Genetic Diversity And Population Structure Of Tomato Yellow Leaf Curl Virus In Shandong Province

Tomato yellow leaf curl virus (TYLCV) is a member of Begomovirus belonging to Geminiviridae transmitted exclusively by the whitefly, Bemisia tabaci. Tomato yellow leaf curl disease(TYLCD) caused by TYLCV made a dramatic damage almost throughout all tropical and subtropical regions. TYLCV had become one of the limiting factors of the world tomato industry. In China, TYLCV was first reported in Shanghai, 2006. After that a large-scale outbreaking of this disease occured in Liaocheng, Shouguang, Linzi of Shandong province in 2008. The affected planting area up to 95% and the yield losses up to 55%. In order to find out the type of virus which infected tomato in Shandong province and offer the theoretical basis for further expansion research concern with defending and controlling of this virus,140 tamato samples showing yellow leaf curl symptoms from 9 different regions of Shandong were collected for study on identification and genetic variability analysis of TYLCVUsing the degenerate primer pair PA and PB of geminivirus, an approximate 500 bp fragment was amplified from 107 samples among all 140 samples. The detection rates of geminivirus in Zaozhuang, Heze, Linyi, Taian, Jinan, Zibo, Weifang, Dezhou and Binzhou was 91.7%,54.5%,66.7%,62.5%,75.0%,71.4%,85.0%,100% and 80.0% respectively. Two positive samples were chosen randomly to clone and sequence. The 2 amplified 500 bp fragments shared 98.5% nucleotide sequence identity. The similarity with the reported TYLCV isolates is more than 94%. Based on the determined sequences, the primer pair SDfull-F/SDfull-R was designed and used to amplify the complete viral DNA-A genome of SD11, SD28, SD39 and SD65, which were chosen randomly in the positive samples from different region of Shandong. PCR products of expected size were cloned and sequenced. The complete nucleotide sequence of these isolate were found to be 2781 nts, with all the characteristic features of begomovirus genome organization. The SD11, SD28, SD39 and SD65 isolate had high sequence identity (more than 98.5%) with TYLCV isolate SH-EU031444, AH-FJ646611 in China, which indicated that they were considered to be isolates of TYLCV. Using TYLCV specific primer pair TYT-F/TYT-R, the 430 bp fagments were detected in 107 of 140 samples by PCR. The detection rate and the numbers of positive samples were exactly same as that of PA/PB amplification result. Neither the DNA-B component nor the satellite DNAβmolecule was found to be associated with TYLCV in those positive isolates.Comparisons of the complete nucleotide sequences of all TYLCV isolates obtained from GenBank indicate that most of the TYLCV isolates shared over 93% nucleotide identity. In order to understand the reason of conservative and provide the basis for the genetic evolutionary of TYLCV and breeding for disease resistance, in this study, using the TYLCV infected field tomato samples collected from Shandong and Shanxi provinces as materials, based on the molecular clone, the genetic structures and population variation of TYLCV DNA-A were analyzed.Our results indicate that the populations of TYLCV were consistent with the natures of "quasispecies" which are not identical but closely related to the consensus sequence. The percentage of mutation clone and the mutation frequency of TYLCV are 21.0% and 2.36×10-4 respectively, which are lower than those reported in the other geminiviruses populations. The diversity level of population of the isolate SD1 obtained in November 2008 from Shouguang in Shandong province is comparable with that of SX15 obtained in July 2011 from Jingyang in Shanxi province. The mutation frequency of SD98 obtained in July 2011 from Shouguang in Shandong province was much lower than the former 2 populations of TYLCV, which is 5.42×10-5. The rules of diversity level of population from different region or in different time are not clear.Mutations were distributed throughout the IR-AC1 region of TYLCV DNA-A and especially concentrated in IR 5’and AC1 non-overlaping region. Substitution mutation was the only mutation form detected in TYLCV populations. Mutation from C to T and T to C were the predominant mutation types in the population of TYLCV populations. Analysis of base mutations and aminoacid mutations in TYLCV populations indicated that aminoacid mutations were non-synonymous mutations in AC1 but synonymous mutations in AC4. Mutations accumulation was found in SX15 population. The variant carried the same mutations at nucleotide site 37 and 59 (IR 5’), and nucleotide site 2152 (non-overlaping region of AC1) were found in several clones, suggesting that these variants possess the selection advantage over the other variants during the infection. Consensus sequences of TYLCV DNA-A obtained from different regions or in different time were quite different. The Consensus sequences of SD1 population were found as variants in SX15 population, which indicated that some advantagous mutations are displaced or even be eliminated in the proceeding of virus evolution under selection pressure.