Lakshman Sahoo

Molecular characterization of two kiss genes and their expression in rohu (Labeo rohita) during annual reproductive cycle.

Kisspeptin is an important regulator of reproduction in mammals and presumably non-mammalian species. In the Indian subcontinent, rohu (Labeo rohita) is a commercially important seasonal breeder freshwater fish species, but till date, the expression of kisspeptin gene during different phases of annual reproductive cycle has not been investigated. To address this, we cloned and characterized kiss1 and kiss2 full-length cDNA by RACE (rapid amplification of cDNA ends), and analyzed their expressions in brain-pituitary-gonad (BPG) axis by quantitative real time PCR (qRT-PCR) assay at various gonadal developmental stages of the annual reproductive cycle. Full-length rohu kiss1 and kiss2 cDNA encodes 116 and 125 amino acids respectively, and in the adult fish, they were widely expressed in brain, pituitary, gonad, liver, muscle, kidney, intestine and eye. In male, kiss1 mRNA in brain and testis showed the highest level of expression during meiosis division of the gonad. The kiss2 mRNA revealed the highest expression during recrudescence stages in the brain, spermiation stages in pituitary and post-spawning stages in testes. In females, significantly (p<0.05) a higher level of kiss1 transcript was expressed in brain and ovary, in the full grown oocyte stages, whereas during pre-vitellogenic and vitellogenic stages in pituitary. The kiss2 gene expression was almost similar at various gonadal developmental stages in the brain and ovary, but, highest expression was detected in full grown oocyte stages in the pituitary. These results together may suggest the involvement of two kiss genes in the control of seasonal gonadal development in rohu.

Complete mitochondrial genome sequence of Catla catla and its phylogenetic consideration

Complete nucleotide sequence of mitochondrial genome (mitogenome) of the Catla catla (Ostariophysi: Cypriniformes: Cyprinidae) was determined in the present study. Its length is 16,594 bp and contains 13 protein coding genes, 22 transfer RNAs, two ribosomal RNAs and one non-coding control region. Most of the genes were encoded on the H-strand, while the ND6 and eight tRNA (Gln, Ala, Asn, Cys, Tyr, Ser (UCN), Glu and Pro) genes were encoded on the L-strand. The reading frames of two pair of genes overlapped: ATPase 8 with 6 and ND4L with ND4 by seven nucleotides each. The main non-coding region was 929 bp, with three conserved sequence blocks (CSB-I, CSB-II, and CSB-III) and an unusual simple sequence repeat, (TA)7. Phylogenetic analyses based on complete mitochondrial genome sequences were in favor of the traditional taxonomy of family Cyprinidae. In conclusion present mitogenome of Catla catla adds more information to our understanding of diversity and evolution of mitogenome in fishes.

Complete mitochondrial genome sequence of Cirrhinus mrigala (Hamilton, 1822)

The complete mitochondrial genome of Cirrhinus mrigala was determined using the polymerase chain reaction. The mitogenome (16,594 bp) has the typical vertebrate mitochondrial gene arrangement, including 13 protein-coding genes, 22 tRNA genes, two rRNA genes and one control region. The overall base composition on the heavy strand was as follows: A: 32.0%, G: 15.5%, C: 28.0%, T: 24.55% and the A+T content: 56.5%. The control region contains a dinucleotide repeat motif, (TA)14, a termination-associated sequence and three conserved sequence blocks. These mitogenome sequence data would play an important role in population genetics and the molecular taxonomy of cultivable cyprinids in India.

Development of polymorphic EST-SSR markers in Macrobrachium rosenbergii by data mining

Expressed sequence tags (EST) provide a valuable and cost effective source of developing simple sequence repeat (SSR) markers. In the present study, genic microsatellite markers were developed for Macrobrachium rosenbergii through mining of EST databases. Twenty-three polymorphic EST-SSR loci were found with number of alleles ranging from 3 to 10, observed and expected heterozygosity per locus ranged from 0.1481 to 0.9565 and from 0.2655 to 0.8741, respectively. Of these SSRs, 7 showed significant homology to known genes by BLASTx (basic local alignment search tool x) search. In cross-species amplification, out of 23 polymorphic loci, there were 21 positive amplifications in M. malcolmsonii, 22 in M. gangeticum and 18 in M. lamarrei. These new EST-SSR markers will be useful for genetic studies and genome mapping of M. rosenbergii and closely related macrobrachium species.

Population divergence and structure of Cirrhinus mrigala from peninsular rivers of India, revealed by mitochondrial cytochrome b gene and truss morphometric analysis

Abstract Genetic diversity and population structure of Cirrhinus mrigala from peninsular riverine systems of India was studied using mitochondrial DNA gene, cytochrome b and truss morphometric analysis. Analysis of 982 bp of the cytochrome b gene from 182 samples collected from six rivers revealed the presence of 28 haplotypes with overall high haplotype diversity value of 0.78981 and nucleotide diversity value of 0.00215. Analysis of molecular variance (AMOVA) showed that genetic variation is mainly harbored within populations rather than among populations. The pairwise Fst values (−0.009 to 0.084) was indicative of a low genetic structure among mrigal populations. Morphometric examination of 243 samples using 10 truss landmarks and principal component analysis showed a similar trend when compared with genetic data. Overall, low genetic and morphometric differences were observed despite those populations from different geographic locations. The results of this study would provide essential information to resource recovery and help in delineating populations for fishery management. Besides, the data will provide a valuable baseline for further investigations on the geographic distribution of this commercially important fish species.

Complete mitochondrial genome sequence of Labeo fimbriatus (Bloch, 1975).

Abstract The complete mitochondrial genome of Labeo fimbriatus, the fringe-lipped carp, was determined for the first time. The genome was 16,614 bp 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 the plus strand was A: 32.35%, G: 15.46%, C: 27.54%, T: 24.647%, and the A + T content 57%. The control region contains a microsatellite, (TA)12, 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 cultivable carps of India.

A comparative study of COI and 16 S rRNA genes for DNA barcoding of cultivable carps in India

Abstract The 5′ region of the mitochondrial DNA gene cytochrome c oxidase subunit I (COI) is the standard marker for DNA barcoding. However, 16 S rRNA has also been advocated for DNA barcoding in many animal species. Herein, we directly compare the usefulness of COI and 16 S rRNA in discriminating six cultivable carp species: Labeo rohita, Catla catla, Cirrhinus mrigala, Labeo fimbriatus, Labeo bata and Cirrhinus reba from India. Analysis of partial sequences of these two gene fragments from 171 individuals indicated close genetic relationship between Catla catla and Labeo rohita. The results of the present study indicated COI to be more useful than 16 S rRNA for DNA barcoding of Indian carps.

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

Abstract 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.

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-β (TGF-β) 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.

Genetic relationship and population structure of three Indian local chicken populations as revealed by mtDNA D-loop.

Abstract The genetic information obtained from the mitochondrial DNA D-loop region has paramount importance in understanding the evolution of closely related individuals, and designing proper breeding or conservation plans. The present study was conducted using partial D-loop sequences of three local poultry populations from Odisha, India. The partial D-loop sequences were found to be highly polymorphic having 164 polymorphic sites with 89 singletons and 75 parsimony informative sites. Furthermore, 25 insertion and deletion sites were observed. High genetic diversity was observed within three local chicken populations. Highest genetic difference was observed between Gujuri and Kalua population (0.2230) followed by Gujuri and Hansli (0.199) and Kalua with Hansli (0.166). The pairwise mismatch distribution showed that all populations are of constant size over time. Phylogenetic tree analysis indicated that the said three populations were close to the referred population of China, Sri Lanka, Indonesia and Japan than Aseel and Kadaknath (Indian native breeds).