High throughput RNA sequencing discovers symptomatic and latent viruses: an example from ornamental hibiscus

Hibiscus rosa-sinensis L. (Hibiscus, Malvaceae) is an ornamental species grown widely in landscape plantings. We collected leaves from a plant on an urban sidewalk near a market in Guangzhou that showed multiple symptoms of leaf rolling, deformation and chlorosis. Initial evaluation by electron microscopy using negative staining of dip preparations revealed the presence of Tobamovirus-like particles. Total RNA was extracted and without any RNA selection based on sequence, was used for cDNA library construction and high-throughput survey sequencing. Chloroplast, ribosomal, and mitochondrial sequences were filtered out of the 814 Mb of clean sequence data (from 2,712,161 paired reads of 150 bp), eliminating 79.1% of reads. 1,135,848×150 bp of the sequence was retained and screened for viral sequences. Assembly of these sequences revealed nine virus species from seven virus genera: tobacco mosaic virus, tobacco mild green mosaic virus and hibiscus latent Singapore virus (Tobamovirus), turnip mosaic virus (Potyvirus), potato virus M (Carlavirus), hibiscus chlorotic ringspot virus (Betacarmovirus), Fabavirus sp. (Fabavirus), cotton leaf curl Multan virus (Begomovirus) and Chenopodium quinoa mitovirus 1 (a putative Mitovirus replicating in mitochondria). Mapping the reads to complete virus reference sequences showed high and uniform coverage of the genomes from 3,729× coverage for turnip mosaic virus to 22× for cotton leaf curl Multan virus. By comparison, nuclear reference genes actin showed 14× coverage and polyubiquitin 27×. Notable variants from reference sequences (SNPs) were identified. With the low cost of sequencing and potential for semi-automated bioinformatic pipelines, the whole-RNA approach has huge potential for identifying multiple undiagnosed viruses in ornamental plants, resulting in the ability to take preventive measures in production facilities against virus spread and to improve product quality for the mutual benefit of producers and consumers.