BAC-FISH Based Physical Map of Endangered Catfish Clarias magur for Chromosome Cataloguing and Gene Isolation through Positional Cloning.

Construction of a physical chromosome map of a species is important for positional cloning, targeted marker development, fine mapping of genes, selection of candidate genes for molecular breeding, as well as understanding the genome organization. The genomic libraries in the form of bacterial artificial chromosome (BAC) clones are also a very useful resource for physical mapping and identification and isolation of full-length genes and the related cis acting elements. Some BAC-FISH based studies reported in the past were gene based physical chromosome maps of Clarias magur (magur) to understand the genome organization of the species and to establish the relationships with other species in respect to genes' organization and evolution in the past. In the present study, we generated end sequences of the BAC clones and analyzed those end sequences within the scaffolds of the draft genome of magur to identify and map the genes bioinformatically for each clone. A total of 36 clones mostly possessing genes were identified and used in probe construction and their subsequent hybridization on the metaphase chromosomes of magur. This study successfully mapped all 36 specific clones on 16 chromosome pairs, out of 25 pairs of magur chromosomes. These clones are now recognized as chromosome-specific makers, which are an aid in individual chromosome identification and fine assembly of the genome sequence, and will ultimately help in developing anchored genes' map on the chromosomes of C. magur for understanding their organization, inheritance of important fishery traits and evolution of magur with respect to channel catfish, zebrafish and other species.

Thermal stress induces expression of Nuclear protein and Parkin genes in endangered catfish, Clarias magur.

Fluctuation in water temperature can create thermal stress, which may impact many aspects of fish life, such as survival, growth, reproduction, disease occurrence etc. The endangered catfish, Clarias magur, has been reported to survive at higher thermal stress, even though the exact mechanism is unknown. The genes coding for Nuclear protein 1 (Nupr1) and Parkin E3 ubiquitin protein ligase (Park2) have been reported to protect cells from stress-induced damage and death. In this study, we characterized both the genes and assessed their quantitative expression in C. magur. Structural features of both the genes were found similar to a related catfish, Ictalurus punctatus, and model fish zebrafish. The genes were fairly conserved in fishes as observed through phylogenetic analysis. The real time expression of the two stress-associated genes were also assessed in brain, kidney, liver and muscle tissues of C. magur exposed to warm (34 °C) and cold (15 °C) water. RT-PCR analysis revealed up-regulation in the relative expression levels of Nupr1 and Park2 genes at both temperatures with maximum positive fold change during stress to cold water, even though the posteriori Dunnett's test after ANOVA revealed that there were significant differences between the control and challenged groups. The study indicated that Nupr1 gene plays role in muscle tissue at both high and low thermal stress, but at high thermal stress in liver, while Park2 plays role in muscle, brain and kidney at low temperature and in liver at high temperature stress in C. magur. The study has generated first-hand information under warm- and cold water, which pave the way to understand the expression response of these genes to thermal vacillations and to establish evolutionary significance in catfishes and other species.

Improved protocols for BAC insert DNA isolation, BAC end sequencing and FISH for construction of BAC based physical map of genes on the chromosomes.

Bacterial artificial chromosome (BAC) library is an important genomic resource useful in targeted marker development, positional cloning, physical mapping and a substrate for genome sequencing for better understanding the genome organization of a species. The present manuscript elucidates the improvement in protocols for economical and efficient BAC insert DNA isolation, BAC end sequencing and FISH for physical localization on the metaphase chromosome complements. BAC clones of Clarias magur, maintained in 384-well plate format in our laboratory, were used in this study. The protocols gave consistent and efficient results. We use routinely these protocols for BAC insert DNA extraction, generating end sequence data of the clone and constructing DNA probes to hybridize on the metaphase spreads of C. magur using FISH for physical their localization.

Isolation and characterization of hypoxia inducible heme oxygenase 1 (HMOX1) gene in Labeo rohita.

The HMOX1 gene plays role in several biological processes and is also responsive to hypoxia stress. Freshwater carp fish, Labeo rohita, is reported as hypoxia sensitive, but the information of annotated hypoxia genes in public domain is very scanty for this species. Here, an attempt was made to isolate and characterize HMOX1 gene in L. rohita using information from zebrafish. HMOX1 gene was obtained by mapping HMOX1 protein of zebrafish over assembled genome of L. rohita. Aligned region was used for designing primers for HMOX1 amplification. Eight overlapping sets of primers were designed for amplifying ~540 bp long successive overlapping fragments. Splicing of overlapping amplicons generated 3715 bp fragment that was confirmed as HMOX1 gene having full coding region with 6 exons between 184 and 2156 bp positions. HMOX1 characterization is an initiative for L. rohita genes annotation to support the characterization of new genes in the important species.

Evolutionary analysis of genus Channa based on karyological and 16S rRNA sequence data.

A wide range of diploid number of chromosomes and the body size of Channa congeners are useful combination of characters for studying the factors controlling the body size. In this study, the karyological information was superimposed on the evolutionary tree generated by 16S rRNA mitochondrial gene sequences. Here, the metaphase chromosome complements stained with Giemsa, AgNO3 and CMA3 were prepared from six snakehead murrel fish species collected from northeast India. The diploid chromosome numbers and the fundamental arms of C. aurantimaculata (2n = 52, NF = 98), C. gachua (2n = 56, NF = 84), C. marulius (2n = 44, NF = 58), C. orientalis (2n = 52, NF = 74), C. punctata (2n = 32, NF = 60) and C. striata (2n = 40, NF = 48) were calculated by the analysis of metaphase chromosome complements. Both methods of nucleolar organizer region (NOR) localization, silver nitrate and chromomycin A3, revealed NOR pairs of 1, 2, 3, 1, 4 and 3 in C. aurantimaculata, C. gachua, C. marulius, C. orientalis, C. punctata and C. striata, respectively. The subject species showed primitive type of asymmetrical chromosomes, except the C. punctata. The variation in 2n for C. orientalis (2n = 52, 78) and C. gachua (2n = 52, 78, 104) of a complete haploid set indicates the possibility of either ploidy change in C. orientalis and C. gachua, if we consider 2n = 52 or the Robertsonian rearrangements in different populations of these two species. The chromosome evolution tree was constructed on 16S rRNA ML-phylogenetic tree using ChromEvol 1.3. The analysis of chromosome evolution explained the loss or gain of chromosome, duplications or semiduplications mechanism. For time scaling the chromosomeevolution, the node age of available 16S rRNA gene of Channa species were estimated, which was also used for estimating the time when chromosomal changes occurred in context of geological time-scale.

Population structuring of Channa striata from Indian waters using control region of mtDNA.

Striped snakehead (Channa striata) is a freshwater species of early Miocene period belonging to family Channidae. The genetic variability of the snakehead populations in India was not well known. Present study was undertaken using 149 sequences of control region of mitochondrial DNA from seven geographically distinct populations of Indian water, which resulted in 46 haplotypes with 137 variable nucleotide sites (60 singletons and 77 parsimony informative) and the nucleotide frequencies was: A = 33.0, T = 28.1, G = 15.4, and C = 23.5%. The presence of low-frequency of younger haplotypes with a large number of singletons indicates the absence of dominant haplotype. Hierarchical AMOVA showed highly significant genetic differentiation (FST = 0.56, p < .00) among the populations. The pattern of genetic differentiation was not consistent with geographical distributions. AMOVA identified three genetically heterogeneous clades. The significant finding of Imphal and Chaliyar was that they were found genetically close to each other, but geographically much isolated from each other. The inference of the study suggests that the hypothesis of the single panmictic population of C. striata in Indian waters is rejected.

Isolation and characterization of hypoxia inducible gene connective tissue growth factor (CTGF) in Labeo rohita.

The connective tissue growth factor gene plays important role in several biological processes and also responsive to hypoxia stress in fishes. The freshwater fish, Labeo rohita, highly cultured in Indian subcontinent for food, is reported as hypoxia sensitive but annotation and sequences of nuclear genes were not available for this species so far in the public domain, except some transcripts. In this study, an attempt was made for isolation and annotation of the CTGF gene in L. rohita using information of zebrafish from the same family. The CTGF gene sequence was obtained by aligning assembled genome of L. rohita, (NCBI BioProject ID: PRJNA437789), with the CTGF protein of zebrafish. Eight overlapping sets of forward and reverse primers from aligned region were designed for amplification of around 600 bp long successive overlapping fragments of CTGF gene in L. rohita. Assembly and annotation of overlapping fragments confirmed a complete 2421 bp long CTGF gene sequence with a full coding region that comprised of five exons between 308 and 1921 positions. This annotated CTGF gene sequence was submitted to GenBank (Acc. No. KY940466). Characterization of CTGF will be an initiative in identification of hypoxia response genes in L. rohita which may further help in understanding the mechanism of hypoxia tolerability in this species.

The complete mitochondrial genome sequence of Osteobrama belangeri (Cyprinidae) and its comparison with other related Cypriniformes fish species.

The complete mitogenome of Osteobrama belangeri is described using Ion Torrent (PGM sequencer), which was 16,609 bp in size comprising 13 mRNAs, teo rRNA genes, 22 tRNAs, and 926 bp as D-Loop control region, in addition to gene order and organization, being similar to most of the other related Cypriniformes fish mitogenome of NCBI databases. The all 22 tRNAs were packed into a typical clover-leaf structure. In the present study, the mitogenome has 99% similarity to the complete mitogenome sequence of O. belangeri mitogenome details previously and also would be helpful in understanding the phylogenetics, population genetics, and evolution of family Cyprinidae fishes.

Deep insights into the mode of ATP-binding mechanism in Zebrafish cyclin-dependent protein kinase-like 1 (zCDKL1): A molecular dynamics approach.

In eukaryotes, the serine/threonine kinases (STKs) belonging to cyclin-dependent protein kinases (CDKs) play significant role in control of cell division and curb transcription in response to several extra and intra-cellular signals indispensable for enzymatic activity. The zebrafish cyclin-dependent protein kinase-like 1 protein (zCDKL1) shares a high degree of sequence and structural similarity with mammalian orthologs and express in brain, ovary, testis, and low levels in other tissues. Regardless of its importance in the developmental process, the structure, function and mode of ATP recognition have not been investigated yet due to lack of experimental data. Henceforth, to gain atomistic insights in to the structural dynamics and mode of ATP binding, a series of computational techniques involving theoretical modeling, docking, molecular dynamics (MD) simulations and MM/PBSA binding free energies were employed. The modeled bi-lobed zCDKL1 shares a high degree of secondary structure topology with human orthologs where ATP prefers to lie in the central cavity of the bi-lobed catalytic domain enclosed by strong hydrogen bonding, electrostatic and hydrophobic contacts. Long range MD simulation portrayed that catalytic domain of zCDKL1 to be highly rigid in nature as compared to the complex (zCDKL1-ATP) form. Comparative analysis with its orthologs revealed that conserved amino acids i.e., Ile10, Gly11, Glu12, Val18, Arg31, Phe80, Glu 130, Cys143 and Asp144 were crucial for ATP binding mechanism, which needs further investigation for legitimacy. MM/PBSA method revealed that van der Waals, electrostatic and polar solvation energy mostly contributes towards negative free energy. The implications of ATP binding mechanism inferred through these structural bioinformatics approaches will help in understanding the catalytic mechanisms of important STKs in eukaryotic system.

The population structure and genetic divergence of Labeo gonius (Hamilton, 1822) analyzed through mitochondrial DNA cytochrome b gene for conservation in Indian waters.

The present study explains the population structure and genetic diversity of medium carp Labeo gonius by analyzing partial sequence of mitochondrial DNA cytochrome b gene. Labeo gonius is a lower risk Near Threatened species, distributed throughout the North Indian major rivers, reservoirs and lakes. This species has a larger scope as an alternative candidate species in carp aquaculture system. In the present investigation, 223 individuals of Labeo gonius were collected from five locations of phylo-geographically isolated riverine ecosystems of India resulted in 12 haplotypes. These haplotypes showed 14 variables, out of which 9 were singletons and 5 were parsimony informative sites of nucleotide positions. The haplotypes H1 was considered as ancestral haplotype. All the haplotypes were connected to each other by 1-4 nucleotide mutations. The Narmada haplotypes (H10; H11 and H12) were isolated from H1 by four nucleotide mutations. The analyses resulted maximum expansion events (τ = 4.13672) in Narmada, with Fst scores more than other population pairs. The analysis of molecular variance (AMOVA) showed significant genetic differentiation among populations (ØST = 0.69470, p < .000). The genetic differentiation patterns were significantly consistence with geographical distributions. This study rejected the null hypothesis of single panmictic population of medium carp, Labeo gonius in Indian water.

Population structure and genetic diversity of Indian Major Carp, Labeo rohita (Hamilton, 1822) from three phylo-geographically isolated riverine ecosystems of India as revealed by mtDNA cytochrome b region sequences.

The population structure and genetic diversity of Rohu (Labeo rohita Hamilton, 1822) was studied by analysis of the partial sequences of mitochondrial DNA cytochrome b region. We examined 133 samples collected from six locations in three geographically isolated rivers of India. Analysis of 11 haplotypes showed low haplotype diversity (0.00150), nucleotide diversity (π) (0.02884) and low heterogeneity value (0.00374). Analysis of molecular variance (AMOVA) revealed the genetic diversity of L. rohita within population is very high than between the populations. The Fst scores (-0.07479 to 0.07022) were the indication of low genetic structure of L. rohita populations of three rivers of India. Conspicuously, Farakka-Bharuch population pair Fst score of 0.0000, although the sampling sites are from different rivers. The phylogenetic reconstruction of unique haplotypes revealed sharing of a single central haplotype (Hap_1) by all the six populations with a point mutations ranging from 1-25 nucleotides.

Genetic variation in wild and hatchery population of Catla catla (Hamilton, 1822) analyzed through mtDNA cytochrome b region.

Catla (Catla catla) is a one of the most harvested Indian major carps and is widely cultured fish species in Indian subcontinent. In the present study, genetic variability between hatchery and wild stocks of Catla was surveyed using sequence data of mitochondrial DNA of partial 307 bp of cytochrome b region. A total of 174 Catla individuals were examined from three different river basins and hatcheries. Significant genetic heterogeneity was observed for the sequence data (FST = 0.308, p ≤ 0.001). However, analysis of molecular variance (AMOVA) resulted in insignificant genetic differentiation among the samples of three rivers and culture zones (FCT = -0.10, p = 0.44). The result suggested a significant genetic variation within different riverine system, low genetic differentiation among samples from river basins and a lack of genetic variation in hatchery populations.

Population genetic structure and geographic differentiation in butter catfish, Ompok bimaculatus, from Indian waters inferred by cytochrome b mitochondrial gene.

Documentation of genetic differentiation among the populations of a species can provide useful information that has roles in conservation, breeding, and management plans. In the present study, we examined the genetic structure and phylogenetic relationships among the 149 individuals of Ompok bimaculatus belonging to 24 populations, collected from Indian waters, using cytochrome b gene. The combined analyses of data suggested that the Indian O. bimaculatus consist of three distinct mtDNA lineages with star-like haplotypes network, which exhibited high genetic variation and haplotypic diversity. Analysis of molecular variance indicated that most of the observed genetic variation was found among the populations suggesting restricted gene flow. Long-term interruption of gene flow was also evidenced by high overall Fst values (0.82367) that could be favored by the discontinuous distributions of the lineages.

The complete mitochondrial genome of the Anabas testudineus (Perciformes, Anabantidae) and its comparison with other related fish species.

In the present study, the complete mitochondrial genome sequence of Anabas testudineusis reported using PGM sequencer (Ion Torrent, Life Technologies, La Jolla, CA). The complete mitogenome of climbing perch, A. testudineusis obtained by the de novo sequences assembly of genomic reads using the Torrent Mapping Alignment Program (TMAP), which is 16 603 bp in length. The mitogenome of A. testudineus composed of 13 protein- coding genes, two rRNA, and 22 tRNAs. Here, 20 tRNAs genes showed typical clover leaf model, and D-Loop as the control region along with gene order and organization, being closely similar to Osphronemidae and most of other Perciformes fish mitogenomes of NCBI databases. The mitogenome in the present study has 99% similarity to the complete mitogenome sequence of earlier reported A. testudineus. The phylogenetic analysis of Anabantidae depicted that their mitogenomes are closely related to each other. The complete mitogenome sequence of A. testudineus would be helpful in understanding the population genetics, phylogenetics, and evolution of Anabantidae.

Complete mitochondrial genome sequence of Indian medium carp, Labeo gonius (Hamilton, 1822) and its comparison with other related carp species.

In the present study, the complete mitochondrial genome sequence of Labeo gonius is reported using PGM sequencer (Ion Torrent). The complete mitogenome of L. gonius is obtained by the de novo sequences assembly of genomic reads using the Torrent Mapping Alignment Program (TMAP) which is 16 614 bp in length. The mitogenome of L. gonius comprised of 13 protein-coding genes, 22 tRNAs, 2 rRNA genes, and D-loop as control region along with gene order and organization, being similar to most of other fish mitogenomes of NCBI databases. The mitogenome in the present study has 99% similarity to the complete mitogenome sequence of Labeo fimbriatus, as reported earlier. The phylogenetic analysis of Cypriniformes depicted that their mitogenomes are closely related to each other. The complete mitogenome sequence of L. gonius would be helpful in understanding the population genetics, phylogenetics, and evolution of Indian Carps.

The complete mitochondrial genome of the Asian stinging catfish, Heteropneustes fossilis (Siluriformes, Heteropneustidae) and its comparison with other related fish species.

The complete mitogenome of Heteropneustes fossilis is described using Ion Torrent (PGM sequencer), which showed it was 16,489 bp in size comprising 13 mRNAs, 22 tRNAs, 2 rRNA genes, and 858 bp as D-Loop control region, along with gene order and organization, being similar to most of the other related Siluriformes fish mitogenome of NCBI databases. The 20 RNAs were packed into a typical cloverleaf structure. The mitogenome in the present study has 99% similarity to the complete mitogenome sequence of H. fossilis mitogenome reported earlier and also would be helpful in understanding the population genetics, phylogenetics, and evolution of catfishes.

Mitochondrial 16S rRNA gene-based evolutionary divergence and molecular phylogeny of Barilius spp.

Barilius is an important genus of fish family Cyprinidae, which comprises 22 species from Indian region. This study aimed to characterize five commonly occurring Bariline species, for example, Barilius bakeri. Barilius gatensis. Barilius vagra. Barilius bendelisis and Barilius tileo, across the country using partial mitochondrial 16 S rRNA gene, to estimate the order of inter- and intra-species divergence among these species and to establish phylogenetic and evolutionary relationships. The average inter-specific divergence was estimated as 7.10%. Intra-specific divergence of 0.20% and 0.10% was observed in B. gatensis and B. bendelisis that exhibited three and two haplotypes with 0.70 and 0.60 haplotype diversities, respectively. The NJ and MP phylogenetic trees were constructed using 16 S rRNA sequences along with sequences of the other five Indian cyprinid species retrieved from NCBI. Phylogenetic trees revealed five discrete branches each as a distinct species of the genus and exhibited identical phylogenetic relationship with other cyprinids. The study provided adequate information to distinguish the five Barilius species and indicated the suitability of 16S rRNA gene sequences in genetic divergence and phylogenetic studies.