P. K. Meher

Genetic variation between four species of Indian major carps as revealed by random amplified polymorphic DNA assay

Information on the genetic structure of cultivable fish species is essential for studying molecular systematics and optimising fisheries management and fish farming. The random amplified polymorphic DNA (RAPD) assay was evaluated for studying genetic relationships and diversities in four species of Indian major carps (Family Cyprinidae). Thirty-four arbitrary primers were screened to identify species-specific RAPD markers among rohu (Labeo rohita), kalbasu (L. calbasu), catla (Catla catla) and mrigal (Cirrhinus mrigala). Distinct and highly reproducible RAPD profiles with a great degree of genetic variability were detected among species. On average, 45% of the scorable RAPD bands were specific to each species. Phylogenetic analysis demonstrated that kalbasu is the closest to rohu and the farthest from mrigal. Data are presented with regard to the applications of RAPD markers for hybrid identification, genetic diversity assessment and studying taxonomic relationships at a molecular level.

Growth and survival in two complete diallele crosses with five stocks of Rohu carp (Labeo rohita)

Abstract The experiment of two 3×3 complete diallele crosses was carried out with four wild river stocks (Ganga, Yamuna, Brahmaputra and Sutlej) and one farmed stock (Local) of Rohu carp ( Labeo rohita ). The Local stock was included in both diallele crosses. In total, 864 fish were individually tagged at 6 months of age and reared for 14 months in three monoculture and two polyculture earthen ponds in Orissa, India. For harvest weight, total heterosis for each of the six stock crosses was low or negative and average heterosis was also low and in most cases not significantly different from zero. For survival, total and average heterosis was negligible and not significantly different from zero. It is concluded that genetic improvement through crossbreeding of Indian stocks of Rohu carp has little practical significance.

Effect of endotoxin on the immunity of Indian major carp, Labeo rohita.

Endotoxin, a lipopolysaccharide component of outer cell wall membrane of the Gram-negative bacteria is a factor responsible for a number of biological effects including immunostimulatory activities in different animal species including fish. In this study, L. rohita yearlings of weight ranging from 80 to 100g were injected intraperitoneally with 0.5, 1, 2, 5, 10 and 20 EU/fish dose of endotoxin to find out its effect on the immunity. The L. rohita yearlings were found to resist the endotoxin dose up to 20 EU/fish and at the lower doses, i.e., at 1 and 2 EU/fish; it acted as an immune potentiator. Different serum and immune parameters like protein, globulin, lysozyme, respiratory burst activity, myeloperoxidase activity, natural agglutination titre were found to be significantly high (p<0.01) at a dose of 1 EU/fish. While at 10 and 20 EU/fish, most of these parameters were lower thereby indicating the immuno-suppressive nature of the endotoxin at these higher doses.

Derivation of rough attenuated variants from smooth virulent Aeromonas hydrophila and their immunogenicity in fish.

Aeromonas hydrophila is a Gram-negative bacteria associated with many disease conditions of aquatic animals. Two rough attenuated variants were derived from two smooth virulent types of this bacterium by continuous passage separately in laboratory over a period of 8 years in brain-heart infusion agar. These variants were checked to be rough lipopolysaccharides (LPS) types and attenuated since they neither produced any disease nor mortality in the injected fishes. The immunogenic potential of these variants remained unaltered as compared to parent smooth types and fish immunized with these variants resisted the infection and mortality upon cross challenge. Because of their intact immunogenic potential and loss of virulence, they may be used as candidates for fish immunization.

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

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Identification of reproduction-related genes and SSR-markers through expressed sequence tags analysis of a monsoon breeding carp rohu, Labeo rohita (Hamilton)

Labeo rohita (Ham.) also called rohu is the most important freshwater aquaculture species on the Indian sub continent. Monsoon dependent breeding restricts its seed production beyond season indicating a strong genetic control about which very limited information is available. Additionally, few genomic resources are publicly available for this species. Here we sought to identify reproduction-relevant genes from normalized cDNA libraries of the brain-pituitary-gonad-liver (BPGL-axis) tissues of adult L. rohita collected during post preparatory phase. 6161 random clones sequenced (Sanger-based) from these libraries produced 4642 (75.34%) high-quality sequences. They were assembled into 3631 (78.22%) unique sequences composed of 709 contigs and 2922 singletons. A total of 182 unique sequences were found to be associated with reproduction-related genes, mainly under the GO term categories of reproduction, neuro-peptide hormone activity, hormone and receptor binding, receptor activity, signal transduction, embryonic development, cell-cell signaling, cell death and anti-apoptosis process. Several important reproduction-related genes reported here for the first time in L. rohita are zona pellucida sperm-binding protein 3, aquaporin-12, spermine oxidase, sperm associated antigen 7, testis expressed 261, progesterone receptor membrane component, Neuropeptide Y and Pro-opiomelanocortin. Quantitative RT-PCR-based analyses of 8 known and 8 unknown transcripts during preparatory and post-spawning phase showed increased expression level of most of the transcripts during preparatory phase (except Neuropeptide Y) in comparison to post-spawning phase indicating possible roles in initiation of gonad maturation. Expression of unknown transcripts was also found in prolific breeder common carp and tilapia, but levels of expression were much higher in seasonal breeder rohu. 3631 unique sequences contained 236 (6.49%) putative microsatellites with the AG (28.16%) repeat as the most frequent motif. Twenty loci showed polymorphism in 36 unrelated individuals with allele frequency ranging from 2 to 7 per locus. The observed heterozygosity ranged from 0.096 to 0.774 whereas the expected heterozygosity ranged from 0.109 to 0.801. Identification of 182 important reproduction-related genes and expression pattern of 16 transcripts in preparatory and post-spawning phase along with 20 polymorphic EST-SSRs should be highly useful for the future reproductive molecular studies and selection program in Labeo rohita.

Isolation and characterization of microsatellite loci in giant freshwater prawn, Macrobrachium rosenbergii

This paper reports on isolation and characterization of 8 microsatellite loci from a partial genomic library of Macrobrachium rosenbergii using DIG labeled dinucleotides, (GT)15 and (CT)15 as probes. Primers were designed and PCR conditions optimized for repeat arrays. Polymorphism was studied using 24 individuals collected from the wild. All the loci were polymorphic with number of alleles ranging from 3 to 5 and observed heterozygosity 0.50–0.85. All the loci except one were in agreement with Hardy–Weinberg equilibrium. No significant pair wise linkage disequilibrium was found among the loci. These markers should prove useful for characterization of natural population as well as brood stock management of this 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.

Construction, De-Novo Assembly and Analysis of Transcriptome for Identification of Reproduction-Related Genes and Pathways from Rohu, Labeo rohita (Hamilton)

Rohu is a leading candidate species for freshwater aquaculture in South-East Asia. Unlike common carp the monsoon breeding habit of rohu restricts its seed production beyond season indicating strong genetic control over spawning. Genetic information is limited in this regard. The problem is exacerbated by the lack of genomic-resources. We identified 182 reproduction-related genes previously by Sanger-sequencing which were less to address the issue of seasonal spawning behaviour of this important carp. Therefore, the present work was taken up to generate transcriptome profile by mRNAseq. 16GB, 72bp paired end (PE) data was generated from the pooled-RNA of twelve-tissues from pre-spawning rohu using IlluminaGA-II-platform. There were 64.97 million high-quality reads producing 62,283 contigs and 88,612 numbers of transcripts using velvet and oases programs, respectively. Gene ontology annotation identified 940 reproduction-related genes consisting of 184 mainly associated with reproduction, 223 related to hormone-activity and receptor-binding, 178 receptor-activity and 355 embryonic-development related-proteins. The important reproduction-relevant pathways found in KEGG analysis were GnRH-signaling, oocyte-meiosis, steroid-biosynthesis, steroid-hormone biosynthesis, progesterone-mediated oocyte-maturation, retinol-metabolism, neuroactive-ligand-receptor interaction, neurotrophin-signaling and photo-transduction. Twenty nine simple sequence repeat containing sequences were also found out of which 12 repeat loci were polymorphic with mean expected-&-observed heterozygosity of 0.471 and 0.983 respectively. Quantitative RT-PCR analyses of 13-known and 6-unknown transcripts revealed differences in expression level between preparatory and post-spawning phase. These transcriptomic sequences have significantly increased the genetic-&-genomic resources for reproduction-research in Labeo rohita.