Use of the asymmetric polymerase chain reaction and DNA sequencing to determine genetic variability of bean golden mosaic geminivirus in the Dominican Republic

Abstract

A combination of the polymerase chain reaction (PCR), asymmetric PCR (A-PCR) and DNA sequencing was used to determine the nucleotide sequence of a hypervariable region of the bipartite genome of bean golden mosaic geminivirus (BGMV). This region, which was part of the intergenic region of the DNA-B component, was amplified using primers designed from the nucleotide sequence of a DNA-B component clone (pDRB1) of an isolate of BGMV from the Dominican Republic (BGMV-DR). pDRB1 is infectious on beans when coinoculated with the DNA-A component of BGMV-DR (pDRA1), and typical bean golden mosaic symptoms are observed on infected plants. Bean leaf tissue infected with BGMV was collected at five separate field locations in the Dominican Republic and the hypervariable region was amplified by PCR, ssDNA was produced using A-PCR, and partial nucleotide sequences were determined. The sequences of the hypervariable region from the field-collected samples ranged from 95% (one sample) to 98% (four samples) identical to the sequence of pDRB1. This contrasts with sequence identities of 86, 75 and 46% between the pDRB1 hypervariable region and the hypervariable regions of BGMV isolates from Guatemala, Puerto Rico and Brazil respectively, and 42% with bean dwarf mosaic geminivirus. These results indicate that Dominican Republic isolates of BGMV are very similar and should be considered isolates of the same virus (BGMV-DR), and that the infectious clones of BGMV-DR are representative of BGMV isolates in the Dominican Republic. The procedures described for DNA extraction from leaf tissue and for production of high quality ssDNA using PCR and A-PCR are rapid and efficient and could be applied to studies of variability and epidemiology of other viruses.