Abstract
Genome-wide in-silico identification of microsatellites or simple sequence repeats (SSRs) in the Orf virus (ORFV), the causative agent of contagious ecthyma has been carried out to investigate the type, distribution and its potential role in the genome evolution. We have investigated eleven ORFV strains, which resulted in the presence of 1,036–1,181 microsatellites per strain. The further screening revealed the presence of 83–107 compound SSRs (cSSRs) per genome. Our analysis indicates the dinucleotide (76.9%) repeats to be the most abundant, followed by trinucleotide (17.7%), mononucleotide (4.9%), tetranucleotide (0.4%) and hexanucleotide (0.2%) repeats. The Relative Abundance (RA) and Relative Density (RD) of these SSRs varied between 7.6–8.4 and 53.0–59.5 bp/kb, respectively. While in the case of cSSRs, the RA and RD ranged from 0.6–0.8 and 12.1–17.0 bp/kb, respectively. Regression analysis of all parameters like the incident of SSRs, RA, and RD significantly correlated with the GC content. But in a case of genome size, except incident SSRs, all other parameters were non-significantly correlated. Nearly all cSSRs were composed of two microsatellites, which showed no biasedness to a particular motif. Motif duplication pattern, such as, (C)-x-(C), (TG)-x-(TG), (AT)-x-(AT), (TC)- x-(TC) and self-complementary motifs, such as (GC)-x-(CG), (TC)-x-(AG), (GT)-x-(CA) and (TC)-x-(AG) were observed in the cSSRs. Finally, in-silico polymorphism was assessed, followed by in-vitro validation using PCR analysis and sequencing. The thirteen polymorphic SSR markers developed in this study were further characterized by mapping with the sequence present in the database. The results of the present study indicate that these SSRs could be a useful tool for identification, analysis of genetic diversity, and understanding the evolutionary status of the virus.
Highlights
Genome-wide in-silico identification of microsatellites or simple sequence repeats (SSRs) in the Orf virus (ORFV), the causative agent of contagious ecthyma has been carried out to investigate the type, distribution and its potential role in the genome evolution
Our study revealed a large number of SSRs scattered throughout the ORFV genomes varying from 1,036 to 1,181 in number with an average of 1,092 per genome
The genome-wide scan revealed the presence of 83–107 compound SSRs (cSSRs), with an average of 93 occurrences per genome
Summary
Genome-wide in-silico identification of microsatellites or simple sequence repeats (SSRs) in the Orf virus (ORFV), the causative agent of contagious ecthyma has been carried out to investigate the type, distribution and its potential role in the genome evolution. We report for the first time a comparative analysis of microsatellites with respect to the abundance, distribution, composition, and polymorphism of SSRs within ORFV through in-silico approach, followed by the development and characterization of thirteen microsatellites markers. Using these tools, we further tested its usefulness by screening the viral genome from an ORFV outbreak and constructing a concatenated phylogenetic tree, which elucidated that the investigated virus closely related to the Chinese isolate. These markers could be used as a tool for making multiplex PCR assays for virus identification, strain demarcation, and evolutionary analysis
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