Institute of Biology, Department of Molecular Biology and Plant Virology, University of Stuttgart, Pfaffenwaldring 57, 70550 Stuttgart, Germany.
Virus Res. 2010 May;149(2):224-33. doi: 10.1016/j.virusres.2010.02.003. Epub 2010 Feb 10.
Begomoviruses cause major diseases of sweet potato worldwide impairing considerably the yields of this important food staple. Since sweet potato plants are vegetatively propagated and globally transported, they are prone to accumulate and disseminate geminiviruses. Effective diagnostic tools are, therefore, desirable. We studied the genomic diversity of geminiviruses present in naturally-infected sweet potato accessions belonging to a Brazilian germplasm bank collection. Fifty-five samples from different sweet potato accessions displaying geminivirus-like symptoms were analyzed by combining rolling circle amplification (RCA) with restriction fragment length polymorphism (RFLP) and sequencing. The restriction enzyme MspI (HpaII) revealed diverse band patterns in 55 samples and digestion with BamHI, SstI or PstI resulted in full-length sweet potato geminivirus DNAs of about 3 kb in 46 samples. In addition, smaller fragments were identified as either viral "Defective DNAs" (D-DNAs) or mitochondrial plasmid DNAs. The diversity of sweet potato-associated geminiviruses was found to be very high under Brazilian conditions. Representative viral full-length DNAs have been cloned and sequenced yielding two new tentative species, three strains and several variants of previously described sweet potato geminiviruses. Sequence comparisons identified footprints of recombination in their genomes underscoring the risk of generating new geminiviruses in vegetatively propagated germplasm bank material. The sites of recombination were found in conjunction with predicted hairpin structures. We propose diagnostic routines to screen germplasm bank material for geminiviruses by the rapid and reliable RCA/RFLP as the technique of choice.
双生病毒引起甘薯的世界性重大病害，极大地影响了这种重要粮食作物的产量。由于甘薯植物是通过营养繁殖和全球运输的，它们容易积累和传播双生病毒。因此，需要有效的诊断工具。我们研究了巴西种质银行收集的自然感染甘薯品系中存在的双生病毒的基因组多样性。对 55 个表现出双生病毒样症状的不同甘薯品系的样品进行了分析，方法是将滚环扩增（RCA）与限制性片段长度多态性（RFLP）和测序相结合。限制性内切酶 MspI（HpaII）在 55 个样品中显示出不同的带型，而用 BamHI、SstI 或 PstI 消化导致 46 个样品中约 3 kb 的全长甘薯双生病毒 DNA。此外，还鉴定出较小的片段为病毒“缺陷 DNA”（D-DNA）或线粒体质粒 DNA。在巴西条件下，发现甘薯相关双生病毒的多样性非常高。代表病毒全长 DNA 已被克隆和测序，产生了两个新的暂定种、三个株系和几种以前描述的甘薯双生病毒的变体。序列比较在它们的基因组中鉴定出重组的痕迹，强调了在营养繁殖的种质银行材料中产生新的双生病毒的风险。重组的位点与预测的发夹结构结合在一起。我们提出了通过快速可靠的 RCA/RFLP 筛选种质银行材料中的双生病毒的诊断程序，作为首选技术。
