烟台市富士苹果上苹果锈果类病毒分子变异分析
Genetic analysis of Apple scar skin viroid from Fuji apple in Yantai, China

为明确苹果锈果类病毒（Apple scar skin viroid，ASSVd）在烟台市富士苹果上的变异情况，通过特异性引物对携带苹果锈果类病毒的富士嫩叶进行ASSVd全长扩增，利用生物学软件DNAMAN对所得变异序列进行分析并构建系统进化树。结果表明，从130个样品中筛选到36个阳性样品，36个阳性样品中共克隆获得52条329~333 nt的ASSVd变异序列，其中30个阳性样品含2条或2条以上的ASSVd序列。对所得变异序列进行序列比对发现，同一样品不同克隆间核苷酸序列相似性为94.0%~100.0%，所有变异序列核苷酸序列相似性为94.0%~99.7%，与GenBank中来自不同国家或地区的10条ASSVd分离物核苷酸序列相似性为88.3%~99.7%。将序列相似性低于97.0%的12条变异序列进行系统进化分析，结果显示除了333 nt变异序列ZS2-6与伊朗苹果分离物在同一分支外，其余11条变异序列位于同一分支。52条变异序列多序列比对发现，变异位点主要集中在TL区、P区和C区。研究表明烟台市富士苹果上ASSVd存在一定的分子变异。
To determine the genetic variation of Apple scar skin viroid (ASSVd) from Fuji apple in Yantai, a pair of specific primers were used to amplify full genome of ASSVd from Fuji young leaves collected in Yantai by RT-PCR, and the sequences were analyzed by software DNAMAN. The results showed that 36 out of the 130 samples arrayed were infected by ASSVd. 52 ASSVd variants were obtained from the 36 positive samples, with the length of 329-333 nt. Among of them, there were 30 positive samples of which obtained two to four variants. The sequence similarities ranged from 94.0% to 100.0% among the clones within each of isolate, while 94.0% to 99.7% among the total 52 variants. Compared with other ten ASSVd sequences from different countries or areas in GenBank, the nucleotide sequence similarity was 88.3%-99.7%. Twelve variants of which nucleotide sequence similarity less than 97.0% were selected to analyze the phylogenetic tree, and the variant ZS2-6 with the length of 333 nt, was clustered with an Iran isolate, and the other 11 variants were clustered together. Multi-alignment analysis showed that the variable sites mainly occurred in the domains of TL, P, and C. The results indicated that the variation in sequence of ASSVd from Fuji apple in Yantai was existed.