Pratap Chandra Das

Development of 21 new microsatellite markers in Labeo rohita (rohu)

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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 of Labeo rohita.

The complete mitochondrial genome of Labeo rohita, an important cultivable fish, was determined for the first time. The genome is 16,611 bp in length and consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and one control region. The gene organisation and its order were similar to other vertebrates. The overall base composition on heavy strand was as follows A: 32.5%, G: 15.2%, C: 27.7%, T: 24.47%, and the A+T content 56.9%. 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 Indian major carps.

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.

Mitochondrial DNA diversity and PCR-based sex determination of Irrawaddy dolphin (Orcaella brevirostris) from Chilika Lagoon, India.

Of the only known two Lagoon populations of Irrawaddy dolphins (Orcaella) in the world, one is residing in the Chilika Lagoon in Orissa state, India. In addition to accidental deaths in gill net fishery and mechanized boat operations, there has been exploitation of the species for their oil. Extreme patchy distribution and vulnerability to becoming entangled in fishing gear has made it a focus of conservation concern. Information on genetic diversity of populations has considerable potential for informing conservation plans. The present paper reports the first genetic study of O. brevirostris from Chilika Lagoon based on mtDNA sequencing and PCR-based sex identification from 11 individuals. Control region sequence comparison showed two haplotypes and cytochrome b a single haplotype in the Chilika population of the species. Phylogenetic analysis indicated distinct clades within the Asian samples, with the Indian population showing closest genetic proximity to the haplotypes from Thailand. Sex of the animal was determined by PCR-based method. It is important to continue to examine the population discreteness and genetic variation of Irrawaddy dolphin in Chilika Lagoon vis-à-vis its global geographic distribution for formulating the conservation plans of the species.

Preliminary genetic linkage map of Indian major carp, Labeo rohita (Hamilton 1822) based on microsatellite markers

Linkage map with wide marker coverage is an essential resource for genetic improvement study for any species. Sex-averaged genetic linkage map of Labeo rohita, popularly known as ‘rohu’, widely cultured in the Indian subcontinent, was developed by placing 68 microsatellite markers generated by a simplified method. The parents and their F 1 progeny (92 individuals) were used as segregating populations. The genetic linkage map spans a sex-averaged total length of 1462.2 cM, in 25 linkage groups. The genome length of rohu was estimated to be 3087.9 cM. This genetic linkage map may facilitate systematic searches of the genome to identify genes associated with commercially important characters and marker-assisted selection programmes of this species.

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.

Isolation and characterization of sixteen microsatellite loci for fringe-lipped carp, Labeo fimbriatus

Labeo fimbriatus, the fringe-lipped peninsula carp, is a commercially important fish species next to Indian major carps. We isolated and characterized polymorphic microsatellite markers to be used as a tool for delineation of genetic stock of this species. Partial genomic libraries enriched for CT and GT repeat motifs generated 103 positive clones out of which 16 loci were found with flanking regions enough for designing primers. Thirty-two individuals of L. fimbriatus collected from wild were used to characterize the polymorphism. All the 16 loci were polymorphic with allele numbers ranging from 3 to 9. The observed and expected heterozygosity ranged from 0.093 to 0.833 and from 0.146 to 0.843, respectively. Nine loci were in agreement with Hardy–Weinberg equilibrium. No significant pair wise linkage disequilibrium was found among the loci. These markers would be very useful for characterization of natural populations of this species.

Use of Rotenone as Piscicide: Toxicity Levels in a Few Common Freshwater Predatory and Weed Fishes

Toxicity of rotenone was studied in a few common freshwater predatory and weed fishes through wet laboratory experiments for its use as a piscicide during pond preparation. Cube root powder (CRP) (ENT-133 Rotenone) containing 9% rotenone was used as the toxicant source. Lethal concentration of CRP for these common predatory and weed fishes varied between 0.75–2.70 mg L−1 (0.068–0.243 mg L−1 of rotenone). Acute toxicity study revealed Puntius sophore to have more susceptibility to rotenone toxicity with 24 h LC50 value of CRP at 0.50 mg L−1 (0.045 mg L−1 rotenone) compared to 1.17 mg L−1 (0.105 mg L−1 rotenone) in Anabas testudineus and 1.90 mg L−1 in Channa punctatus (0.171 mg L−1 rotenone); while Heteropneustes fossilis showed higher tolerance with 24 h LC50 value at 2.42 mg L−1 (0.218 mg L−1 rotenone). Such result suggested rotenone toxicity to depend on the respiratory behavior of fish. The marginal reduction in 48 h LC50 of CRP compared to its 24 h value and no fish mortality beyond 48-h in all tested species suggested faster degradation of the toxicant in water. Since application of the piscicide aims at eradication of all commonly available species of predatory and weed fishes in the culture pond, the study suggested a dose of 2.5 mg L−1 of CRP (0.225 mg L−1 rotenone) for pond application.