The Detection Of Two Kinds Of Geminivirus Disease Complexes And Molecular Variability Of Satellite DNAs

Two kinds of geminivirus disease complexes, Malvastrum yellow vein virus (MYVV) and Malvastrum yellow vein Yunnan virus (MYVYNV), which are associated with their satellite DNAβ called Malvastrum yellow vein betasatellite (MYVB) and Malvastrum yellow vein Yunnan betasatellite (MYVYNB), have caused serious damage in Yunnan and Sichuan provinces. Studies have shown that the pathogenesis of MYVV/MYVB and MYVYNV/MYVYNB are different. MYVB is needed for induction of symptoms. While MYVYNB is not necessary for pathogenicity but intensifies symptoms, and some MYVYNV isolates were found to be associated with DNAβ in the field. To identify the occurrence, development and genetic variation of these two kinds of begomovirus/DNAβ complexes,255samples with severe symptoms were collected from three weed species in Sichuan province, which then were addressed for detections of these two kinds of begomovirus/DNAp complexes with specific primers of MYVV/MYVB and MYVYNV/MYVYNB. The genome organization of DNA-A and DNAβ, and genetic diversity of viruses and betasatellites were analyzed.The result of PCR detection using universal primer pair PA/PB for geminiviruses showed that195of255samples were positive (positive ratio was76.5%), and500bp specific DNA segments were amplified from them. Among these195samples,11samples were infected with MYVV and162samples were infected with MYVYNV. The infection ratio of MYVV was4.3%, and the infection ratio of MYVYNV was63.5%. The mixed-infection ratio of these two viruses was3.5%. These results suggested that MYVV and MYVYNV commonly occurred in Malvastrum coromandelianum. The results of PCR detection using universal primer pair βO1/βO2for betasatellite showed that159of255samples were positive (positive ratio was62.4%), and1300bp specific DNA segments were amplified from them. The infection ratio of MYVB was20.0%, and the infection ratio of MYVYNB was37.3%.Two isolates of MYVV, five isolates of MYVYNV, one isolate of MYVB, four isolates of MYVYNB from different hosts were chosen to be were sequenced and the genome organization and genetic diversity were analyzed in this study. Results showed that MYVV isolates and MYVYNV isolates all were of typical DNA-A genomic structure of geminiviruses which encoded six open reading frames, AVI, AV2, ACl, AC2, AC3and AC4. AV2and ACl were separated by IR region which contains many kinds of conservative cis component, such as stem loop structure, TATA box, repetitive sequence and so on. There are three results by analyzing total nucleotide similarity, nucleotide similarity of regions and amino acid sequence similarity of ORFs. Firstly, variation of IR region is the biggest, while AC4region is the smallest. Secondly, nucleotide similarity of AC4region is the highest than others while IR region is the lowest. Lastly, the major mutation occurred in AC1region is synonymous while is non-synonymous in AC3and AC4region. MYVB isolates and MYVYNB isolates all are of typical DNAβ structure of geminiviruses which encoded only one ORF in complementary strand and contained satellite conserved region with conservative stem-loop structure of TAATATT/AC and A-rich region that is rich in adenine.It has been reported that pathogenesis of MYVB and MYVYNB are different. In order to understand the genetic structure and population variation of MYVB and MYVYNB in different hosts, the populations of MYVB and MYVYNB were chosen as research objects in this study. Results showed that about55.6%of MYVB clones mutated and with1.4×10-3of mutation frequency in natural hosts. While it was found that about100%of MYVB clones mutated in laboratory-maintained population and with2.4×10"3of mutation frequency in N. benthamiana or with2.4×10-3of mutation frequency in N. glutinosa. It showed that the level of mutation in natural-maintained population was lower than laboratory-maintained population. And it showed that mutations existed in natural-maintained population because of insertions or deletions of bases but existed in laboratory-maintained population because of insertions of bases (mainly was A). And it showed that the main regions in which mutations occurred were satellite conserved region (SCR) and A-rich region in the natural hosts and laboratory hosts respectively by analyzing the distribution of mutations in MYVB population. In conclusion, MYVB population in natural hosts and laboratory hosts were different.It showed that about84.2%MYVYNB clones mutated and with1.5×10-3of mutation frequency in YN65population from the host Capsicum,35.0%MYVYNB clones mutated and with2.6×10-4of mutation frequency in the population of MY382from the host Malva and76.2%MYVYNB clones mutated and with9.9×10-4of mutation frequency in the host Malvastrum. Results showed that the level of mutations of MYVYNB population from the three hosts were different. The number of base transition was more than base transversion in MYVYNB population from the host Capsicum while just the opposite in Malva and Malvastrum by analyzing the type of base mutations. There were two bases insertion occurred in Capsicum and one base deletion occurred in Malva while no deletion or insertion occurred in Malvastrum in MYVYNB population. And it showed that the main areas in which mutations occurred were SCR, A-rich region and βC1region in the host Capsicum, Malvastrum and Malva respectively by analyzing the distribution of mutations in MYVYNB population. Results showed that the mutation frequency, types of base mutations and distribution of mutations of MYVYNB population in the three hosts from different families were different. All these results showed that the selection pressures from hosts might play an important role in evolution of betasatellite.