Development of novel SSR markers and validation by assessing the genetic diversity of endangered Deccan mahseer, Tor khudree.

BACKGROUND: The Deccan mahseer, Tor khudree (Sykes, 1839) is a potential game and food fish species belonging to the family cyprinidae and is categorized as endangered. Its distribution is restricted to southern part of India, specifically to Peninsular Rivers. This study is first to assess the genetic diversity and differentiation in Tor khudree by developing novel simple sequence repeat (SSR) markers. METHODS AND RESULTS: Low depth next generation sequencing followed by sequence analysis in MISA software identified 187,649 SSRs. The novel fourteen validated SSR loci were used for population genetic analysis. All of the SSR loci were highly informative with mean PIC > 0.5. High mean allelic richness (9.29) observed heterozygosity (0.98) and expected heterozygosity (0.79) were observed across the loci. However, genetic differentiation was low but significant (0.052). Negative FIS values were observed in both locus-wise and populations indicating the presence of high heterozygosity. Intrapopulation variation was found to be high (96.29%). The population structure revealed two genetic stocks. CONCLUSIONS: The results from the present study including the highly polymorphic markers developed would be a useful resource for further research on population genetics and conservation genetics of the species.

Activin receptor type IIB in rohu (Labeo rohita): molecular characterization, tissue distribution and immunohistochemical localization during different stages of gonadal maturation.

Activin receptor type IIB (ActRIIB) is a transmembrane serine/threonine kinase receptor which plays a pivotal role in regulating the reproduction in vertebrates including teleost. Earlier studies have documented its importance in governing gonadal maturation in higher vertebrates. However, reports on the regulation of fish reproductive system by ActRIIB gene are still limited. Here, we report the identification and characterization of ActRIIB cDNA of Labeo rohita, a commercially important fish species of the Indian subcontinent. The full-length gene encoding rohu ActRIIB was cloned and found to be of 1674 bp in length. Functional similarities were evident from evolutionary analysis across vertebrates. Real-time PCR to measure the expression of ActRIIB transcript in rohu revealed significant mRNA levels in gonads followed by non-reproductive tissues, including the brain, pituitary and muscle. With respect to different gonadal maturation stages, predominant expression of ActRIIB mRNA was observed during the pre-spawning phase of both sexes. To further delineate its role in rohu reproduction, a recombinant protein of the extracellular domain of ActRIIB (rECD-ActRIIB) was produced, and polyclonal antibody is raised against the protein for its immuno-localization studies during different gonadal maturation stages. Strong immunoreactivity was noticed in the pre-vitellogenic oocytes which decreased dramatically in the fully mature oocytes. Similarly, the strong and intense immunoreactivity was found in the spermatids and spermatocytes of the immature testis, and eventually the intensity reduced with the progression of the maturation stage. These results provide the first evidence of the presence of ActRIIB in rohu gonadal tissues. Taken together, our observations lay the groundwork for further understanding and investigating on the potential role of ActRIIB in fish reproduction system in the event of gonadal maturation.

Genome size estimation and its associations with body length, chromosome number and evolution in teleost fishes.

Precise estimation of genome size (GS) is vital for various genomic studies, such as deciding genome sequencing depth, genome assembly, biodiversity documentation, evolution, genetic disorders studies, duplication events etc. Animal Genome Size Database provides GS of over 2050 fish species, which ranges from 0.35 pg in pufferfish (Tetraodon nigroviridis) to 132.83 pg in marbled lungfish (Protopterus aethiopicus). The GS of majority of the fishes inhabiting waters of Indian subcontinent are still missing. In present study, we estimated GS of 51 freshwater teleost (31 commercially important, 7 vulnerable and 13 ornamental species) that ranged from 0.58 pg in banded gourami (Trichogaster fasciata) to 1.92 pg in scribbled goby (Awaous grammepomus). Substantial variation in GS was observed within the same fish orders (0.64-1.45 pg in cypriniformes, 0.70-1.41 pg in siluriformes and 0.58-1.92 pg in perciformes). We examined the relationship between the GS, chromosome number and body length across all the fishes. Body length was found to be associated with GS, whereas no relationship was noticed between the GS and the chromosome number. The analysis using ancestral information revealed haploid chromosome number 25, 27 and 24 for the most recent common ancestor of cypriniformes, siluriformes and perciformes, respectively. The study led to generation of new records on GS of 43 fish species and revalidated records for 8 species. The finding is valuable resource for further research in the areas of fish genomics, molecular ecology and evolutionary conservation genetics.

Assembling mitogenome of Himalayan Black Bear (U. t. laniger) from low depth reads and its application in drawing phylogenetic inferences.

The complete mitogenome of Himalayan black bear (Ursus thibetanus laniger) from Indian Himalayan region was assembled following the modified approach of mitochondrial baiting and mapping using the next-generation sequencing reads. The complete mitogenome was of 16,556 bp long, consisted of 37 genes that contained 13 protein-coding genes, 22 tRNAs, 2 rRNAs and 1 control region. The complete base composition was 31.33% A, 15.24% G, 25.45%C, and 27.98%T and gene arrangement was similar to the other sub-species of Asiatic black bear. The relative synonymous codon usage analysis revealed the maximum abundance of Isoleucine, Tyrosine, Leucine and Threonine. The assembled mitogenome of U. t. laniger exhibited 99% similarity with the mitogenomes of Himalayan black bear available from Nepal and Tibetan Plateau-Himalaya region. The findings of the present study has proven low depth sequencing data, adequate and highly efficient in rapid recovering the mitochondrial genome by overcoming the conventional strategies of obtaining long-range PCR and subsequently drawing phylogenetic inferences.

The genome of walking catfish Clarias magur (Hamilton, 1822) unveils the genetic basis that may have facilitated the development of environmental and terrestrial adaptation systems in air-breathing catfishes.

The walking catfish Clarias magur (Hamilton, 1822) (magur) is an important catfish species inhabiting the Indian subcontinent. It is considered as a highly nutritious food fish and has the capability to walk to some distance, and survive a considerable period without water. Assembly, scaffolding and several rounds of iterations resulted in 3,484 scaffolds covering ∼94% of estimated genome with 9.88 Mb largest scaffold, and N50 1.31 Mb. The genome possessed 23,748 predicted protein encoding genes with annotation of 19,279 orthologous genes. A total of 166 orthologous groups represented by 222 genes were found to be unique for this species. The Computational Analysis of gene Family Evolution (CAFE) analysis revealed expansion of 207 gene families and 100 gene families have rapidly evolved. Genes specific to important environmental and terrestrial adaptation, viz. urea cycle, vision, locomotion, olfactory and vomeronasal receptors, immune system, anti-microbial properties, mucus, thermoregulation, osmoregulation, air-breathing, detoxification, etc. were identified and critically analysed. The analysis clearly indicated that C. magur genome possessed several unique and duplicate genes similar to that of terrestrial or amphibians' counterparts in comparison to other teleostean species. The genome information will be useful in conservation genetics, not only for this species but will also be very helpful in such studies in other catfishes.

Molecular characterization of Activin Receptor Type IIA and its expression during gonadal maturation and growth stages in rohu carp.

Activin receptor type IIA (ActRIIA), a transmembrane serine/threonine kinase receptor is an important regulator of physiological traits, viz., reproduction and body growth in vertebrates including teleosts. However, existing knowledge of its role in regulating fish physiology is limited. To address this, we have cloned and characterized the ActRIIA cDNA of Labeo rohita (rohu), an economically important fish species of the Indian subcontinent. Comparative expression profiling of the receptor gene at various reproductive and growth stages supports to its role in promoting oocyte maturation, spermatogenesis and skeletal muscle development via interaction with multiple ligands of transforming growth factor-ß (TGF-ß) family. The full-length cDNA of rohu ActRIIA was found to be of 1587bp length encoding 528 amino acids. The three-dimensional structure of the intracellular kinase domain of rohu ActRIIA has also been predicted. Phylogenetic relationship studies showed that the gene is evolutionarily conserved across the vertebrate lineage implicating that the functioning of the receptor is more or less similar in vertebrates. Taken together, these findings could be an initial step towards the use of ActRIIA as a potential candidate gene marker for understanding the complex regulatory mechanism of fish reproduction and growth.

Complete mitochondrial genome sequence of E. suratensis revealed by next generation sequencing.

The complete mitochondrial genome of Etroplus suratensis, the Green chromide cichlid, was determined for the first time through NGS method. The genome is 16,467 bp (Accession no. KU301747) in length and consisted of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and one control region. Organization of genes and their order are in accordance with other vertebrates. The overall base composition on plus strand was A: 28.3%, G: 15.2%, C: 30.9%, T: 25.6%, and the A + T content 53.9%. The control region contains a putative termination-associated sequence and three conserved sequence blocks. This mitogenome sequence data would play an important role in population genetics and phylogenetics of cichlid fish of India.

Complete mitochondrial genome sequence of Heteropneustes fossilis obtained by paired end next generation sequencing.

In the present study, the complete mitochondrial genome sequence of Heteropneustes fossilis is reported using massive parallel sequence technology. The complete mitogenome of H. fossilis is obtained by de novo assembly of paired end Illumina sequences using CLC Genomics Workbench version 7.0.4, which is 16,489 bp in length. It comprised of 13 protein- coding genes, 22 tRNAs, 2 rRNA genes and a putative control region along with the gene order and organization, being similar to most of vertebrates. The mitogenome in the present study has 99% similarity to the complete mitogneome sequence of H. Fossilis, as reported earlier. Phylogenetic analysis of Siluriformes depicted that Heteropneustids were closer to Clariids. The mitogenome sequence of H. fossilis contributes better understanding of population genetics, phylogenetics and evolution of Indian catfish species.

Low-depth shotgun sequencing resolves complete mitochondrial genome sequence of Labeo rohita.

Labeo rohita, popularly known as rohu, is a widely cultured species in whole Indian subcontinent. In the present study, we used in-silico approach to resolve complete mitochondrial genome of rohu. Low-depth shotgun sequencing using Roche 454 GS FLX (Branford, Connecticut, USA) followed by de novo assembly in CLC Genomics Workbench version 7.0.4 (Aarhus, Denmark) revealed the complete mitogenome of L. rohita to be 16 606 bp long (accession No. KR185963). It comprised of 13 protein-coding genes, 22 tRNAs, 2 rRNAs and 1 putative control region. The gene order and organization are similar to most vertebrates. The mitogenome in the present investigation has 99% similarity with that of previously reported mitogenomes of rohu and this is also evident from the phylogenetic study using maximum-likelihood (ML) tree method. This study was done to determine the feasibility, accuracy and reliability of low-depth sequence data obtained from NGS platform as compared to the Sanger sequencing. Thus, NGS technology has proven to be competent and a rapid in-silico alternative to resolve the complete mitochondrial genome sequence, thereby reducing labors and time.

Assembly and variation analyses of Clarias batrachus mitogenome retrieved from WGS data and its phylogenetic relationship with other catfishes.

Whole genome sequencing (WGS) using next generation sequencing technologies paves the way to sequence the mitochondrial genomes with greater ease and lesser time. Here, we used the WGS data of Clarias batrachus, generated from Roche 454 and Ion Torrent sequencing platforms, to assemble the complete mitogenome using both de novo and reference based approaches. Both the methods yielded almost similar results and the best assembled mitogenome was of 16,510 bp size (GenBank Acc. No. KM259918). The mitogenome annotation resulted in 13 coding genes, 22 tRNA genes, 2 rRNA genes and one control region, and the gene order was found to be identical with other catfishes. Variation analyses between assembled and the reference (GenBank Acc. No. NC_023923) mitogenome revealed 51 variations. The phylogenetic analysis of coding DNA sequences and tRNA supports the monophyly of catfishes. Two SSRs were identified in C. batrachus mitogenome, out of which one was unique to this species. Based on the relative rate of gene evolution, protein coding mitochondrial genes were found to evolve at a much faster pace than the d-loop, which in turn are followed by the rRNAs; the tRNAs showed wide variability in the rate of sequence evolution, and on average evolve the slowest. Among the coding genes, ND2 evolves most rapidly. The variations present in the coding regions of the mitogenome and their comparative analyses with other catfish species may be useful in species conservation and management programs.