A unique single nucleotide polymorphism in Agouti Signalling Protein (ASIP) gene changes coat colour of Sri Lankan leopard (Panthera pardus kotiya) to dark black.

The Sri Lankan leopard (Panthera pardus kotiya) is an endangered subspecies restricted to isolated and fragmented populations in Sri Lanka. Among them, melanistic leopards have been recorded on a few occasions. Literature suggests the evolution of melanism several times in the Felidae family, with three species having distinct mutations. Nevertheless, the mutations or other variations in the remaining species, including Sri Lankan melanistic leopard, are unknown. We used reference-based assembled nuclear genomes of Sri Lankan wild type and melanistic leopards and de novo assembled mitogenomes of the same to investigate the genetic basis, adaptive significance, and evolutionary history of the Sri Lankan melanistic leopard. Interestingly, we identified a single nucleotide polymorphism in exon-4 Sri Lankan melanistic leopard, which may completely ablate Agouti Signalling Protein (ASIP) function. The wild type leopards in Sri Lanka did not carry this mutation, suggesting the cause for the occurrence of melanistic leopords in the population. Comparative analysis of existing genomic data in the literature suggests it as a P. p. kotiya specific mutation and a novel mutation in the ASIP-gene of the Felidae family, contributing to naturally occurring colour polymorphism. Our data suggested the coalescence time of Sri Lankan leopards at ~0.5 million years, sisters to the Panthera pardus lineage. The genetic diversity was low in Sri Lankan leopards. Further, the P. p. kotiya melanistic leopard is a different morphotype of the P. p. kotiya wildtype leopard resulting from the mutation in the ASIP-gene. The ability of black leopards to camouflage, along with the likelihood of recurrence and transfer to future generations, suggests that this rare mutation could be environment-adaptable.

Genetic features of Sri Lankan elephant, Elephas maximus maximus Linnaeus revealed by high throughput sequencing of mitogenome and ddRAD-seq.

Elephas maximus maximus Linnaeus, the Sri Lankan subspecies is the largest and the darkest among Asian elephants. Patches of depigmented areas with no skin color on the ears, face, trunk, and belly morphologically differentiate it from the others. The elephant population in Sri Lanka is now limited to smaller areas and protected under Sri Lankan law. Despite its ecological and evolutionary importance, the relationship between Sri Lankan elephants and their phylogenetic position among Asian elephants remains controversial. While identifying genetic diversity is the key to any conservation and management strategies, limited data is currently available. To address such issues, we analyzed 24 elephants with known parental lineages with high throughput ddRAD-seq. The mitogenome suggested the coalescence time of the Sri Lankan elephant at ~0.2 million years, and sister to Myanmar elephants supporting the hypothesis of the movement of elephants in Eurasia. The ddRAD-seq approach identified 50,490 genome-wide SNPs among Sri Lankan elephants. The genetic diversity within Sri Lankan elephants assessed with identified SNPs suggests a geographical differentiation resulting in three main clusters; north-eastern, mid-latitude, and southern regions. Interestingly, though it was believed that elephants from the Sinharaja rainforest are of an isolated population, the ddRAD-based genetic analysis clustered it with the north-eastern elephants. The effect of habitat fragmentation on genetic diversity could be further assessed with more samples with specific SNPs identified in the current study.

Raw transcriptomics data to gene specific SSRs: a validated free bioinformatics workflow for biologists.

Recent advances in next-generation sequencing technologies have paved the path for a considerable amount of sequencing data at a relatively low cost. This has revolutionized the genomics and transcriptomics studies. However, different challenges are now created in handling such data with available bioinformatics platforms both in assembly and downstream analysis performed in order to infer correct biological meaning. Though there are a handful of commercial software and tools for some of the procedures, cost of such tools has made them prohibitive for most research laboratories. While individual open-source or free software tools are available for most of the bioinformatics applications, those components usually operate standalone and are not combined for a user-friendly workflow. Therefore, beginners in bioinformatics might find analysis procedures starting from raw sequence data too complicated and time-consuming with the associated learning-curve. Here, we outline a procedure for de novo transcriptome assembly and Simple Sequence Repeats (SSR) primer design solely based on tools that are available online for free use. For validation of the developed workflow, we used Illumina HiSeq reads of different tissue samples of Santalum album (sandalwood), generated from a previous transcriptomics project. A portion of the designed primers were tested in the lab with relevant samples and all of them successfully amplified the targeted regions. The presented bioinformatics workflow can accurately assemble quality transcriptomes and develop gene specific SSRs. Beginner biologists and researchers in bioinformatics can easily utilize this workflow for research purposes.