Application of sequence-independent amplification (SIA) for the identification of RNA viruses in bioenergy crops

Miscanthus x giganteus, energycane, and Panicum virgatum (switchgrass) are three potential biomass crops being evaluated for commercial cellulosic ethanol production. Viral diseases are potentially significant threats to these crops. Therefore, identification of viruses infecting these bioenergy crops is important for quarantine purposes, virus resistance breeding, and production of virus-free planting materials. The application is described of sequence-independent amplification, for the identification of RNA viruses in bioenergy crops. The method involves virus partial purification from a small amount of infected leaf tissue (miniprep), extraction of viral RNA, amplification of randomly primed cDNAs, cloning, sequencing, and BLAST searches for sequence homology in the GenBank. This method has distinct advantage over other virus characterization techniques in that it does not require reagent specific to target viruses. Using this method, a possible new species was identified in the genus Marafivirus in switchgrass related to Maize rayado fino virus, its closest relative currently in GenBank. Sugarcane mosaic virus (SCMV), genus Potyvirus, was identified in M.xgiganteus, energycane, corn (Zea mays), and switchgrass. Other viruses identified were: Maize dwarf mosaic virus (MDMV), genus Potyvirus, in johnsongrass (Sorghum halepense); Soil borne wheat mosaic virus (SBWMV), genus Furovirus, in wheat (Triticum aestivum); and Bean pod mottle virus (BPMV), genus Comovirus, in soybean (Glycine max). The method was as sensitive as conventional RT-PCR. This is the first report of a Marafivirus infecting switchgrass, and SCMV infecting both energycane and M. x giganteus.