Optimization Of Multiple RT-PCR For Detection Of Garlic Viruses And Identification Of A Novel Viral Sequences In Garlic

Garlic is one of vegetable widely cultivated in the world,which has unique flavor and high medicinal value.Asexual reproduction is the main mode of production of garlic.This way of reproduction leads to the accumulation of viruses in garlic bulbs,causing harm to garlic.Virus-free garlic culture requires rapid and accurate virus detection methods in order to determine the status of virus infection and effect of the cultivate virus-free garlic.At present,reverse transcription PCR（RT-PCR）detection methods for garlic viruses are usually for one or several single viruses,and there are few studies to newly viruses in garlic.In the research,Allexiviruses,Carlaviruses,and Potyviruses were detected.3-plex RT-PCR system were built for detection of above three genera related viruses in garlic.The concentration of Mg²⁺,d NTP,primer and Taq enzyme and annealing temperature in the triple RT-PCR system were optimized.The complete genomic sequence of a novel foveavirus,identified in garlic from Huanggang in China,was determined by using RNA-seq,reverse transcription polymerase chain reaction（RT-PCR）and rapid amplification of c DNA ends（RACE）PCR.The entire genomic RNA（Gen Bank accession MT981417）is 8748 nucleotides long excluding the 3’-terminal poly（A）tail and comprises five open reading frames（ORFs）.These ORFs encode the viral replicase,a triple gene block（TGB）and coat protein（CP）.The virus was tentatively named garlic yellow stripe associated virus（Gar YSa V）.Pairwise comparisons of protein sequences show that Gar YSa V encodes proteins that share less than 47.2%identity with those of other foveaviruses suggesting that it represents a new species in the genus.Similarly,phylogenetic analysis of amino acid sequences of the replicase and CP showed that Gar YSa V was a member of the genus Foveavirus.Confocal microscope results showed that Gar YSa V CP protein was found to be localized in the nucleus and peripheral cells.TGB1 protein was localized in the nucleus,cell periphery and chloroplast.TGB2 protein aggregates in the cytoplasm.TGB3 protein is localized in the cell periphery and aggregates near plasmodesmata.To our knowledge,this is the first report of a foveavirus in a monocot plant.In summary,we established a triple RT-PCR system for the detection of Allexivirus,Carlavirus and Potyvirus in garlic.The complete genomic sequence of a novel foveavirus was obtained from garlic.The sequence of the foveavirus was analyzed by bioinformatics,and the localization of CP protein and TGB proteins of foveavirus in tobacco cells were investigated.