Simultaneous Detection Of Three Wheat Viruses By Multiplex PCR And Analyses Of Wheat Yellow Mosaic Virus Complete Genomic Sequences

Huang-Huai-Hai region is one of major wheat growing area in China. Viruses especially causing soil-borne mosaic and green dwarf are important problem affecting wheat production. It showed that Wheat yellow mosaic virus(WYMV) and Chinese wheat mosaic virus(CWMV) transmitted by Polymyxa graminis L. are the pathogen causing soil-borne mosaic symptom. Rice black-streaked dwarf virus(RBSDV) mainly transmitted by Laodelphax striatellus is the one causing green dwarf symptom.Rapid detection of plant virus plays an important role in virus identification, prediction and virus control. Moreover, known viral disease epidemic, virus variation and evolution could help us understand viral occurrence, pathogenic mechanism and breeding resistant cultivars.This study, a optimized multiplex reverse transcription-polymerase chain reaction(RT-PCR), in which Taq DNA polymerase(0.025 U/?L), dNTP(0.25 mmol/L), annealing temperature 56 ℃, extension time 60 second and 30 cycles were selected, is developed to simultaneous detect WYMV, CWMV and RBSDV.The occurrence of these three viruses in wheat in Shandong province 17 cities were investigated during 2013 and 2014. No virus-like samples was found in Jinan, Dongying, Heze, Laiwu and Dezhou. In total, 165 samples were collected from the rest 12 cities. Based on results of multiplex PCR, 93 samples collected from Zibo, Zaozhuang, Tai’an, Yantai, Jining, Rizhao, Linyi, Liaocheng and Weihai contain WYMV. It’s the first report that the occurrence of WYMV in Zibo, Rizhao and Liaocheng. 14 samples contain CWMV, and 11 contain RBSDV. CWMV is very serious problem in Yantai and Weihai, and is spreading to Linyi. RBSDV was detected in Jining and Linyi. Mixed infection of WYMV and CWMV was found in Yantai, Weihai and Linyi. Mixed infection of WYMV and RBSDV was found in Jining and Linyi. None of these three viruses was detected in sample collected from Qingdao, Bingzhou and Weifang.To study the genetic diversity of WYMV, 17 complete genomes of WYMV were obtained. Sequence analysis showed that there are 7635 and 3651 nucleotides in RNA 1 and RNA 2, respectively, excluding poly(A) tail. The open reading frame in RNA 1 and RNA 2 encode 2404 and 903 amino acids, respectively. Recombination events were found in RNA 1 of LY-1, LY-2, LY-3 and LY-4 isolates, and in RNA 2 of Yamato, Yangzhou, QF-2, and LY-2 isolates. Phylogenetic analysis based on RNA 1 and RNA 2 indicated that all isolates were clustered into two groups. RNA 1 of isolates Date, Kyowa, Ya’an, Zhouzhi, TA-2, LY-6, QF-4 and LY-5 were clustered to group II. In contrast, RNA 2 of isolates Date, Kyowa, Zhouzhi and TA-2 were clustered to group I. Moreover, RNA 1 of RZ belongs to group I but RNA 2 belongs to group II. It suggested that genome reassortmant existed in isolates Date, Kyowa, Zhouzhi, TA-2 and RZ.