The complete mitochondrial genome of the medicinal fish, Cyprinion semiplotum: Insight into its structural features and phylogenetic implications.

Assamese kingfish (Cyprinion semiplotum) belonging to the subfamily Barbinae is an important food as well as aquarium fish having identified pharmacological benefits. The species has a complex taxonomic history and its phylogenetic position remains uncertain. Molecular data employed in earlier phylogenetic studies was inadequate for its phylogenetic placement. Therefore, we characterized 16,671 bp long complete mitogenome of C. semiplotum using next-generation sequencing. The mitogenome encodes the typical set of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and two non-coding regions. Its gene organization, distribution pattern, nucleotide composition, tRNA secondary structure and codon usage was similar to other Cyprinid mitogenomes. However, a distinctive 90 bp insertion was found in 3' periphery of the AT-rich control region. This can be a tool for identification of the species at the population level. Further, we reconstructed the most comprehensive phylogenetic trees of Cyprinidae based on complete mitogenome. In the resulting phylogenetic trees, C. semiplotum clustered tightly with other Barbinae species and exhibited a sister relationship with the species of the genera Aulopye, Barbus, Luciobarbus and Capoeta. The results presented herein will support future investigations on molecular taxonomy, population genetics, evolution and molecular phylogeny of C. semiplotum and its relatives.

Data on solute carrier transporter genes of a threatened Himalayan fish species - Schizothorax richardsonii.

The Snowtrout, Schizothorax richardsonii, is a vulnerable fish species found in different rivers and rivulets of the Himalayan region. The species is also a suitable poikilotherm to study the low-temperature tolerance as it dwells well at a temperature range of 5-20 °C. The solute carrier (SLC) group of membrane transport proteins play an integral role in cellular acclimation response. The present RNA sequencing was done to identify solute carrier transporter which are the major gene cascades responsible for transport of sugars, amino acids, oligonucleotides, ions, drugs, etc. to and from the cell organelles. A reference transcriptome database was created from liver tissue of Schizothorax richardsonii through RNA sequencing on Illumina HiSeq 2000 platform. The sequences were annotated and characterized under various solute carrier families in the species. So far, 113 transcripts were identified as solute carrier transporter genes categorized under 13 different families. This data will be useful for many researchers working on gene cloning and differential expression of solute carriers.

Solute carriers (SLCs) identified and characterized from kidney transcriptome of golden mahseer (Tor putitora) (Fam: Cyprinidae).

The solute carriers (SLC) are trans-membrane proteins, those regulate the transport of various substances (sugars, amino acids, nucleotides, inorganic cations/anions, metals, drugs etc.) across the cell membrane. There are more than 338 solute carriers (slc) reported in fishes that play crucial role in cellular influx and efflux. The study of solute carrier families may reveal many answers regarding the function of transporter genes in the species and their effect in the existing environment. Therefore, we performed RNA sequencing of kidney tissue of the golden mahseer (Tor putitora) using Illumina platform to identify the solute carrier families and characterized 24 putative functional genes under 15 solute carrier families. Out of 24 putative functional genes, 11 genes were differentially expressed in different tissues (head kidney, trunk kidney, spleen, liver, gill, muscle, intestine and brain) using qRT-PCR assay. The slc5a1, slc5a12, slc12a3, slc13a3, slc22a13 and slc26a6 were highly expressed in kidney. The slc15a2, slc25a47, slc33a1 and slc38a2 were highly expressed in brain and slc30a5 was over-expressed in gill. The unrooted phylogenetic trees of slc2, slc5, slc13 and slc33 were constructed using amino acid sequences of Homo sapiens, Salmo salar, Danio rerio, Cyprinus carpio and Tor putitora. It appears that all the putative solute carrier families are very much conserved in human and fish species including the present fish, golden mahseer. This study provides the first hand database of solute carrier families particularly transporter encoding proteins in the species.

Transcriptional response to heat shock in liver of snow trout (Schizothorax richardsonii)--a vulnerable Himalayan Cyprinid fish.

The snow trout (Schizothorax richardsonii) belonging to family Cyprinidae, is an endemic fish of the Himalayan region. The species is tagged as vulnerable species in the IUCN red list of threatened species. The fish thrives well in snowmelt water of several streams and rivers in the region but are occasionally exposed to more than 20 °C during the summer season. Therefore, we have used deep RNA sequencing to decipher the transcriptome of snow trout and characterize the genes and molecular pathways involved in heat shock response. In this study 72,601,298 and 65,428,283 raw reads for heat-shocked and control, respectively, were obtained by Illumina paired-end sequencing technology. The de novo assembled transcriptome was tested for differential gene expression across the treatment groups. The quality of assembly was high with N75 and N50 lengths of 461 and 1274 bases, respectively. A total of 65 unique transcripts were differentially expressed in liver under heat shock and control. Annotated blast matches reveal that differentially expressed transcripts correspond to critical chaperones and molecular pathways, previously shown to be important for thermal stress in other fish species. Eight randomly selected heat-stressed responsive transcripts were also observed to be upregulated during qRT-PCR analysis. This study is the preliminary step to understanding the responses during sudden environmental changes like heat shock. The reference transcriptome database would also aid further studies on biological and physiological aspects of the snow trout under abiotic stresses.

De novo assembly and characterization of tissue-specific transcriptome in the endangered golden mahseer, Tor putitora.

The golden mahseer (Tor putitora) graces most of the Himalayan Rivers of India and neighboring South Asian countries. Despite its several importance as a research model, as food, and in sport fishing, knowledge on transcriptome database is nil. Therefore, it was targeted to develop reference transcriptome databases of the species using next-generation sequencing. In the present study, 100,540,130 high-quality paired-end reads were obtained from six cDNA libraries of spleen, liver, gill, kidney, muscle, and brain with 28.4 GB data using Illumina paired-end sequencing technology. Tissue-specific transcriptomes as well as complete transcriptome assembly were analyzed for concise representation of the study. In brief, the de novo assembly of individual tissue resulted in an average of 31,829 (18,512-46,348) contigs per sample, while combined transcriptome comprised 77,907 unique transcript fragments (unigenes) assembled from reads of six tissues. Approximately 75,407 (96.8%) unigenes could be annotated according to their homology matches in the nr, SwisseProt, GO, or KEGG databases. Comparative analysis showed that 84% of the unigenes have significant similarity to zebra fish RefSeq proteins. Tissue-specific-dominated genes were also identified to hypothesize their localization and expression in individual tissue. In addition, 2485 simple sequence repeats (SSRs) were detected from 77,907 transcripts in the combined transcriptome of the golden mahseer. This study has generated organ-specific transcriptome profiles, which will be helpful to understand the local adaptation, genome evolution, and also future functional studies on immune system of the golden mahseer.

Complete mitochondrial genome organization of Schizothorax plagiostomus (Heckel, 1838).

The complete mitochondrial genome of Schizothorax plagiostomus, an endemic coldwater fish of Himalayas, was determined for the first time. The genome is 16,576 bp in length and consists of 13 protein coding genes, 22 tRNAs, 2rRNA genes and 1 putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was A 30%, G 17.4%, C 26.8%, T 25.8%, A + T content 55.8% and the G + C content 44.2%. The control region consists of a microsatellite locus (TA)12 between 16,460 and 16,483 bp. The present study will provide the rationale for the management and conservation of S. plagiostomus.

Phenotypic differentiation of Barilius bendelisis (Cypriniformes: Cyprinidae) in four rivers from Central Indian Himalaya.

Barilius bendelisis, commonly known as Indian Hill Trout is an upland water fish of South East Asia. It belongs to the family Cyprinidae and dwells in shallow, clear and cold water. In this study, the intraspecific variation of Barilius bendelisis, on the basis of morphometric characters, was investigated. Altogether, 402 specimens were collected from four rivers in the Central Indian Himalaya. A truss network was constructed by interconnecting 12 landmarks to yield 30 distance variables that were extracted from digital images of specimens using tpsDig2 and PAST software. Allometric transformed truss measurements were subjected to univariate analysis of variance, factor analysis and discriminant analysis. All variables exhibited significant differences between the populations. Altogether 88% of the specimens were classified into their original populations (81.98% under a 'leave-one-out' procedure). With factor analysis measurements of the head region, the middle portion and the caudal region had high loadings on the first and second axis. The results indicated that B. bendelisis has significant phenotypic heterogeneity between the geographically isolated regions of Central Indian Himalaya. We hypothesize that the marked interspecific variation in B. bendelisis is the result of local ecological conditions.

The complete mitochondrial genome of threatened chocolate mahseer (Neolissochilus hexagonolepis) and its phylogeny.

The chocolate mahseer (Neolissochilus hexagonolepis) is an important food and game fish of North Eastern India. To study the phylogenetic status we sequenced the complete mitochondrial genome of N. hexagonolepis. The mitogenome is 16,563 bp in length and composed of 13 protein coding genes, 22 tRNAs, 2 rRNAs and one putative control region. The overall base composition was A 31.8%, T 25.0%, G 15.8%, C 27.4% and A+T content 56.9%, G+C content 43.1%. The phylogenetic analysis using the complete mitochondrial genome revealed that the chocolate mahseer belonged to same clade of mahseer group of fishes but different from genera Barbus and Acrossocheilus. The present study will be helpful for the evolution and conservation genetic studies of N. hexagonolepis.

Molecular cloning and characterization of mannose binding lectin homologue from snow trout (Schizothorax richardsonii).

The teleost fishes depend on their innate immune system for first line of defense. Mannose binding lectin (MBL) is a member of the collectin family of proteins that plays an important role in the innate immune response in many vertebrates including fish. The present study was thus carried out to identify and characterize MBL homologue from snow trout. The cDNA of snow trout MBL homologue (galactose binding; Sch-GBL) was cloned and characterized. The obtained cDNA (778-bp) contains 762 bp long ORF (open reading frame) and a 16 bp partial 3'UTR (untranslated region); transcripts are ubiquitously expressed in all the tissues under study. Sch-GBL encodes a 253-aa protein that clusters with that of common carp, goldfish and contain several common structural motifs. In addition, the K(a)/K(s) ratios of the C-type lectin domain sequences depicts that they are under negative selection. Sch-GBL transcript level increases in response to challenge to Aeromonas hydrophila infection and decreases in response to downshift in temperature. Altogether, these findings suggest that Sch-GBL play an important role in the first line of fish defense mechanisms.

Complete mitochondrial genome organization of Schizothorax esocinus (Heckel, 1838).

The complete mitochondrial genome of Schizothorax esocinus, an endemic coldwater fish of Himalayas, was determined for the first time. The genome is 16,583 bp in length and consists of 13 protein-coding genes, 22 tRNAs, 2rRNA genes and 1 putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was; A 29.8%, G 17.8%, C 27%, T 25.4%, A + T content 55.2% and the G + C content 44.8%. The control region was also consisted of a microsatellite locus (TA) 13 between 16,463 to 16,488 bp. The present study will provide the rationale for the management and conservation of S. esocinus.

Complete mitochondrial genome organization of Schizopyge niger (Heckel, 1838).

The complete mitochondrial genome of Schizopyge niger, an endemic coldwater fish of Himalayas was determined for the first time. The genome is 16,585 bp in length and consists of 13 protein-coding genes, 22 tRNAs, 2rRNA genes and 1 putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was; A: 29.9%, G: 17.7%, C 27.1%, T 25.3%, A + T content 55.2% and the G + C content 44.8%. The control region was also consisted of a microsatellite locus (TA)13 between 16,471 to 16,496 bp. The present study will provide the rationale for the management and conservation of S. niger.

Genetic characterization of Golden mahseer (Tor putitora) populations using mitochondrial DNA markers.

Golden Mahseer (Tor putitora) is an economically important fish of India and Southeast Asia. The present study examined the genetic variations between seven geographically isolated populations of T. putitora using Cyt b (Cytochrome b) and ATPase6/8 gene sequences of mitochondrial DNA. Analysis of 133 sequences of Cyt b (1141 bp) and 130 sequences of ATPase6/8 gene (842 bp) revealed 47 and 44 haplotypes, respectively. The estimated haplotype and nucleotide diversity was high in River Jia Bhoreli (Bhalukpong) population (h = 1.00000, π = 0.007121 for Cyt b and h = 0.90441 π = 0.004867 for ATPase6/8). Results of AMOVA indicated that majority of the genetic variations in both genes were due to variation among populations (60.79% for Cyt b and 51.41% for ATPase6/8 gene). The pairwise F(ST) comparison and neighbor-joining tree revealed high genetic divergence of River Jia Bhoreli population from other populations. The understanding of genetic variations of T. putitora populations will play a key role in conservation and management of this endangered fish species.

Complete mitochondrial genome organization of Schizothorax labiatus (McClelland, 1842).

The complete mitochondrial genome of Schizothorax labiatus, an endemic coldwater fish of Himalayas was determined for the first time. The genome is 16,582 bp in length and consists of 13 protein coding genes, 22 tRNAs, 2rRNA genes and one putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was; A: 29.8%, G: 17.7%, C 27%, T 25.5%, A+T content 55.3% and the G+C content 44.7%. The control region was also consisted of a microsatellite locus (TA)12 between 16,467 and 16,490 bp. The present study will provide the rationale for the management and conservation of S. labiatus.

Complete mitochondrial genome organization of Schizothorax progastus (McClelland, 1839).

The complete mitochondrial genome of Schizothorax progastus, an endemic coldwater fish of Himalayas, was determined for the first time. The genome was 16,575 bp in length and consists of 13 protein coding genes, 22 tRNAs, 2rRNA genes and one putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was--A: 29.8%, G: 17.8%, C: 27%, T: 25.4%, A+T content: 55.2% and the G+C content: 44.8%. The control region also consisted of a microsatellite locus (TA)12 between 16,466 and 16,489 bp. The present study will provide the rationale for the management and conservation of S. progastus.

Complete mitochondrial genome organization of Schizothorax richardsonii (Gray, 1832).

The complete mitochondrial genome of Schizothorax richardsonii, an endemic coldwater fish of Himalayas, was determined for the first time. The genome is 16,592 bp in length and consists of 13 protein coding genes, 22 tRNAs, 2 rRNA genes and one putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was as follows: A: 29.9%, G: 17.7%, C: 26.9%, T: 25.5%, A + T content 55.4% and the G + C content 44.6%. The control region contains a microsatellite; (TA)13 exists between 16,469 and 16,494 bp. This study will provide the rationale for the management and conservation of the snow trout.

Complete mitochondrial genome organization of Tor putitora.

The complete mitochondrial genome of Tor putitora, an endemic coldwater fish of Himalayas was determined for the first time. The genome is 16,576 bp in length and consists of 13 protein coding genes, 22 tRNAs, 2 rRNA genes and 1 putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was; A: 31.9%, G: 15.6%, C: 27.5%, T: 25%, A + T content 56.9% and the G + C content 43.1%. The control region was also consisted of a microsatellite locus (TA) 13 between 16,456 to 16,481 bp. The present study will provide the rationale for the management and conservation of T. putitora.

Phylogenetic relationship and molecular identification of five Indian Mahseer species using COIsequence.

This study examined the phylogenetic relationship and identification of five Mahseer species (Tor putitoro, Tor tor, Tor khudree, Tor chelynoides and Neolissochilus hexogonolopis) using partial sequencing of a Cytochrome Oxidase I (COl) DNA barcodes. The sequence analysis data showed that 134 (21.61%) sites out of 628 sites were variable without insertion or deletion. Rate of transition (70.5%) were higher than transversion (29.41%). There was a high inter-species divergence (range 4.1% to 12.2%) in Mahseer species as compared to intra-specific sequence divergence (1.7% for T. putitora, 1.2% for T tor, 1.4% for T. khudree, 3.0% for T chelynoides, 0.26 % for N. hexagonolopis). The phylogenetic tree, constructed by maximum parsimony, maximum likelihood and unweighted pair group average methods revealed similar results suggesting that T. putitoro, T. tor and T. khudree had a close relationship to each other while maximum divergence was observed in T. chelynoides, which was also confirmed by the genetic distance data. The results indicate that COl sequencing or bar-coding is useful in unravelling phylogenetic relationship and identification of Mahseer species.