Molecular Detection And Sequence Analysis Of Tomato Yellow Leaf Curl Virus In Three Provinces Of Northern China

Tomato yellow leaf curl virus （TYLCV） is the virus causing the tomato yellow leaf curl disease. In recent years, this disease occurred extensively in north China, causing a serious impact on tomato yield. It has become the primary limiting factor for tomato production in China. However, research for the TYLCV molecular mutate is imperfect. Besides, the detection of virus only stays on a qualitative level, which is not conducive to carrying out the prevention and research of TYLCV. During2010and2011, we’ve collected samples suspected to have been infected by TYLCV in the tomato growing areas of Shandong, Henan, Hebei and other places. By using the augmentation of the geminivirus’universal primer and the full-length primer of TYLCV, we have got30DNA-A full-length sequences of the TYLVC. Meanwhile, we’ve also optimized the detection system of PCR and fluorescence quantitative PCR. The main results are as follows:1. By comparing with other sequences in the GENBANK, the report says that the isolates of TYLCV do not have an obvious recombination in northern area. While according to the homology comparison, the TYLCV isolates have the highest homology with the TYLCV-IL, whose rate is98.8%-99.4%; according to the phylogenic analysis, the TYLCV isolates belong to the TYLCV-IL and in cladogram it can be divided into three groups, of which group II comes mainly from Shandong while group III mainly from Henan. The three groups of TYLCV isolates have presented a negative selection, in which the nucleotide shows a bigger variation. According to the mismatch analysis, TYLCV has existed in a state of equilibrium in these three provinces. The genetic exchange report says that the genes exchange most frequently between Shandong and Hebei province.2. Based on the genetic sequence of TYLCV on GENBANK, we designed three pairs of detection primer in different positions. We have got the best primer and detection system by optimizing the sensitivity of Taq polymerase, annealing temperature and primers of different positions. The best primers are SY1:5’-GTT GAG GAA ACT TAC GAG CC-3’; SY2:5’-CAT TCT TCA CTG TTG CGG T-3’, the annealing temperature is55℃, the elements of the system includes2.5μL of10xPCR buffer solution (including Mg²⁺),1μL for each10mmol/L forward and reverse prime10.5μL of Taq polymerase, and3μL of sample DNA, then adding redistilled water to25μL. We found TYLCV by detecting these diseased tomatoes using this system.3. Based on the genetic sequence of TYLCV and the sequence of tomato’s internal reference gene on GENBANK, we designed qPCR purpose primer and internal reference primer. The final annealing temperature was fixed on58℃and the consistency of primer is0.2μmol/L by analyzing the melting curve and optimizing the annealing temperature and consistency of primer. The detection of levels of virus on different parts of the infected plant shows that the highest one occurs on the phloem. According to the detection of different cultivars of infected plants, the81st plant has the worst virus antagonism. We also build the standard quality plasmid. Comparing the sensitivity of normal PCR, we found the qPCR of SYBR Green is keener100times than the normal one and its pertinence of the standard curve is good. By means of detecting the levels of virus on the tomatoes that had been transplanted in different times, we found the levels of virus had increased rapidly within22-28days.