The use of rolling circle amplification (RCA) for diagnosis and characterization of geminiviruses

Geminiviridae is the largest family of plant DNA viruses that infect a broad range of plants causing a limitation to the production of economically important or staple food crops within tropical and subtropical countries. Increasing knowledge about its epidemiology, sequence diversity and biodiversity is highly important in order to implement preventative strategies. With the use of rolling circle amplification (RCA) combined with restriction fragment length polymorphism (RFLP), in addition to other methods applied in molecular biology, technical improvements on direct sequencing, shot gun cloning and pyrosequencing were achieved in this work. In the first two parts of this study RCA/RFLP was used to diagnose geminiviral infection in symptomatic plant samples originally collected in South America. All samples from Bolivia, in total 7 weeds and 21 of 22 samples from Brazil, including beans samples, were diagnosed virus positive when analyzed using RCA/RFLP. RCA products were sequenced using two different methods and the complete genome sequences of all detected viruses were obtained. For the Bolivian samples, a novel and efficient cloning strategy with tandem repeat inserts obtained by limited Sau3AI digestion was used and for the Brazilian samples, in order to develop a faster detection and characterization system for geminiviral infection, the RCA products were pooled and sequenced by the commercially available 454 method. The sequences analysis showed that all viruses had a genomic organization of bipartite New World begomoviruses. Phylogenetic analysis revealed amongst the detected viruses, five distinct new virus species, two new strains and five variants of previously described viruses. In the third part of the study, two variants of Tomato golden mosaic virus (TGMV), common strain (cs) and yellow vein (yv), originally extracted from tomato but never detected in tomatoes again, were completely sequenced for the first time and successfully bombarded back into tomato plants. The infection rate was extremely low and symptoms induction was very mild. Finally, in the last part of this work, Asystasia gangetica plants, collected in West Africa in the 1980s with segregation of symptoms, including mosaic and yellow veining, typical of geminiviral infections were analyzed. Also in this case using the RCA/RFLP technique followed by deep sequencing of the RCA products, the results confirm the idea of infection by two distinct geminiviruses, showing a remarkable segregation of geminiviruses in a single plant.