Identification of fig mosaic disease by NGS

Fig mosaic (FMD) is a common disease in fig orchards in Iran with high economic importance its etiology is not fully clarified. Association of different viruses and variation of symptoms suggest that FMD may be caused by the synergistic effects in mixed-infection of several viruses. Although FMD has been seen in Iran since some decades ago, high current incidence in fig orchards of Fars province, as one of the main fig production regions, create a national alarm which necessitates a better understanding of the viral complex associated with the disease. Nextgeneration sequencing (NGS) offers a beneficial tool for studying the virome profile of diseases. The main challenge in identifying the virome profile of infected fig by NGS is that separation of RNAs of viruses from those of the host plant is difficult. Consequently, the virome metagenome needs to be analyzed with the host RNA (fig). Unavailability of the full genome of fig adds another level of complexity to the analysis. In this study, we developed a pipeline for detection of viral sequences based on RNASeq de novo assembly of short reads in healthy fig (to make reference genome). To this end, RNA samples were extracted from leaf pools of infected and healthy fig plants in three biological replicates and total RNA from leaves of healthy and fig-mosaic-infected plants were sequenced using Illumina HiSeq 2000 high throughput sequencing (as 100 bp, paired-end short reads) to determine the pathogenic virome profile of FMD in Fars province. The obtained sequences from total reads (4,852 megabases) in infected plants were compared with those obtained from healthy fig (5,116 megabases). Then, viral + fig sequences were mapped to the constructed reference genome. Fig infecting viral sequences were collected from unmapped reads. The obtained viral short reads were used to construct contigs and were subjected to blast analysis against NCBI to identify the virome profile in infected figs. The results demonstrated the existence of fig fleckassociated virus (FFKaV), fig badnavirus-1 (FBV-1), and fig mosaic virus (FMV) in fig leaves of the infected plant. This method provides a rapid and cost-effective pipeline for detection of virome profile of infected fig using the mixed RNA sequences of viruses and fig.

Keywords:

Fig mosaic disease , Next-generation sequencing