Rolling circle amplification to screen yam germplasm for badnavirus infections and to amplify and characterise novel badnavirus genomes
Since the first discovery of badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 200...
| Main Authors: | , , , , , |
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| Format: | Journal Article |
| Language: | Inglés |
| Published: |
Bio-Protocol, LLC
2018
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| Subjects: | |
| Online Access: | https://hdl.handle.net/10568/108679 |
| _version_ | 1855513244732489728 |
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| author | Bömer, M. Turaki, A. Rathnayake, A. Silva, G. Kumar, P. Lava Seal, S. |
| author_browse | Bömer, M. Kumar, P. Lava Rathnayake, A. Seal, S. Silva, G. Turaki, A. |
| author_facet | Bömer, M. Turaki, A. Rathnayake, A. Silva, G. Kumar, P. Lava Seal, S. |
| author_sort | Bömer, M. |
| collection | Repository of Agricultural Research Outputs (CGSpace) |
| description | Since the first discovery of badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 2008; Bousalem et al., 2009), but only a few complete Dioscorea bacilliform virus (DBV) genome sequences have been reported (Phillips et al., 1999; Seal and Muller, 2007; Bömer et al., 2016 and 2017; Sukal et al., 2017; Umber et al., 2017). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm. We have employed this approach successfully revealing three novel episomal yam badnaviruses (Bömer et al., 2016). We proposed this to be a complementary method to denaturing gradient gel electrophoresis, which enables a rapid indication of badnavirus diversity as well as the identification of potentially integrated badnavirus sequences in the host genome (Turaki et al., 2017). Here, we describe the step-by-step protocol to screen yam germplasm for badnavirus infections using RCA as an efficient research tool in the amplification and characterization of novel badnavirus genomes. |
| format | Journal Article |
| id | CGSpace108679 |
| institution | CGIAR Consortium |
| language | Inglés |
| publishDate | 2018 |
| publishDateRange | 2018 |
| publishDateSort | 2018 |
| publisher | Bio-Protocol, LLC |
| publisherStr | Bio-Protocol, LLC |
| record_format | dspace |
| spelling | CGSpace1086792023-09-08T08:58:39Z Rolling circle amplification to screen yam germplasm for badnavirus infections and to amplify and characterise novel badnavirus genomes Bömer, M. Turaki, A. Rathnayake, A. Silva, G. Kumar, P. Lava Seal, S. yams dioscorea dna disgnostic techniques pcr germplasm genomes Since the first discovery of badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 2008; Bousalem et al., 2009), but only a few complete Dioscorea bacilliform virus (DBV) genome sequences have been reported (Phillips et al., 1999; Seal and Muller, 2007; Bömer et al., 2016 and 2017; Sukal et al., 2017; Umber et al., 2017). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm. We have employed this approach successfully revealing three novel episomal yam badnaviruses (Bömer et al., 2016). We proposed this to be a complementary method to denaturing gradient gel electrophoresis, which enables a rapid indication of badnavirus diversity as well as the identification of potentially integrated badnavirus sequences in the host genome (Turaki et al., 2017). Here, we describe the step-by-step protocol to screen yam germplasm for badnavirus infections using RCA as an efficient research tool in the amplification and characterization of novel badnavirus genomes. 2018 2020-07-03T11:39:46Z 2020-07-03T11:39:46Z Journal Article https://hdl.handle.net/10568/108679 en Limited Access Bio-Protocol, LLC Bömer, M., Turaki, A., Rathnayake, A., Silva, G., Kumar, P.L. & Seal, S. (2018). Rolling circle amplification to screen yam germplasm for badnavirus infections and to amplify and characterise novel badnavirus genomes. Bio-protocol, 8(1), 1-16. |
| spellingShingle | yams dioscorea dna disgnostic techniques pcr germplasm genomes Bömer, M. Turaki, A. Rathnayake, A. Silva, G. Kumar, P. Lava Seal, S. Rolling circle amplification to screen yam germplasm for badnavirus infections and to amplify and characterise novel badnavirus genomes |
| title | Rolling circle amplification to screen yam germplasm for badnavirus infections and to amplify and characterise novel badnavirus genomes |
| title_full | Rolling circle amplification to screen yam germplasm for badnavirus infections and to amplify and characterise novel badnavirus genomes |
| title_fullStr | Rolling circle amplification to screen yam germplasm for badnavirus infections and to amplify and characterise novel badnavirus genomes |
| title_full_unstemmed | Rolling circle amplification to screen yam germplasm for badnavirus infections and to amplify and characterise novel badnavirus genomes |
| title_short | Rolling circle amplification to screen yam germplasm for badnavirus infections and to amplify and characterise novel badnavirus genomes |
| title_sort | rolling circle amplification to screen yam germplasm for badnavirus infections and to amplify and characterise novel badnavirus genomes |
| topic | yams dioscorea dna disgnostic techniques pcr germplasm genomes |
| url | https://hdl.handle.net/10568/108679 |
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