Molecular and phylogenetic identification of unique isolates of hammerhead viroid-like RNA from 'Pacific Gala' apple (Malus domestica) in Canada

Abstract

[EN] Ten isolates of apple hammerhead viroid-like RNA (AHVd RNA) were detected by next-generation sequencing (NGS) and/or by RT-PCR analysis of apple (Malus domestica) 'Pacific Gala' (PG) from Canada. All consisted of 433 nucleotides in size, and their circular structure and sequence were supported by reverse transcription amplification with two separate pairs of abutted primers, cloning and sequencing. Two regions were defined for the computer-predicted secondary structure of AHVd RNA-PG isolates - a hammerhead region (HHR) and a loop-rich region (LRR). The HHR contained the core and flanking nucleotides typical of natural hammerhead ribozymes. The secondary structure associated with the LRR was distinct from that predicted for the corresponding region of AHVd RNA isolates previously described in 'Fuji' apple in China. However, both structures share conserved stem-loops in the LRR, identified as S1, S2, S3 and S4.