Hybridization in Carps and Early Detection of Carp Hybrids Using PCR-Based Kit

Hybridization is the mating of genetically differentiated individuals or groups and may involve crosses within a species or between separate species. Hybridization can be natural or human-mediated. Reproductive barriers prevent excessive introgression in the former, whereas more often introgression and genetic pollution happen in the latter. Hybridization is more widespread among members of Cyprinidae than any other groups of freshwater fishes. In many carp hatcheries in India, breeders of catla (Catla catla) and rohu (Labeo rohita) are kept in the same tank for breeding, resulting in production of hybrid seeds. Fish hybrids can pose a serious threat to the aquatic environment biodiversity. Consequently, genetic monitoring of organisms is entailed to unambiguously identify parental species and their hybrids. Adopting a multiplex PCR using β-actin gene primers, a kit has been developed to distinguish between the hybrids from their parental species. Agarose electrophoresis revealed one band of about 100 bp in size specific for rohu, another at 300 bp specific for catla, and both bands in hybrid. The kit was tested successfully with the samples collected from many hatcheries located in four Indian states. The rohu-catla early hybrid identification PCR kit could serve as a stepping stone for carp seed certification and hatchery accreditation

Identification and Characterization of Differentially Expressed Transcripts in the Gills of Freshwater Prawn (Macrobrachium rosenbergii) under Salt Stress

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important species. It is a euryhaline shrimp, surviving in wide-range salinity conditions. A change in gene expression has been suggested as an important component for stress management. To better understand the osmoregulatory mechanisms mediated by the gill, a subtractive and suppressive hybridization (SSH) tool was used to identify expressed transcripts linked to adaptations in saline water. A total of 117 transcripts represented potentially expressed under salinity conditions. BLAST analysis identified 22% as known genes, 9% as uncharacterized showing homologous to unannotated ESTs, and 69% as unknown sequences. All the identified known genes representing broad spectrum of biological pathways were particularly linked to stress tolerance including salinity tolerance. Expression analysis of 10 known genes and 7 unknown/uncharacterized genes suggested their upregulation in the gills of prawn exposed to saline water as compared to control indicating that these are likely to be associated with salinity acclimation. Rapid amplification of cDNA ends (RACE) was used for obtaining full-length cDNA of MRSW-40 clone that was highly upregulated during salt exposure. The sequenced ESTs presented here will have potential implications for future understanding about salinity acclimation and/or tolerance of the prawn

Elucidation of Novel Structural Scaffold in Rohu TLR2 and Its Binding Site Analysis with Peptidoglycan, Lipoteichoic Acid and Zymosan Ligands, and Downstream MyD88 Adaptor Protein

Toll-like receptors (TLRs) play key roles in sensing wide array of microbial signatures and induction of innate immunity. TLR2 in fish resembles higher eukaryotes by sensing peptidoglycan (PGN) and lipoteichoic acid (LTA) of bacterial cell wall and zymosan of yeasts. However, in fish TLR2, no study yet describes the ligand binding motifs in the leucine rich repeat regions (LRRs) of the extracellular domain (ECD) and important amino acids in TLR2-TIR (toll/interleukin-1 receptor) domain that could be engaged in transmitting downstream signaling. We predicted these in a commercially important freshwater fish species rohu (Labeo rohita) by constructing 3D models of TLR2-ECD, TLR2-TIR, and MyD88-TIR by comparative modeling followed by 40 ns (nanosecond) molecular dynamics simulation (MDS) for TLR2-ECD and 20 ns MDS for TLR2-TIR and MyD88-TIR. Protein (TLR2-ECD)–ligands (PGN, LTA, and zymosan) docking in rohu by AutoDock4.0, FlexX2.1, and GOLD4.1 anticipated LRR16–19, LRR12–14, and LRR20-CT as the most important ligand binding motifs. Protein (TLR2-TIR)—protein (MyD88-TIR) interaction by HADDOCK and ZDOCK predicted BB loop, αB-helix, αC-helix, and CD loop in TLR2-TIR and BB loop, αB-helix, and CD loop in MyD88-TIR as the critical binding domains. This study provides ligands recognition and downstream signaling

Identification of Deleterious Mutations in Myostatin Gene of Rohu Carp (Labeo rohita) Using Modeling and Molecular Dynamic Simulation Approaches

The myostatin (MSTN) is a known negative growth regulator of skeletal muscle. The mutated myostatin showed a double-muscular phenotype having a positive significance for the farmed animals. Consequently, adequate information is not available in the teleosts, including farmed rohu carp, Labeo rohita. In the absence of experimental evidence, computational algorithms were utilized in predicting the impact of point mutation of rohu myostatin, especially its structural and functional relationships. The four mutations were generated at different positions (p.D76A, p.Q204P, p.C312Y, and p.D313A) of MSTN protein of rohu. The impacts of each mutant were analyzed using SIFT, I-Mutant 2.0, PANTHER, and PROVEAN, wherein two substitutions (p.D76A and p.Q204P) were predicted as deleterious. The comparative structural analysis of each mutant protein with the native was explored using 3D modeling as well as molecular-dynamic simulation techniques. The simulation showed altered dynamic behaviors concerning RMSD and RMSF, for either p.D76A or p.Q204P substitution, when compared with the native counterpart. Interestingly, incorporated two mutations imposed a significant negative impact on protein structure and stability. The present study provided the first-hand information in identifying possible amino acids, where mutations could be incorporated into MSTN gene of rohu carp including other carps for undertaking further in vivo studies

Statistics of microsatellite search in <i>Labeo rohita</i>.

<p>Statistics of microsatellite search in <i>Labeo rohita</i>.</p

QC report for fastQ files.

<p>¹* >70% of bases in a read with >20 phred score</p><p>QC report for fastQ files.</p

Distribution of reproduction-relevant transcripts identified in <i>Labeo rohita</i>.

<p>A total of 940 reproduction relevant genes were distributed among 184 reproduction related proteins (GO: 0000003), 223 related to hormone activity (GO: 0005179) and receptor binding related proteins (GO: 0005102), 178 receptor activity related proteins (GO: 0004872) and 355 embryonic development related proteins (GO: 0009792).</p

Distribution of rohu transcripts and exons on zebra fish chromosome.

<p>rohu transcripts found distributed in all 25 chromosomes of zebrafish.</p

Microsatellite Locus, repeat type motif and PCR product size, amplification temp, Number of alleles, observed heterozygosity (HO), expected Heterozygosity (HE) and polymorphic in content (PIC) of 12 rohu microsatellite loci.

<p>***—Highly significant (p≤0.001)</p><p>** significant difference (p≤0.05)</p><p>* different (p≤0.1)</p><p>^NE- Not Estimated</p><p>Microsatellite Locus, repeat type motif and PCR product size, amplification temp, Number of alleles, observed heterozygosity (HO), expected Heterozygosity (HE) and polymorphic in content (PIC) of 12 rohu microsatellite loci.</p