Genetic Diversity Of Grapevine Viroids And The Construction Of Their Infectious Clones

Viroids are the smallest known plant pathogens and grapevine is one of the most popular cultivated fruit crops in China. In 2006, it ranks fourth and fifth in the world in acreage and production respectively. Up to now, five viroids have been identified with the ability to infect grapevines: Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd), Australian grapevine viroid (AGVd) and two Grapevine yellow speckle viroids (GYSVd-1 and GYSVd-2). AGVd has originated from extensive RNA recombination involving other viroids; therefore, it is a typical material for investigating the evolution and recombination of viroids. However, the pathogenesis mechanism of AGVd hasn't been clear. To date, AGVd has been found in only three countries including Australia, Tunisia and China. GYSVd-2 is a viroid found only in grapevines in China and Australia. It is associated with disease symptoms such as yellow speckle and cause adverse effects on production and quality of grapevines. Due to the low infection rate of cultivated grapevines by GYSVd-2, little is known about this viroid. Therefore, the studies on them are of great significance to guide basic research work of viroids as well as production practice.The present studies is intend to identify and isolate AGVd and GYSVd-2 from grapevines in China, do sequencing analysis after cloning, and then analyze their populative structure and genetic diversity. After that, the information content of the grapevines viroids would be enriched in GenBank. Our studies would also lay a solid foundation for the investigation of the pathogenesis mechanism, evolution and recombination of viroids and provide theoretical base for the transmission, popular rule and control of the viroids.The present work detected AGVd and GYSVd-2 from the 89 grapevine samples collected from Beijing, Shenyang, Xinjiang and Fujian areas. The results demonstrated that: (1) AGVd was detected in three samples: Jingchuan, Zaoyu and Thomson Seedless. (2) GYSVd-2 was positive in only three samples: Black Olympia, Zaoyu and Thomson Seedless. The Jingchuan, Black Olympia and Zaoyu samples were collected from Beijing, while the Thomson Seedless sample estimated to be older than 150 years was collected from Xinjiang autonomous region. Specific primers for AGVd and GYSVd-2 were designed for cloning and sequencing. The results of sequence analysis showed that: (1) The genetic diversity of AGVd and GYSVd-2 were lower and each isolate was a mixture of RNA species, in agreement with the concept of quasispecies. (2) Compared to the sequences reported in other countries, the sequences from China revealed characteristic variations. All the variant bases were located in the domain out of the conserved region including the center conserved region (CCR) and the terminal conserved region (TCR). The results of the predicated second structure and phylogenetic analysis demonstrated that these changed bases influenced the second structures of the viroids more or less, and whether these changes are associated with the pathogenicity of viroids needs further study. (3) The sequences of GYSVd deposited in GenBank and the GYSVd-2 sequences we have obtained were analyzed by a series of biological software and the results demonstrated that there was a new species of grapevine viroids, tentatively named CGVd. The homology between the first reported sequence of GYSVd-1 or GYSVd-2 and the CGVd sequence variants was <90%, According to the molecular characterization of CGVd and the classification benchmarks of the International Committee on Taxonomy of Viruses (ICTV), we propose that CGVd is a new species and a member of the Apscaviroid group, and we suggest naming it Grapevine yellow speckle viroid (GYSVd-3).In this study, 123 sequences of AGVd and 37 sequences of GYSVd-2 have been deposited in GenBank and these sequences enriched the information of the GenBank infinitely.In addition, through molecular biological methods including restriction enzyme digestion, ligation and transformation et al., the infectious clones of AGVd, HSVd and (AGVd + HSVd) were constructed successfully and they lay the foundation for further study of the cross-protection mechanism between HSVd and AGVd as well as their pathogenicity.