Gene annotation and small RNA characterization in the salt water crocodile: Crocodylus porosus

Crocodilian genomes have been evolving very slowly over the past several million years, even when compared to their closest extant relatives, the birds. Understanding the evolution, regulation and adaptive capabilities of the crocodilian genome and its genetic diversity can therefore provide information on how slowly evolving genomes manage to stay viable in the face of competition from other taxa and changing environmental conditions. Transposable elements (TEs) are genetic features that can modulate gene expression through multiple processes including disruption of coding and non-coding regions, promotion of non-homologous recombination and exaptation, potentially resulting in speciation. Small RNAs- including microRNAs (miRNA) and PIWI-interacting RNAs (piRNA) are short 18-35-nt sequences capable of regulating gene and TE expression through mRNA cleavage, translational repression as well as silencing through methylation. They are therefore important for understanding how gene expression phenotypes evolve and the TE landscape is maintained in the genome. However, a well annotated crocodile genome as well as information on crocodilian small RNAs are both currently unavailable. A highly contiguous saltwater crocodile genome assembly was constructed using the Chicago library and signature HiRise software pipeline of Dovetail Genomics. Overall, the genome had an 82 fold improvement of minimum scaffold N50 size from the previous assembly in the same species. 23,242 genes were annotated with >96% of these genes possessing a functional protein domain. Over 40 genes under potential selection were also identified in both crocodile and the American alligator. Deep sequencing of libraries from 11 tissues was done using Illumina’s Hiseq 2500 platform. Utilizing in-house pipelines and established software tools, 643 (known and novel) miRNAs and novel piRNAs in the saltwater crocodile were also characterized. miRNA targets and homologous miRNAs in ten vertebrates were identified. When analyzed in the context of TEs, the miRNAs showed a high association with type II TEs - DNA transposons, while the piRNAs (ping-pong types) were mostly associated with Type I retrotransposon (LINEs) followed by type II DNA transposons. With an improved, contiguous and annotated genome- these small RNAs can provide novel insights into the evolutionary impact of miRNAs and piRNAs in reptiles as well as regulation of TEs in the crocodilian genome.