Gilbertson R L, Rojas M R, Russell D R, Maxwell D P
Department of Plant Pathology, University of California, Davis 95616.
J Gen Virol. 1991 Nov;72 ( Pt 11):2843-8. doi: 10.1099/0022-1317-72-11-2843.
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.
采用聚合酶链反应（PCR）、不对称PCR（A-PCR）和DNA测序相结合的方法，测定了菜豆金色花叶双生病毒（BGMV）二分体基因组高变区的核苷酸序列。该区域是DNA-B组分基因间隔区的一部分，使用根据来自多米尼加共和国的BGMV分离株（BGMV-DR）的DNA-B组分克隆（pDRB1）的核苷酸序列设计的引物进行扩增。当与BGMV-DR的DNA-A组分（pDRA1）共接种时，pDRB1对菜豆具有感染性，在受感染的植株上可观察到典型的菜豆金色花叶症状。在多米尼加共和国的五个不同田间地点采集感染BGMV的菜豆叶片组织，通过PCR扩增高变区，使用A-PCR产生单链DNA（ssDNA），并测定部分核苷酸序列。田间采集样本的高变区序列与pDRB1序列的同一性在95%（一个样本）至98%（四个样本）之间。这与pDRB1高变区与分别来自危地马拉、波多黎各和巴西的BGMV分离株的高变区序列同一性86%、75%和46%形成对比，与菜豆矮化花叶双生病毒的序列同一性为42%。这些结果表明，多米尼加共和国的BGMV分离株非常相似，应被视为同一病毒（BGMV-DR）的分离株，并且BGMV-DR的感染性克隆代表了多米尼加共和国的BGMV分离株。所描述的从叶片组织中提取DNA以及使用PCR和A-PCR产生高质量ssDNA的方法快速且高效，可应用于其他病毒的变异性和流行病学研究。
