Genome analysis of Geminivirus in pepper cultivation from West Sumatera

Jamsari, JMS (2010) Genome analysis of Geminivirus in pepper cultivation from West Sumatera. Proceeding od ISFAS-2010. ISSN 9786029630107

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Abstract

Pepper yellow leaf curl virus (PepYLCV) has been regarded as one of the notorious pathogen in pepper (Capsicum annuum) production. No chemical and cultural practices that could be used to prevent and manage this pathogen so far. Furthermore no resistant cultivar is available currently. Classical breeding is hindered by lacking of resistant genes resources that could be used for developing of new pepper cultivar that is resistant against this pathogen. For that reason, biotechnological approach is one alternative that could be chosen to solve this problem. This project was aimed to create a transgenic pepper that could be resistant against PepYLCV infection. For this purpose, analysis of DNA components that compose genome of geminivirus is a prerequisite. Number of genes involved in the genome and the detail structure of gene sequences must be elucidated. In order to achieve the goal, a strategy which is called primer walking was performed. The strategy involved some steps namely designing of primer combinations, testing of primer combinations, and followed by sequencing analysis of PCR products. Finally, all partial sequences were joined by their overlapping region to develop an integrated contig. Forty-six primers in total or twenty three primer combinations has been synthesized and tested for the identification of DNA components in the geminivirus genome. They consisted of 26, 12, 1, primers that should amplify DNA-A, DNA B and satellite DNA respectively. The primers were designed and could be combined in different orientation so they could amplify in partial or complete length of geminivirus genome. All the designed primers has been tested in six different isolates. The result successfully identified of DNA-A, and satellite DNA but no DNA-B in geminivirus from West Sumatera could be amplified. An initial fragment of 1600 bp in length as produced by PAL1v1978 and PAR1c715 primer pair was used as starting point in the sequence analysis. Sequencing was performed bidirectionally by applying both primers. After editing the sequence data, 1464 bases was collected and analyzed. The length is approximately 54% from the total of gemini virus genome. Furthermore it is successfully identified the complete sequence of C4 and V2 genes. C4 gene is responsible in the expression of the virus gene, and V2 gene is necessary for the movement of the virus in the infected cells. However C1-gene which is needed for virus replication and V1-gene which is important in the coat protein production could be identified partially. Extension of the sequence elucidation is currently still on going. Analysis of the β-component (satellite DNA) was successfully done from isolate TD-21. The satellite DNA is composed by 1351 bases, which is almost as long as Cotton Leaf curl virus (CLCV) and Mesta yellow vein mosaic virus (MYVMV) which is 1359 bases and 1354 bases respectively. However, the satellite DNA isolated from geminivirus of West Sumatera isolates is longer almost twice that the satellite DNA from tomatoe leaf curl virus (ToLCV) which is only about 632 bases. In order to be able to compare the sequence variability among some isolates cloning and sequencing of satellite DNA from PSS-14 is still in progress. In conclusion some facet could be mentioned that, genome structure of geminivirus from West Sumatera is monopartite as proved by the absence of DNA component and the presence of β-component. About 54% of the DNA-A component was successfully sequenced and analyzed and the other 46% is still in progress.

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