Kuldeep K. Lal

DNA barcoding Indian marine fishes.

DNA barcoding has been adopted as a global bio‐identification system for animals in recent years. A major national programme on DNA barcoding of fish and marine life was initiated in India by the authors during 2006 and 115 species of marine fish covering Carangids, Clupeids, Scombrids, Groupers, Sciaenids, Silverbellies, Mullids, Polynemids and Silurids representing 79 Genera and 37 Families from the Indian Ocean have been barcoded for the first time using cytochrome c oxidase I gene (COI) of the mtDNA. The species were represented by multiple specimens and a total of 397 sequences were generated. After amplification and sequencing of 707 base pair fragment of COI, primers were trimmed which invariably generated a 655 base pair barcode sequence. The average Kimura two parameter (K2P) distances within species, genera, families, orders were 0.30%, 6.60%, 9.91%, 16.00%, respectively. In addition to barcode‐based species identification system, phylogenetic relationships among the species have also been attempted. The neighbour‐joining tree revealed distinct clusters in concurrence with the taxonomic status of the species.

Genetic variation and population structure of endemic yellow catfish, Horabagrus brachysoma (Bagridae) among three populations of Western Ghat region using RAPD and microsatellite markers

Random amplified polymorphic DNA (RAPD) and microsatellite markers were applied to evaluate the genetic variation in endemic and endangered yellow catfish, Horabagrusbrachysoma sampled from three geographic locations of Western Ghat, South India river systems. In RAPD, of 32 10-mer RAPD primers screened initially, 10 were chosen and used in a comparative analysis of H. brachysoma collected from Meenachil, Chalakkudy and Nethravathi River systems. Of the 124 total RAPD fragments amplified, 49 (39.51%) were found to be shared by individuals of all 3 populations. The remaining 75 fragments were found to be polymorphic (60.48%). In microsatellites, six polymorphic microsatellite loci were identified by using primers developed for Pangasiushypophthalmus, Clariasmacrocephalus and Clariasgariepinus. The identified loci were confirmed as microsatellite by sequencing after making a clone. The nucleotide sequences of 6 loci were published in NCBI genbank. The number of alleles across the six loci ranged from 4 to 7 and heterozygosities ranged from 0.07 to 0.93. The mean number of alleles and effective number of alleles per locus were 5.00 and 3.314, respectively. The average heterozygosity across all investigated samples was 0.72, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods reported a high degree of gene diversity and genetic distances depicted by UPGMA dendrograms among the populations of H. brachysoma.

Fish genetics and conservation research in India: status and perspectives

Recent years have witnessed a rise in sensitivity towards protection of natural genetic resources and the environment at the international level. This is evident from the implementation of the convention on biodiversity (CBD). To conserve and optimize utilization of its bioresources, India enacted the Biological Diversity Act 2002. The conservation and management of aquatic resources is important for sustainable utilization of fisheries potential for the economic advancement of farmers and fishery workers, today as well as for posterity. As the biotechnological and genomic revolutions are ready to take off, the sovereign rights of native inhabitants to intellectual property rights (IPR) related to aquatic genetic resources and associated traditional knowledge are also integral components of management strategies for genetic resources.

Molecular identification of five Indian sciaenids (pisces: perciformes, sciaenidae) using RAPD markers

The randomly amplified polymorphic DNA (RAPD) markers were used to detect interspecific genetic variability and genetic relatedness among five Indian sciaenids namely Otolithes cuvieri, Johnieops sina, Johnieops macrorhynus, Johnieops vogleri and Protonibea diacanthus for the first time. Eight RAPD primers (OPA01, OPA06, OPA07, OPA18, OPP12, OPP14, OPP16 and OPP11) generated 40 species specific diagnostic bands. The highest genetic divergence was detected between J. macrorhynus and P. diacanthus (0.586) where as the lowest one was observed between J. sina and J. vogleri (0.274).

Hypoxia induced altered expression of heat shock protein genes (Hsc71, Hsp90α and Hsp10) in Indian Catfish, Clarias batrachus (Linnaeus, 1758) under oxidative stress

Heat shock proteins (Hsps) are typically associated with stress response and tolerance. The Indian catfish, Clarias batrachus, is a freshwater air-breathing hypoxia tolerant teleost and is potentially important catfish species for aquaculture and for its economic value as food. The present study aimed at determining the transcriptional response of three Hsps, CbHsc71, CbHsp90α and CbHsp10 in hypoxia tolerant Indian catfish, C. batrachus, under experimental and natural hypoxia. The expression profile of above three genes were studied under different periods of hypoxia, through qRT-PCR. Primers were designed from ESTs obtained through SSH libraries constructed from hypoxia treated fishes. The Hsp10 ESTs and deduced protein was in silico characterized for its ORF and for its physical and chemical properties, respectively, using GeneScan, blastp, scanprosite, superfamily and other softwares. A phylogenetic tree was constructed based on deduced amino acid sequences of Hsc71, Hsp90α, Hsp90β of Homo sapiens and other fishes along with CbHsp10 protein in MEGA4. The deduced protein sequences of CbHsp10 was found to have characteristic Hsp10 family signatures, and it is proposed for inclusion of methionine in the consensus sequences of Hsp10 family signature, after the “proline” residue. At transcription level, these genes were found to be differentially regulated under hypoxia stress, in different tissues of C. batrachus. The CbHsc71 and CbHsp90α were up-regulated after short and long-term hypoxia, whereas CbHsp10 was significantly down-regulated after short-term hypoxia. The differential expression of these Hsps may play a role in protection and survival under hypoxia induced oxidative stress in C. batrachus.

Molecular evidence to reconcile taxonomic instability in mahseer species (Pisces: Cyprinidae) of India

The mahseers are an important group of fishes endemic to Asia with most species considered threatened. Conservation plans to save declining wild populations are hindered by unstable taxonomy, and detailed systematic review could form a solid platform for future management and conservation. D-loop and cytochrome c oxidase I (COI) mtDNA sequences were examined in nine mahseer species of Tor, Neolissochilus, and Naziritor. Pseudogenes amplified in a portion of the species limited the utility of the D-loop region. ABGD analysis, NJ, ML, and MP methods and genetic distance (TrN + I + G) using COI data revealed concordant species delimiting patterns. The three genera were monophyletic, separated as distinct clades (TrN + I + G 0.064 to 0.106), and Naziritor was flagged as a separate genus, distinct from Puntius (TrN + I + G 0.196). Out of seven nominal species known for Tor cogeners from India, only five were recovered with mtDNA data (TrN + I + G 0.000 to 0.037) and two species could not be distinguished with the molecular data set employed. Tor mosal, synonymized as Tor putitora, was rediscovered as a distinct species (TrN + I + G 0.031) based on its type locality. Tor mussulah was confirmed as a separate species (TrN + I + G 0.019 to 0.026). Two valid species, Tor macrolepis and T. mosal mahanadicus, were not distinct from T. putitora (TrN + I + G 0.00). The high divergence with mtDNA data failed to validate T. mosal mahanadicus as a subspecies of T. mosal (TrN + I + G 0.031). Morphological outliers discovered within the distribution range of Tor tor (TrN + I + G 0.022 to 0.025) shared the same lineage with T. putitora (TrN + I + G 0.002 to 0.005), indicating a new extended distribution of the Himalayan mahseer T. putitora in the rivers of the Indian central plateau. The findings indicate the need for integrating molecular and morphological tools for taxonomic revision of the Tor and Naziritor genera, so that taxa are precisely defined for accurate in situ and ex situ conservation decisions.

Evaluation of Genetic Variation in the Clown Knifefish, Chitala chitala, Using Allozymes, RAPD, and Microsatellites

Twenty-seven enzyme systems, six random amplified polymorphic DNA (RAPD) primers, and two microsatellite loci were tested to determine intraspecific divergence in the natural population of the endangered Indian featherback fish, Chitala chitala, for the first time. The 262 samples of C. chitala were collected from six riverine locations in India: the Satluj, Ganga (Ghagra, Bhagirathi, and Brahmaputra), Mahanadi, and Narmada river systems. The analysis revealed population subdivisions, with an FST value from 0.1235 (95% confidence 0.0868–0.1621) for RAPD and a combined FST of 0.0344 (95% confidence 0.0340–0.0350) for microsatellite loci. An analysis of 38 allozyme loci did not reveal any polymorphism in the samples from any of the riverine localities; a possible explanation for this could be that the ancestors of Chitala could have faced a population reduction in prehistoric periods, as low allozyme variation is also reported for other species of Chitala from south Asia.

DNA barcoding Indian freshwater fishes

Abstract DNA barcoding is a promising technique for species identification using a short mitochondrial DNA sequence of cytochrome c oxidase I (COI) gene. In the present study, DNA barcodes were generated from 72 species of freshwater fish covering the Orders Cypriniformes, Siluriformes, Perciformes, Synbranchiformes, and Osteoglossiformes representing 50 genera and 19 families. All the samples were collected from diverse sites except the species endemic to a particular location. Species were represented by multiple specimens in the great majority of the barcoded species. A total of 284 COI sequences were generated. After amplification and sequencing of 700 base pair fragment of COI, primers were trimmed which invariably generated a 655 base pair barcode sequence. The average Kimura two-parameter (K2P) distances within-species, genera, families, and orders were 0.40%, 9.60%, 13.10%, and 17.16%, respectively. DNA barcode discriminated congeneric species without any confusion. The study strongly validated the efficiency of COI as an ideal marker for DNA barcoding of Indian freshwater fishes.

Development of EST derived SSRs and SNPs as a genomic resource in Indian catfish, Clarias batrachus

Clarias batrachus, an Indian catfish species, is endemic to the Indian subcontinent and potential cultivable species. The genomic resources in C. batrachus in the form of ESTs containing microsatellite repeats (EST-SSR) and single nucleotide polymorphisms (SNPs) that are associated with the expressed genes from spleen were mined. From a total of 1,937 ESTs generated, 1,698 unique sequences were obtained, out of which 221 EST-SSRs were identified and 54% could be functionally annotated by similarity searches. A total of 23 contigs containing 3 or more ESTs were found to contain 31 SNP loci, out of which 8 ESTs showed similarity to genes of known function and 1 for hypothetical protein. Nine ESTs with SSRs and/or SNPs identified in this study were reported to be associated with diseases in human and animals. These identified loci can be developed into markers in C. batrachus, which can be useful in linkage mapping, comparative genomics studies and for its genetic improvement programmes.

Complete mitochondrial genome sequences of two endangered Indian catfish species, Clarias batrachus and Pangasius pangasius.

Abstract Complete mitogenome sequences for Clarias batrachus (magur) and Pangasius pangasius (family Claridae and Pangasiidae, respectively) were generated, which were 16,511 and 16,476 bp, respectively. Both the mitogenomes contained 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs and 1 non-coding (control) region. The order of the genes was identical to that observed in most other vertebrates. The genome resource of complete mitogenome sequence of Indian catfish species generated during this study would be useful in phylogenetic studies.