Identification and characterization of a viroid resembling apple dimple fruit viroid in fig (Ficus carica L.) by next generation sequencing of small RNAs

• A viroid sharing 85% of nucleotide identity with apple dimple fruit viroid is present in fig.
• Deep sequencing of a small RNA library from the fig accession produced a complete coverage of the viroid sequence.
• A first characterization of small RNAs from an ADFVd fig variant was done.
• A nested RT-PCR was developed allowing viroid detection in several fig accessions.

Viroids are small (246–401 nt) circular and non coding RNAs infecting higher plants. They are targeted by host Dicer-like enzymes (DCLs) that generate small RNAs of 21–24 nt (sRNAs), which are involved in the host RNA silencing pathways. The accumulation in plant tissues of such viroid-derived small RNAs (vd-sRNAs) is a clear sign of an ongoing viroid infection. In this study, next generation sequencing of a sRNAs library and assembling of the sequenced vd-sRNAs were instrumental for the identification of a viroid resembling apple dimple fruit viroid (ADFVd) in a fig accession. After confirming by molecular methods the presence of this viroid in the fig tree, its population was characterized, showing that the ADFVd master sequence from fig diverges from that of the ADFVd reference variant from apple. Moreover, since this viroid accumulates at a low level in fig, a semi-nested RT-PCR assay was developed for detecting it in other fig accessions. ADFVd seems to have a wider host range than thought before and this poses questions about its epidemiology. A further characterization of ADFVd-sRNAs showed similar accumulation of (+) or (−) vd-sRNAs that mapped on the viroid genome generating hotspot profiles. Moreover, similarly to other nuclear-replicating viroids, vd-sRNAs of 21, 22 and 24 nt in size prevailed in the distribution profiles. Altogether, these data support the involvement of double-stranded RNAs and different DCLs, targeting the same restricted viroid regions, in the genesis of ADFVd-sRNAs.