrum intermedium translocation lines with resistance to barley yellow dwarf virus.Euphytica.2001 119:161-165), finds that middle couchgrass karyomit(e) 7Ai#1 long-armed (7Ai# 1L) the end small segment that carries resistance to yellow dwarf gene Bdv2 translocates to the long-armed end (Zhang Zengyan of chromosome of wheat 7D; Xin Zhiyong, horse is strong-willed etc., Mapping of a BYDV resistancegene from Thintermedium intermedium in wheat background by molecular markers; Science in China (Series C), 1999,42 (6): 663-668).Discover, carry wheat-middle couchgrass translocation line YW642, YW443, YW243, TC14 of resistance to yellow dwarf gene Bdvv2 etc., high resistance to yellow dwarf can be used as the important anti-source of wheat resistance to yellow dwarf breeding.
Summary of the invention
The method and the primer special thereof that the purpose of this invention is to provide a kind of assistant identification wheat with resistance to yellow dwarf are right.
Assistant identification provided by the invention is carried the method for wheat of TiSTK1 gene, comprises the steps: that the genomic dna with wheat to be measured is a template, and the primer of forming with DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table is to carrying out pcr amplification; If obtain the dna fragmentation of 1016bp, wheat to be measured is candidate's the wheat that carries the TiSTK1 gene; If do not obtain the dna fragmentation of 1016bp, wheat to be measured is candidate's the wheat that does not carry the TiSTK1 gene;
Said TiSTK1 gene is following 1) to 6) in arbitrary described dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 171st to 1448 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 674th to 2032 Nucleotide;
4) dna molecular shown in the sequence 1 in the sequence table;
5) under stringent condition with 1) or 2) or 3) or 4) the dna sequence dna hybridization that limits and the dna molecular of coded plant resistance to yellow dwarf GAP-associated protein GAP;
6) with 1) or 2) or 3) or 4) dna sequence dna that limits has 90% above homology, and the dna molecular of coded plant yellow dwarf resistance-associated protein.
The program of said pcr amplification is following: 94 ℃ of 3min; 94 ℃ of 45s, 68 ℃ of 35s, 72 ℃ of 45s-1min30s, 5 circulations; 94 ℃ of 45s, 66 ℃ of 35s, 72 ℃ of 45s-1min30s, 5 circulations; 94 ℃ of 45s, 64 ℃ of 35s, 72 ℃ of 45s-1min30s, 25 circulations; 72 ℃ of 10min.The program of said pcr amplification is specific as follows: 94 ℃ of 3min; 94 ℃ of 45s, 68 ℃ of 35s, 72 ℃ of 1min30s, 5 circulations; 94 ℃ of 45s, 66 ℃ of 35s, 72 ℃ of 1min30s, 5 circulations; 94 ℃ of 45s, 64 ℃ of 35s, 72 ℃ of 1min30s, 25 circulations; 72 ℃ of 10min.
The present invention also protects a kind of assistant identification to carry the method for wheat of TiSTK1 gene, comprises the steps: that the cDNA with wheat to be measured is a template, and the primer of forming with DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table is to carrying out pcr amplification; If obtain the dna fragmentation of 512bp, wheat to be measured is candidate's the wheat that carries the TiSTK1 gene; If do not obtain the dna fragmentation of 512bp, wheat to be measured is candidate's the wheat that does not carry the TiSTK1 gene;
Said TiSTK1 gene is following 1) to 6) in arbitrary described dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 171st to 1448 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 674th to 2032 Nucleotide;
4) dna molecular shown in the sequence 1 in the sequence table;
5) under stringent condition with 1) or 2) or 3) or 4) the dna sequence dna hybridization that limits and the dna molecular of coded plant resistance to yellow dwarf GAP-associated protein GAP;
6) with 1) or 2) or 3) or 4) dna sequence dna that limits has 90% above homology, and the dna molecular of coded plant yellow dwarf resistance-associated protein.
The program of said pcr amplification is following: 94 ℃ of 3min; 94 ℃ of 45s-1min30s, 68 ℃ of 35s, 72 ℃ of 45s-1min30s, 5 circulations; 94 ℃ of 45s, 66 ℃ of 35s, 72 ℃ of 45s-1min30s, 5 circulations; 94 ℃ of 45s, 64 ℃ of 35s, 72 ℃ of 45s-1min30s, 25 circulations; 72 ℃ of 10min.Said pcr amplification program is specific as follows: 94 ℃ of 3min; 94 ℃ of 45s, 68 ℃ of 35s, 72 ℃ of 45s, 5 circulations; 94 ℃ of 45s, 66 ℃ of 35s, 72 ℃ of 45s, 5 circulations; 94 ℃ of 45s, 64 ℃ of 35s, 72 ℃ of 45s, 25 circulations; 72 ℃ of 10min.
More than in two kinds of methods, the primer that pcr amplification adopts is to identical, but because template is respectively genomic dna or cDNA, so amplified fragments is big or small variant.
Said wheat to be measured specifically can be disomic addition line L1, the resistance to yellow dwarf of resistance to yellow dwarf both-end system 7Ai# 1L, resistance to yellow dwarf translocation line YW642, resistance to yellow dwarf translocation line YW443, resistance to yellow dwarf translocation line YW243, resistance to yellow dwarf translocation line TC14, in 8601, the wheat-middle couchgrass addition line Z1 or the PmV of China spring, YW641S, resistance to yellow dwarf.
The present invention also protects the primer that DNA forms shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table right.
Said primer is to can be used for preparing the test kit that assistant identification is carried the wheat of TiSTK1 gene;
Said TiSTK1 gene is following 1) to 6) in arbitrary described dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 171st to 1448 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 674th to 2032 Nucleotide;
4) dna molecular shown in the sequence 1 in the sequence table;
5) under stringent condition with 1) or 2) or 3) or 4) the dna sequence dna hybridization that limits and the dna molecular of coded plant resistance to yellow dwarf GAP-associated protein GAP;
6) with 1) or 2) or 3) or 4) dna sequence dna that limits has 90% above homology, and the dna molecular of coded plant yellow dwarf resistance-associated protein.
The present invention also protects a kind of assistant identification to carry the test kit of the wheat of TiSTK1 gene, comprises that said primer is right;
Said TiSTK1 gene is following 1) to 6) in arbitrary described dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 171st to 1448 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 674th to 2032 Nucleotide;
4) dna molecular shown in the sequence 1 in the sequence table;
5) under stringent condition with 1) or 2) or 3) or 4) the dna sequence dna hybridization that limits and the dna molecular of coded plant resistance to yellow dwarf GAP-associated protein GAP;
6) with 1) or 2) or 3) or 4) dna sequence dna that limits has 90% above homology, and the dna molecular of coded plant yellow dwarf resistance-associated protein.
The said primer of said method to or said test kit can be used for the assistant identification wheat with resistance to yellow dwarf; Carry the wheat with resistance to yellow dwarf of the wheat of TiSTK1 gene for the candidate; Do not carry the sense BYDV wheat of the wheat of TiSTK1 gene for the candidate.Disease resistance identifies that classification adopts the domestic standard of yellow stunt of wheat severity, i.e. the standard of IT, and 0-3 is a wheat with resistance to yellow dwarf, 6-8 is sense BYDV wheat.
Said method, said primer to or said test kit can be used for wheat breeding.
Primer of the present invention to method based on couchgrass in the middle of coming from, the nucleotide sequence (1822bp) of the resistance to yellow dwarf important gene TiSTK1 that wheat-middle couchgrass translocation line YW642 is total.The present invention successfully develops the As-PCR mark of resistance to yellow dwarf important gene TiSTK1.Carry the wheat with resistance to yellow dwarf of the wheat of TiSTK1 gene for the candidate.Use method of the present invention, accuracy is high, can realize early screening, saves time and resource, and breeding is significant for wheat with resistance to yellow dwarf.
Description of drawings
Fig. 1 is the electrophorogram of embodiment 2.
Fig. 2 is the electrophorogram of embodiment 3.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment like no specified otherwise, is the quality percentage composition.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
Middle couchgrass (Ti):, be numbered Z1146 available from Institute of Crop Science, Chinese Academy of Agricultural Science resource germplasm storehouse.
In 8601: available from Institute of Crop Science, Chinese Academy of Agricultural Science.
China spring: available from Institute of Crop Science, Chinese Academy of Agricultural Science.
Barley yellow dwarf virus (BYDV-GAV strain system): available from Plant Protection institute, Chinese Academy of Agricultral Sciences.
Resistance to yellow dwarf translocation line YW642 (HW642): reference: Zhang Zengyan, horse is strong-willed, and hot will is brave etc., and 1998, applying gene group hybridization in situ technique is identified wheat with resistance to yellow dwarf new germ plasm, Scientia Agricultura Sinica, 31 (3): 1-4; Zhang Z; Xin Z; Ma Y, Chen X, Xu Q; Lin Z.1999, Mapping of a BYDV resistance gene from Thinopyrum intermedium in wheat background by molecular markers.Sci China C Life Sci.42 (6): 663-668.; This translocation line is that the hot will of Institute of Crop Science, Chinese Academy of Agricultural Science in 1991 bravely waits initiative, and 1996 yearbooks such as Zhang Zengyan are fixed; Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public.
Resistance to yellow dwarf translocation line YW443: reference: Zhang Zengyan, Xin Zhiyong, old filial piety etc., the molecular cytogenetics of wheat with resistance to yellow dwarf new lines YW443 is identified, Acta Genetica Sinica, 2000,27 (7): 614～620; This translocation line is that the hot will of 1991-1995 Institute of Crop Science, Chinese Academy of Agricultural Science bravely waits initiative, and 1998-1999 yearbooks such as Zhang Zengyan are fixed; Institute of Crop Science, Chinese Academy of Agricultural Science's wheat molecular breeding group has preservation; Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public.
Resistance to yellow dwarf translocation line YW243: reference: Xie Hao, old filial piety, Zhang Zengyan, Xin Zhiyong; Lin Zhishan, Du Lipu, horse is strong-willed, Xu Huijun; The seed selection of wheat with resistance to yellow dwarf new lines YW243 and cellular elements biological assay, Acta Agronomica Sinica, 2000,26 (6): 687-691; This translocation line is that the hot will of 1991-1995 Institute of Crop Science, Chinese Academy of Agricultural Science bravely waits initiative, and 1998-1999 yearbooks such as Xie Hao are fixed; Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public.
Resistance to yellow dwarf translocation line TC14: reference: Banks, P., Larkin, P., Bariana, H.; Lagudah, E., Appels, R., Waterhouse, P.; Brettell, R., Chen, X., Xu, H.; Xin, Z., Qian, Y., Zhou, M.; Cheng, Z., and Zhou, G.The use of cell culture for sub-chromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.Genome.1995,38:395-405; This translocation line is 1991-1994 Australia scientist Banks, initiatives such as Larkin and brave, the old filial piety of the hot will of Chinese science man, Xu Huijun, and Banks, Larkin waits nineteen ninety-five to identify out; Institute of Crop Science, Chinese Academy of Agricultural Science introduces, has preservation; Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public.
The disomic addition line L1 of resistance to yellow dwarf: reference: Cauderon, Y., Saigne; B., and Dauge, M. (1973) The resistance to wheat rusts of Agropyron intermedium and its use inwheat improvement.Proc Int Wheat Genet Symp Wheat Improvement; Vol.4, E.R.Sears and L.M.S.Sears eds (Univ of Missouri, Columbia; MD), pp 401-407; L1 be 1973 by initiatives such as French scientist Cauderon; Institute of Crop Science, Chinese Academy of Agricultural Science introduces, has preservation; Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public.
The both-end system 7Ai# 1L of resistance to yellow dwarf: reference: Banks, P., Larkin, P., Bariana, H.; Lagudah, E., Appels, R., Waterhouse, P.; Brettell, R., Chen, X., Xu, H.; Xin, Z., Qian, Y., Zhou, M.; Cheng, Z., and Zhou, G.The use of cell culture for sub-chromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.Genome.1995,38:395-405; This translocation line is 1991-1994 Australia scientist Banks, initiatives such as Larkin and brave, the old filial piety of the hot will of Chinese science man, Xu Huijun, and Banks, Larkin waits nineteen ninety-five to identify out; Institute of Crop Science, Chinese Academy of Agricultural Science introduces, has preservation; Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public.
YW641S: reference: Xiaodong Liu; ZengYan Zhang (communication author); Zhiyong Xin, 2005, Molecular evidence of barley yellow dwarf virus replication/movement suppressed by the resistance gene Bdv2 derived from Th.intermedium; Journal of Genetic and Genomics (Yi Chuan Xue Bao), 932:942-947; This is to be the susceptible sisters system of HW642, is seed selection such as the Zhang Zengyan of 1996-1998 Institute of Crop Science, Chinese Academy of Agricultural Science, identifies; Wheat molecule seminar of Institute of Crop Science, Chinese Academy of Agricultural Science (Zhang Zengyan researcher) preserves; Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public.
PmV: reference: Li H; Chen X, Xin Z Y, Ma Y Z; Chen X Y; Jia X.Development and identification of wheat-Haynaldia villosa T6DL.6VS chromosome translocation lines conferring resistance to powdery mildew.Plant Breeding, 2005,124:203-205; This is to be initiatives such as the old filial piety of the scientist of 1997-2001 Institute of Crop Science, Chinese Academy of Agricultural Science, hot will is brave, and evaluations such as Li Hui are come out; There is preservation in wheat molecule seminar of Institute of Crop Science, Chinese Academy of Agricultural Science; Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public.
The wheat of resistance to yellow dwarf-middle couchgrass addition line Z1 (carrying Bdv4): reference: Banks, P., Larkin, P., Bariana, H.; Lagudah, E., Appels, R., Waterhouse, P.; Brettell, R., Chen, X., Xu, H.; Xin, Z., Qian, Y., Zhou, M.; Cheng, Z., and Zhou, G.The use of cell culture for sub-chromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.Genome.1995,38:395-405; Zhang; Z.Y.; Xin, Z.Y., and Larkin; P.J.2001.Molecular characterization of a Thinopyrum intermedium group 2chromosome (2Ai-2) conferring resistance to barley yellow dwarf virus.Genome, 44:1129-1135; Z1 is couchgrass karyomit(e) 2Ai-2 in the middle of in the wheat cdna group, having added 1 couple; The latter carries resistance to yellow dwarf gene Bdv4; By Australian scientist Banks; Larkin and brave, the old filial piety of the hot will of Chinese science man, Xu Huijun equal the 1992-1995 initiative, identify that there is preservation in Institute of Crop Science, Chinese Academy of Agricultural Science; Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public.
Embodiment 1, the right discovery of primer
Utilize a pair of primer of resistance to yellow dwarf important gene (TiSTK1) cDNA sequences Design; Sequencing and compare of analysis to 62 STK gene cDNA clones that extension increasing sequence obtained of resistance to yellow dwarf material (Ti, YW642), sense BYDV wheat cDNA; In the couchgrass 3 transcripts are arranged in the middle of finding; Wherein 2 are positioned on the chromosomal middle couchgrass 7Ai#1L of the wheat 7D that translocates to anti-disease wheat; Wherein this primer is to promptly being the middle couchgrass TiSTK1 gene order that translocates in the anti-disease wheat; In the susceptible in addition wheat sequence of very high homology is arranged, and find that ' there is 2 successive SNPs (SNPs) difference in non-coding region, these 2 successive base differences is designed to the 3 ' end of upstream primer TISTK1-ASF 5 for the two; Utilize the PRIMER5 primer-design software to design the downstream primer TISTK1-ASR that matees with upstream primer then, primer promptly of the present invention is right.With primer of the present invention to increasing; If obtain with sequence 1 in corresponding fragment; This wheat is disease-resistant material because the TiSTK1 gene is positioned on the middle couchgrass karyomit(e) 7Ai#1L that contains Bdv2, this section karyomit(e) and wheat be difficult to the exchange; As long as therefore carrying the anti-disease wheat translocation line of Bdv2, middle couchgrass, anti-disease wheat cross materials all can amplify this special band, and susceptible wheat all can not increase with the powdery-mildew-resistance wheat that carries PmV.
TISTK1-ASF:5 '-CGCTCCCCTCCTTCCCCTTC-3 ' (sequence 3);
TISTK1-ASR:5 '-TGAGTATCTCCGTCGGATGAGTTGG-3 ' (sequence 4).
Embodiment 2, application primer are to the assistant identification wheat with resistance to yellow dwarf
Application implementation example 1 designed primer detects following sample to (primer of being made up of TISTK1-ASF and TISTK1-ASR to):
The middle couchgrass (Ti) of resistance to yellow dwarf; The disomic addition line L1 of resistance to yellow dwarf; The both-end system 7Ai# 1L of resistance to yellow dwarf; Resistance to yellow dwarf translocation line: YW642 (HW642), YW443 and YW243, above resistance to yellow dwarf material all carries the TiSTK1 gene; In 8601 (senses BYDV wheats); China spring (CS; Sense BYDV wheat); YW641S (the susceptible sisters of YW642 are).
One, disease resistance is identified
Plant above-mentioned biomaterial (every part of material is planted 60 strains) respectively, inoculate the aphid of BYDV virus (BYDV-GAV strain system) in seedling stage.The aphid that BYDV-GAV is carried in tweezer places on the plant of wheat or middle couchgrass, and about 10 aphids of every strain inoculate the 30-40 days above-mentioned biomaterials of " Invest, Then Investigate " to yellow dwarf resistance.
Disease resistance is identified the domestic standard of classification employing yellow stunt of wheat severity, and promptly the standard of IT is seen table 1, reference: " Li Guangbo, Ceng Shimai, Li Zhenqi chief editor. the sick Chinese caterpillar fungus plague of rats comprehensive regulation [M] of wheat. Beijing: Chinese agriculture science and technology press, 1990 ".
Table 1 yellow stunt of wheat severity grade scale
| Rank (level value) | Domestic standard (11 grades) method |
| 0 | Healthy tree |
| 1 | The yellow of part blade tip |
| 2 | The following 1 leaf yellow of boot leaf |
| 3 | The following 2 leaf yellows of boot leaf |
| 4 | Boot leaf yellow 1/4, the following 1 leaf yellow of boot leaf |
| 5 | Boot leaf yellow 1/4, the following 2 leaf yellows of boot leaf |
| 6 | The boot leaf yellow |
| 7 | The boot leaf yellow, the following 1 leaf yellow of boot leaf |
| 8 | The following 2 leaf yellows of boot leaf and boot leaf |
| 9 | Plant is downgraded, but can ear |
| 10 | Plant is downgraded significantly, does not ear |
The result shows: it is anti-that Ti, L1,7Ai# 1L, YW642 (HW642), YW443 and YW243 infect the performance height to BYDV-GAV strain system, and they are 0 to the yellow stunt of wheat severity, promptly all are healthy tree; In the wheat line 8601; It is susceptible that China spring (CS), YW641S infect performance to wheat BYDV-GAV strain system, and its BYDV severity is the 6-8 level.
Two, use primer to assistant identification
1, extracts the genomic dna (gDNA) of each biomaterial blade.
2, with the genomic dna be template, the primer of forming with TISTK1-ASF and TISTK1-ASF TISTK1-ASR obtains pcr amplification product to carrying out pcr amplification.
Pcr amplification system: ddH
2017.3 μ l, 10 * PCR Buffer, 2.5 μ l, 25mM MgCl
21.5 μ l, 2.5mMdNTP mix 1.5 μ l, TISTK1-ASF (10 μ M) 0.5 μ l, TISTK1-ASR (10 μ M) 0.5 μ l, 100ng/uL gDNA 1 μ l, rTaq (5U/ μ l) 0.2 μ l.
Pcr amplification program: 94 ℃ of 3min; 94 ℃ of 45s, 68 ℃ of 35s, 72 ℃ of 1min30s, 5 circulations; 94 ℃ of 45s, 66 ℃ of 35s, 72 ℃ of 1min30s, 5 circulations; 94 ℃ of 45s, 64 ℃ of 35s, 72 ℃ of 1min30s, 25 circulations; 72 ℃ of 10min.
3, pcr amplification product is carried out 1% agarose gel electrophoresis.
The result sees Fig. 1.All can amplify a disease-resistant special band (1016bp) in the wheat lines of the disomic addition line L1 of middle couchgrass, resistance to yellow dwarf, the both-end system 7Ai# 1L of resistance to yellow dwarf and resistance to yellow dwarf translocation line; The susceptible wheat lines that does not carry the TiSTK1 gene can not amplify this special band.
4, respectively each pcr amplification product is checked order
All can amplify a disease-resistant special band (1016bp) in the wheat lines of the disomic addition line L1 of middle couchgrass, resistance to yellow dwarf, the both-end system 7Ai# 1L of resistance to yellow dwarf and resistance to yellow dwarf translocation line, and the sequence of this special band all like the sequence 1 of sequence table from (DNA shown in the sequence 1 is the genome sequence of TiSTK1 gene) shown in 5 ' the terminal 52-1067 position Nucleotide.Explain that this amplified band can be used as the PCR mark of this TiSTK1 gene specific.
Three, the tracking of disease resistance and TiSTK1 gene
Disease resistance to the plant of future generation of disease-resistant strain that the specific amplified band is arranged is followed the tracks of.The result shows, utilizes the TISTK1-ASF of this invention and TISTK1-ASR primer to carry out pcr amplification, and the plant to be measured that every genomic dna can amplify the distinguished sequence of 1016bp is and contains the disease-resistant individual plant of TiSTK1.Therefore, utilize primer of the present invention right, according to this specific band, can identify having or not of resistance to yellow dwarf important gene TiSTK1 in the test plant, assisted Selection resistance to yellow dwarf individual plant is used for assist-breeding wheat with resistance to yellow dwarf new lines (kind); In addition; Utilize the special functional label assisted Selection of TiSTK1 of this invention, need not per generation all carries out artificial inoculation, carries out disease-resistant evaluation, helps carrying out the procedure of breeding that breeding adds generation, realizes 1 year 2-3 generation; Shorten the cycle of wheat with resistance to yellow dwarf breeding, accelerate breeding process.
Embodiment 3, application primer are to the assistant identification wheat with resistance to yellow dwarf
Application implementation example 1 designed primer detects following sample to (primer of being made up of TISTK1-ASF and TISTK1-ASR to):
The disomic addition line L1 of resistance to yellow dwarf; The both-end system 7Ai# 1L of resistance to yellow dwarf; Resistance to yellow dwarf translocation line: YW642 (HW642), YW443, YW243 and TC14; In 8601 (senses BYDV wheats); The wheat of resistance to yellow dwarf-middle couchgrass addition line Z1 (carries Bdv4; Resistance to yellow dwarf), PmV (carries PmV; Mildew-resistance; The sense BYDV).
One, disease resistance is identified
Method is with the step 1 of embodiment 2.
The result shows: it is anti-that L1,7Ai# 1L, YW642 (HW642), YW443, YW243, TC14 and Z1 infect the performance height to BYDV-GAV strain system, and they are 0 to the yellow stunt of wheat severity, promptly all are healthy tree; In 8601 with PmV that performance is infected by wheat BYDV-GAV strain system is susceptible, its BYDV severity is the 6-8 level.
Two, use primer to assistant identification
1, extract the RNA of each biomaterial blade, reverse transcription is cDNA.
2, with cDNA be template, the primer of forming with TISTK1-ASF and TISTK1-ASR obtains pcr amplification product to carrying out pcr amplification.
Pcr amplification system: ddH
2O 17.3 μ l, 10 * PCR Buffer, 2.5 μ l, 25mM MgCl
21.5 μ l, 2.5mMdNTP mix 1.5 μ l, TISTK1-ASF (10 μ M) 0.5 μ l, TISTK1-ASR (10 μ M) 0.5 μ l, cDNA1 μ l, rTaq (5U/ μ l) 0.2 μ l.
Pcr amplification program: 94 ℃ of 3min; 94 ℃ of 45s, 68 ℃ of 35s, 72 ℃ of 45s, 5 circulations; 94 ℃ of 45s, 66 ℃ of 35s, 72 ℃ of 45s, 5 circulations; 94 ℃ of 45s, 64 ℃ of 35s, 72 ℃ of 45s, 25 circulations; 72 ℃ of 10min.
Simultaneously, as internal standard gene the total cDNA concentration of sample room is consistent with conservative wheat house-keeping gene (18SrRNA, F:5 '-GTGACGGGTGACGGAGAATT-3 ', R:5 '-GACACTAATGCGCCCGGTAT-3 ').
3, pcr amplification product is carried out 1% agarose gel electrophoresis.
The result sees Fig. 2.L1,7Ai# 1L, YW642 (HW642), YW443, YW243 and TC14 all increase and obtain the specific band of 512bp, Z1, in 8601 and PmV do not obtain this band.
4, respectively each pcr amplification product is checked order
All can amplify a disease-resistant special band (512bp) in the wheat lines of the disomic addition line L1 of resistance to yellow dwarf, the both-end system 7Ai# 1L of resistance to yellow dwarf and resistance to yellow dwarf translocation line, and the sequence of this special band all like the sequence 2 of sequence table from (DNA shown in the sequence 2 is the cDNA sequence of TiSTK1 gene) shown in 5 ' the terminal 53-564 position Nucleotide.
The TiSTK1 gene is only expressed in the both-end system 7Ai# 1L of the disomic addition line L1 of the resistance to yellow dwarf that carries the Bdv2 gene, resistance to yellow dwarf and resistance to yellow dwarf translocation line; In the PmV (carrying PmV) of Z1 that does not carry the sense BYDV wheat lines of TiSTK1 gene, resistance to yellow dwarf and mildew-resistance, all do not express.Explain that TiSTK1 is the gene that carries the resistance to yellow dwarf material specifically expressing of Bdv2.The above results further proof primer of the present invention can assisted Selection resistance to yellow dwarf gene TiSTK1 and Bdv2 to (As-PCR mark), thus assistant identification with select the wheat with resistance to yellow dwarf material.
Claims (10)
1. the method for wheat that assistant identification is carried the TiSTK1 gene comprises the steps: that the genomic dna with wheat to be measured is a template, and the primer of forming with DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table is to carrying out pcr amplification; If obtain the dna fragmentation of 1016bp, wheat to be measured is candidate's the wheat that carries the TiSTK1 gene; If do not obtain the dna fragmentation of 1016bp, wheat to be measured is candidate's the wheat that does not carry the TiSTK1 gene;
Said TiSTK1 gene is following 1) to 6) in arbitrary described dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 171st to 1448 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 674th to 2032 Nucleotide;
4) dna molecular shown in the sequence 1 in the sequence table;
5) under stringent condition with 1) or 2) or 3) or 4) the dna sequence dna hybridization that limits and the dna molecular of coded plant resistance to yellow dwarf GAP-associated protein GAP;
6) with 1) or 2) or 3) or 4) dna sequence dna that limits has 90% above homology, and the dna molecular of coded plant yellow dwarf resistance-associated protein.
2. the method for claim 1, it is characterized in that: the program of said pcr amplification is following: 94 ℃ of 3min; 94 ℃ of 45s, 68 ℃ of 35s, 72 ℃ of 45s-1min30s, 5 circulations; 94 ℃ of 45s, 66 ℃ of 35s, 72 ℃ of 45s-1min30s, 5 circulations; 94 ℃ of 45s, 64 ℃ of 35s, 72 ℃ of 45s-1min30s, 25 circulations; 72 ℃ of 10min.
3. the method for wheat that assistant identification is carried the TiSTKi gene comprises the steps: that the cDNA with wheat to be measured is a template, and the primer of forming with DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table is to carrying out pcr amplification; If obtain the dna fragmentation of 512bp, wheat to be measured is candidate's the wheat that carries the TiSTK1 gene; If do not obtain the dna fragmentation of 512bp, wheat to be measured is candidate's the wheat that does not carry the TiSTK1 gene;
Said TiSTK1 gene is following 1) to 6) in arbitrary described dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 171st to 1448 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 674th to 2032 Nucleotide;
4) dna molecular shown in the sequence 1 in the sequence table;
5) under stringent condition with 1) or 2) or 3) or 4) the dna sequence dna hybridization that limits and the dna molecular of coded plant resistance to yellow dwarf GAP-associated protein GAP;
6) with 1) or 2) or 3) or 4) dna sequence dna that limits has 90% above homology, and the dna molecular of coded plant yellow dwarf resistance-associated protein.
4. method as claimed in claim 3 is characterized in that: the program of said pcr amplification is following: 94 ℃ of 3min; 94 ℃ of 45s, 68 ℃ of 35s, 72 ℃ of 45s-1min30s, 5 circulations; 94 ℃ of 45s, 66 ℃ of 35s, 72 ℃ of 45s-1min30s, 5 circulations; 94 ℃ of 45s, 64 ℃ of 35s, 72 ℃ of 45s-1min30s, 25 circulations; 72 ℃ of 10min.
5. like arbitrary described method in the claim 1 to 4, it is characterized in that: the both-end system 7Ai#1L of the disomic addition line L1 that said wheat to be measured is a resistance to yellow dwarf, resistance to yellow dwarf, resistance to yellow dwarf translocation line YW642, resistance to yellow dwarf translocation line YW443, resistance to yellow dwarf translocation line YW243, resistance to yellow dwarf translocation line TC14, in 8601, the wheat-middle couchgrass addition line Z1 or the PmV of China spring, YW641S, resistance to yellow dwarf.
6. the primer of the composition of DNA shown in the sequence 4 of DNA and sequence table shown in the sequence 3 of sequence table is right.
7. the said primer of claim 6 is to the application in the test kit of wheat that carries the TiSTK1 gene in the preparation assistant identification;
Said TiSTK1 gene is following 1) to 6) in arbitrary described dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 171st to 1448 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 674th to 2032 Nucleotide;
4) dna molecular shown in the sequence 1 in the sequence table;
5) under stringent condition with 1) or 2) or 3) or 4) the dna sequence dna hybridization that limits and the dna molecular of coded plant resistance to yellow dwarf GAP-associated protein GAP;
6) with 1) or 2) or 3) or 4) dna sequence dna that limits has 90% above homology, and the dna molecular of coded plant yellow dwarf resistance-associated protein.
8. an assistant identification is carried the test kit of the wheat of TiSTK1 gene, comprises that the said primer of claim 6 is right;
Said TiSTK1 gene is following 1) to 6) in arbitrary described dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 171st to 1448 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 674th to 2032 Nucleotide;
4) dna molecular shown in the sequence 1 in the sequence table;
5) under stringent condition with 1) or 2) or 3) or 4) the dna sequence dna hybridization that limits and the dna molecular of coded plant resistance to yellow dwarf GAP-associated protein GAP;
6) with 1) or 2) or 3) or 4) dna sequence dna that limits has 90% above homology, and the dna molecular of coded plant yellow dwarf resistance-associated protein.
9. arbitrary said method in the claim 1 to 5; The said primer of claim 6 to or the application of the said test kit of claim 8 in the assistant identification wheat with resistance to yellow dwarf; Wherein, Carry the wheat with resistance to yellow dwarf of the wheat of TiSTK1 gene, do not carry the sense BYDV wheat of the wheat of TiSTK1 gene for the candidate for the candidate.
10. arbitrary said method in the claim 1 to 5, the said primer of claim 6 to or the application of the said test kit of claim 8 in wheat breeding.
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| CN201010226688.7A CN101892315B (en) | 2010-07-14 | 2010-07-14 | Aided identification method of wheat with resistance to yellow dwarf and special primer pair thereof |
| PCT/CN2011/001120 WO2012006866A1 (en) | 2010-07-14 | 2011-07-06 | BOTANICAL YELLOW DWARF DISEASE RESISTANT PROTEIN TiSTKI, CODING GENE AND APPLICATION THEREOF |
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| CN201010226688.7A CN101892315B (en) | 2010-07-14 | 2010-07-14 | Aided identification method of wheat with resistance to yellow dwarf and special primer pair thereof |
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| CN104365471B (en) * | 2014-11-06 | 2016-06-15 | 南京农业大学 | One soft, the selection-breeding of mildew-resistance Triticum aestivum-Haynaldia villosa translocation line, authentication method |
| CN110643731B (en) * | 2019-10-30 | 2023-06-09 | 山西省农业科学院作物科学研究所 | Rapid detection of Elytrigia intermedium 6J S Specific molecular marker of chromosome and application |
| CN115785235B (en) * | 2022-09-19 | 2023-11-17 | 隆平生物技术(海南)有限公司 | Vip3Aa truncated protein variant and vector and application thereof |
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| CN101704882A (en) * | 2009-11-16 | 2010-05-12 | 中国农业科学院作物科学研究所 | Plant yellow dwarf resistance-associated protein, coding gene and application thereof |
| CN101704883A (en) * | 2009-11-26 | 2010-05-12 | 中国农业科学院作物科学研究所 | Plant yellow dwarf resistance-associated protein TiDPK1, coding gene and application thereof |
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| CN101704882A (en) * | 2009-11-16 | 2010-05-12 | 中国农业科学院作物科学研究所 | Plant yellow dwarf resistance-associated protein, coding gene and application thereof |
| CN101704883A (en) * | 2009-11-26 | 2010-05-12 | 中国农业科学院作物科学研究所 | Plant yellow dwarf resistance-associated protein TiDPK1, coding gene and application thereof |
Non-Patent Citations (2)
| Title |
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| Research progress in BYDV resistance genes derived from wheat and its wild relatives;ZHANG, Z. Y. 等;《Journal of Genetics and Genomics》;200909;第36卷(第9期);第567-573页，参见摘要，第568页右栏第三段 * |
| ZHANG, Z. Y. 等.Research progress in BYDV resistance genes derived from wheat and its wild relatives.《Journal of Genetics and Genomics》.2009,第36卷(第9期),第567-573页，参见摘要，第568页右栏第三段. |
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