Abstract
Columnea latent viroid (CLVd) is one of the most serious tomato diseases. In general, viroids have high mutation rates. This generates a population of variants (so-called quasi-species) that co-exist in their host and exhibit a huge level of genetic diversity. To study the population of CLVd in individual host plants, we used amplicon sequencing using specific CLVd primers linked with a sample-specific index sequence to amplify libraries. An infectious clone of a CLVd isolate Chaipayon-1 was inoculated on different solanaceous host plants. Six replicates of the amplicon sequencing results showed very high reproducibility. On average, we obtained 133,449 CLVd reads per PCR-replicate and 79 to 561 viroid sequence variants, depending on the plant species. We identified 19 major variants (>1.0% mean relative abundance) in which a total of 16 single-nucleotide polymorphisms (SNPs) and two single nucleotide insertions were observed. All major variants contained a combination of 4 to 6 SNPs. Secondary structure prediction clustered all major variants into a tomato/bolo maka group with four loops (I, II, IV and V), and a chili pepper group with four loops (I, III, IV and V) at the terminal right domain, compared to the CLVd Chaipayon-1 which consists of five loops (I, II, III, IV and V).
Highlights
Columnea latent viroid (CLVd), a member of the Pospiviroid genus, is one of the smallest plant pathogens, consisting of a non-coding, naked, circular single-stranded RNA molecule of 367 to 374 nucleotides [1,2,3]
Secondary structure prediction clustered all major variants into a tomato/bolo maka group with four loops (I, II, IV and V), and a chili pepper group with four loops (I, III, IV and V) at the terminal right domain, compared to the CLVd Chaipayon-1 which consists of five loops (I, II, III, IV and V)
Our study focuses on the evolution of distinct CLVd populations across a range of host species, and the finding that the quasi-species sequence spectrum consistently depends on the host species
Summary
Columnea latent viroid (CLVd), a member of the Pospiviroid genus, is one of the smallest plant pathogens, consisting of a non-coding, naked, circular single-stranded RNA molecule of 367 to 374 nucleotides [1,2,3]. In Thailand, CLVd was first discovered in an open-field tomato seed production facility in the Khon Kaen province in 2004 [14]. This was caused by CLVd contamination of imported tomato seeds that were post-entry quarantine regulated and eradicated shortly afterwards [8,15]. CLVd-Chaipayon-1 (GenBank Accession No KM214216) was first found in a tomato hybrid-seed production site in the northeastern region of Thailand in 2014. This CLVd isolate causes intermediate symptoms in tomato, bolo maka and Thai round eggplant compared to most of the currently known Asian isolates [8]
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