Molecular Identification And Variation Analysis Of Tomato Yellow Leaf Curl Virus In Henan Province

Tomato yellow leaf curl virus (TYLCV) is a geminivirus transmitted by the white fly Bemisia tabaci. It can cause Tomato yellow leaf curl disease (TYLCD) which is a serious disease for tomato production The typical symptoms include severely stunted, upward curling and yellow wing along margins. Firstly TYLCV was spread to southern province of China; recently, it broke out in Jiangsu, Shandong, and Hebei province, etc. In2007, some tomato plants showing typical yellow leaf curl symptom were found in green houses in rural areas of Zhoukou for the first time; the disease broke out in Henan in2009.This disease was a serious impact on tomato yield and quality.With total DNA isolated from diseased samples of Henan, a pair of degenerate primers which named CPF and CPR was designed according to the conserved sequence of coat protein genes of TYLCV. Then the770bp fragment was obtained by PCR procedure. PCR products were cloned into the vector pMD18-T. Sequence analysis indicated all tomato plants sampled from16districts in Henan have been infected by TYLCV. They share99.7%homology.According to the nucleotide sequence of the770bp with the CPR/CPF primer, we designed a pair of adjacent back primers, and using total DNA of the tomato leaves as a template. Then the HX of DNA-A sequence was amplified by PCR. By the HX whole genome amplification, recovery, ligation, transformation, screening, identification and sequence analysis, it was revealed that HX genome length is2780nucleotides encoding six open reading frames. Virus-sense part encodes AV1and AV2. Complementary sense part encodes AC1, AC2, AC3and AC4. In addition, the structure of inter genic region is fairly conservative. Sequence analysis showed that the whole genome sequence identity with isolates of Tomato yellow leaf curl virus from other places was more than92%, and it has99.4%sequence similarity with the TYLCV isolate reported in Shandong (JN990928).At last, DNAβ was found in the isolates from Zhongmou by PCR procedure with the universal primer Beta01and Beta02.