Location: National Germplasm Resources LaboratoryTitle: Discovery of uncharacterized sugarcane viruses by next generation sequencing technology: the case of Ramu stunt Author
|Marron-lango, Clarissa - Animal And Plant Health Inspection Service (APHIS)|
|Kuniata, Lastus - New Britain Palm Oil|
Submitted to: American Society of Sugar Cane Technologists
Publication Type: Abstract Only
Publication Acceptance Date: 4/15/2015
Publication Date: N/A
Technical Abstract: Ramu stunt disease of sugarcane was first reported in Papua New Guinea in the mid 1980's. The disease can reduce sugarcane yields significantly and causes severe stunting and mortality in highly susceptible cultivars. The causal agent of Ramu stunt has been investigated but its characterization has not been completed. Sugarcane cv. Ragnar plant material, with symptoms of Ramu stunt, was received from Papua New Guinea through the USDA APHIS PPQ Plant Germplasm Quarantine Program and used in this study. Total RNA was extracted and subjected to next generation sequencing (NGS) using whole transcriptome shotgun sequencing method on the Illumina platform. Approximately forty million reads with an average length of 100 bp were obtained. More than eighteen thousand contigs were assembled and subjected to BLASTX analysis. Twenty-one contigs were virus related and five were associated with plant viruses. The BLAST algorithms revealed sequence similarity to Tenuiviruses, a genus of viruses whose members contain genomes consisting of four to six RNA segments. The five contigs derived from the RNA sequencing data correspond to the five RNAs that compose the Ramu stunt virus genome. Primers were designed for each of the five RNAs and RT-PCR amplicons were obtained only from the symptomatic sugarcane. There was concordance between the sequence data of the contigs obtained from the NGS and that of the amplicons obtained by RT-PCR. The NGS approach allowed us to determine the complete genomic sequence of Ramu stunt virus. It is likely that this virus is the causal agent of Ramu stunt disease. Characterizing the genomic organization and developing improved diagnostic methods for Ramu stunt virus will aid in quarantine and disease management efforts.