Development of novel SSR markers and validation by assessing the genetic diversity of endangered Deccan mahseer, Tor khudree.

BACKGROUND: The Deccan mahseer, Tor khudree (Sykes, 1839) is a potential game and food fish species belonging to the family cyprinidae and is categorized as endangered. Its distribution is restricted to southern part of India, specifically to Peninsular Rivers. This study is first to assess the genetic diversity and differentiation in Tor khudree by developing novel simple sequence repeat (SSR) markers. METHODS AND RESULTS: Low depth next generation sequencing followed by sequence analysis in MISA software identified 187,649 SSRs. The novel fourteen validated SSR loci were used for population genetic analysis. All of the SSR loci were highly informative with mean PIC > 0.5. High mean allelic richness (9.29) observed heterozygosity (0.98) and expected heterozygosity (0.79) were observed across the loci. However, genetic differentiation was low but significant (0.052). Negative FIS values were observed in both locus-wise and populations indicating the presence of high heterozygosity. Intrapopulation variation was found to be high (96.29%). The population structure revealed two genetic stocks. CONCLUSIONS: The results from the present study including the highly polymorphic markers developed would be a useful resource for further research on population genetics and conservation genetics of the species.

Morphometric and genetic characterization of cultured and wild populations of tilapia, Oreochromis niloticus in India.

To study genetic variation in Indian populations of tilapia, Oreochromis niloticus, both truss morphometrics and genetic characterization have been performed. In the present study, 88 individuals from two farm populations (GIFT and West Bengal) and one reservoir population (Gujarat) were selected to analyse variations at ten morphometric landmarks and eight microsatellite loci. Truss morphometric analysis showed PCI, PCII, and PCIII expressing 29.1%, 21.36%, and 15.48% of the variance, respectively. Results showed no clear shift in shape between the studied populations of O. niloticus, indicating low morphological variability among them. The number of microsatellite alleles ranged from 3 to 9, while expected heterozygosity (HE) and observed heterozygosity (HO) values ranged from 0.56 (WB) to 0.68 (Guj) and 0.59 (GIFT) to 0.72 (Guj), respectively. The Gujarat and West Bengal populations had the smallest pairwise distance (0.0123) between them, indicating that they were genetically closer. Individuals from GIFT, however, showed the largest distance from the other populations. DNA marker variations revealed the highest genetic variability in the Gujarat population and the lowest variability in the GIFT population. The results of this study will help establish a base population for genetic improvement program and conservation of wild populations.

High Genetic Differentiation and Genetic Diversity in Endangered Mahseer Tor khudree (Sykes, 1839) as Revealed from Concatenated ATPase 6/8 and Cyt b Mitochondrial Genes.

The Deccan mahseer, Tor khudree (Sykes 1839), belonging to family Cyprinidae is an important food and a game fish distributed in peninsular India. Due to overfishing and habitat destruction, the species is declared endangered and placed on the IUCN red list. Therefore, a well-designed conservation program may be essential to get this species protected in its natural habitat. We used a total of 152 samples from four rivers of peninsular India to assess the genetic diversity and structure of the mahseer using concatenated sequences of two mitochondrial genes, ATPase 6/8 (790 bp) and Cyt b (1000 bp). High haplotypic diversity was seen with 44 haplotypes. Individual gene wise haplotypes included 10 and 21 haplotypes for ATPase6/8 and Cyt b, respectively. AMOVA revealed most of the genetic variations (71.02%) to be within the populations. Significant genetic differentiation was observed between all population pairs, with FST values ranging from 0.121 to 0.372, with minimum between Tunga and Tungabhadra population and maximum between Tunga and Periyar population. Haplotype network showed one ancestral haplotype (TKACH04). Significant negative Fu's F and unimodal mismatch distribution suggested recent demographic expansion. The results of the present study would serve as a useful resource for further research on population genetics and conservation programs of the species.

Genetic diversity and genome-scale population structure of wild Indian major carp, Labeo catla (Hamilton, 1822), revealed by genotyping-by-sequencing.

Labeo catla (catla) is the second most commercially important and widely cultured Indian major carp (IMC). It is indigenous to the Indo-Gangetic riverine system of India and the rivers of Bangladesh, Nepal, Myanmar, and Pakistan. Despite the availability of substantial genomic resources in this important species, detailed information on the genome-scale population structure using SNP markers is yet to be reported. In the present study, the identification of genome-wide single nucleotide polymorphisms (SNPs) and population genomics of catla was undertaken by re-sequencing six catla populations of riverine origin from distinct geographical regions. DNA isolated from 100 samples was used to perform genotyping-by-sequencing (GBS). A published catla genome with 95% genome coverage was used as the reference for mapping reads using BWA software. From a total of 472 million paired-end (150 × 2 bp) raw reads generated in this study, we identified 10,485 high-quality polymorphic SNPs using the STACKS pipeline. Expected heterozygosity (He) across the populations ranged from 0.162 to 0.20, whereas observed heterozygosity (Ho) ranged between 0.053 and 0.06. The nucleotide diversity (π) was the lowest (0.168) in the Ganga population. The within-population variation was found to be higher (95.32%) than the among-population (4.68%) variation. However, genetic differentiation was observed to be low to moderate, with Fst values ranging from 0.020 to 0.084, and the highest between Brahmani and Krishna populations. Bayesian and multivariate techniques were used to further evaluate the population structure and supposed ancestry in the studied populations using the structure and discriminant analysis of principal components (DAPC), respectively. Both analyses revealed the existence of two separate genomic clusters. The maximum number of private alleles was observed in the Ganga population. The findings of this study will contribute to a deeper understanding of the population structure and genetic diversity of wild populations of catla for future research in fish population genomics.

Genome size estimation and its associations with body length, chromosome number and evolution in teleost fishes.

Precise estimation of genome size (GS) is vital for various genomic studies, such as deciding genome sequencing depth, genome assembly, biodiversity documentation, evolution, genetic disorders studies, duplication events etc. Animal Genome Size Database provides GS of over 2050 fish species, which ranges from 0.35 pg in pufferfish (Tetraodon nigroviridis) to 132.83 pg in marbled lungfish (Protopterus aethiopicus). The GS of majority of the fishes inhabiting waters of Indian subcontinent are still missing. In present study, we estimated GS of 51 freshwater teleost (31 commercially important, 7 vulnerable and 13 ornamental species) that ranged from 0.58 pg in banded gourami (Trichogaster fasciata) to 1.92 pg in scribbled goby (Awaous grammepomus). Substantial variation in GS was observed within the same fish orders (0.64-1.45 pg in cypriniformes, 0.70-1.41 pg in siluriformes and 0.58-1.92 pg in perciformes). We examined the relationship between the GS, chromosome number and body length across all the fishes. Body length was found to be associated with GS, whereas no relationship was noticed between the GS and the chromosome number. The analysis using ancestral information revealed haploid chromosome number 25, 27 and 24 for the most recent common ancestor of cypriniformes, siluriformes and perciformes, respectively. The study led to generation of new records on GS of 43 fish species and revalidated records for 8 species. The finding is valuable resource for further research in the areas of fish genomics, molecular ecology and evolutionary conservation genetics.

Population genetic structure of fringe-lipped carp, Labeo fimbriatus from the peninsular rivers of India.

Labeo fimbriatus is a medium carp species found throughout India's peninsular river basins and is regarded as a valuable aquaculture resource alongside Indian major carps due to its taste and nutritional value. This species has recently declined dramatically due to habitat degradation and overfishing. Because of its enormous economic importance, a selective breeding programme is likely to be in place to improve performance traits. Knowledge of genetic variation among the base population from which the broodstock will be selected is an important step in this process. A diverse genetic base of broodstock is required to achieve the best response to selection for long-term aquaculture management practices. Consequently, using mitochondrial DNA (ATPase 6 and Control region) and microsatellite markers, we have made the first step toward estimating the level of genetic variation and how it is distributed among the four populations of L. fimbriatus found in peninsular rivers in India. The ATPase 6 gene analysis in four populations revealed 15 haplotypes and 51 variable sites, in contrast to the Control region, which had 60 haplotypes together with 73 variable sites and a haplotype diversity of 0.941. Twelve microsatellite loci displayed estimated allele numbers (N A) ranging from 3 to 19, observed heterozygosity (H O), and expected heterozygosity (H E), respectively, of 0.705 to 0.753 and 0.657 to 0.914. Each marker type showed a significant F ST value, indicating the presence of low to moderate genetic differentiation across entire wild populations. The Godavari, Kaveri, and Mahanadi populations formed one cluster according to the UPGMA, which was based on genetic distance matrix, while the Krishna population formed a separate cluster. The comparative genetic analysis of data from different markers utilized in the current study would enable the identification of the genetic stocks of L. fimbriatus and facilitate conservation measures and selective breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03369-y.

Revelation of candidate genes and molecular mechanism of reproductive seasonality in female rohu (Labeo rohita Ham.) by RNA sequencing.

BACKGROUND: Carp fish, rohu (Labeo rohita Ham.) is important freshwater aquaculture species of South-East Asia having seasonal reproductive rhythm. There is no holistic study at transcriptome level revealing key candidate genes involved in such circannual rhythm regulated by biological clock genes (BCGs). Seasonality manifestation has two contrasting phases of reproduction, i.e., post-spawning resting and initiation of gonadal activity appropriate for revealing the associated candidate genes. It can be deciphered by RNA sequencing of tissues involved in BPGL (Brain-Pituitary-Gonad-Liver) axis controlling seasonality. How far such BCGs of this fish are evolutionarily conserved across different phyla is unknown. Such study can be of further use to enhance fish productivity as seasonality restricts seed production beyond monsoon season. RESULT: A total of ~ 150 Gb of transcriptomic data of four tissues viz., BPGL were generated using Illumina TruSeq. De-novo assembled BPGL tissues revealed 75,554 differentially expressed transcripts, 115,534 SSRs, 65,584 SNPs, 514 pathways, 5379 transcription factors, 187 mature miRNA which regulates candidate genes represented by 1576 differentially expressed transcripts are available in the form of web-genomic resources. Findings were validated by qPCR. This is the first report in carp fish having 32 BCGs, found widely conserved in fish, amphibian, reptile, birds, prototheria, marsupials and placental mammals. This is due to universal mechanism of rhythmicity in response to environment and earth rotation having adaptive and reproductive significance. CONCLUSION: This study elucidates evolutionary conserved mechanism of photo-periodism sensing, neuroendocrine secretion, metabolism and yolk synthesis in liver, gonadal maturation, muscular growth with sensory and auditory perception in this fish. Study reveals fish as a good model for research on biological clock besides its relevance in reproductive efficiency enhancement.

Activin receptor type IIB in rohu (Labeo rohita): molecular characterization, tissue distribution and immunohistochemical localization during different stages of gonadal maturation.

Activin receptor type IIB (ActRIIB) is a transmembrane serine/threonine kinase receptor which plays a pivotal role in regulating the reproduction in vertebrates including teleost. Earlier studies have documented its importance in governing gonadal maturation in higher vertebrates. However, reports on the regulation of fish reproductive system by ActRIIB gene are still limited. Here, we report the identification and characterization of ActRIIB cDNA of Labeo rohita, a commercially important fish species of the Indian subcontinent. The full-length gene encoding rohu ActRIIB was cloned and found to be of 1674 bp in length. Functional similarities were evident from evolutionary analysis across vertebrates. Real-time PCR to measure the expression of ActRIIB transcript in rohu revealed significant mRNA levels in gonads followed by non-reproductive tissues, including the brain, pituitary and muscle. With respect to different gonadal maturation stages, predominant expression of ActRIIB mRNA was observed during the pre-spawning phase of both sexes. To further delineate its role in rohu reproduction, a recombinant protein of the extracellular domain of ActRIIB (rECD-ActRIIB) was produced, and polyclonal antibody is raised against the protein for its immuno-localization studies during different gonadal maturation stages. Strong immunoreactivity was noticed in the pre-vitellogenic oocytes which decreased dramatically in the fully mature oocytes. Similarly, the strong and intense immunoreactivity was found in the spermatids and spermatocytes of the immature testis, and eventually the intensity reduced with the progression of the maturation stage. These results provide the first evidence of the presence of ActRIIB in rohu gonadal tissues. Taken together, our observations lay the groundwork for further understanding and investigating on the potential role of ActRIIB in fish reproduction system in the event of gonadal maturation.

Complete mitochondrial genome of nearly threatened freshwater ornamental fish, Microphis deocata and its phylogenetic relationship within Syngnathidae.

Microphis deocata (deocata pipefish), belonging to family Syngnathidae, is one of the important indigenous ornamental fish species listed as near threatened in the IUCN red list. Here, we first report the complete mitochondrial genome of deocata pipefish using Illumina next-generation sequencing platform. The total length of the mitogenome is 16,526 bp. It encompasses 13 protein coding genes, 2 ribosomal rRNAs, and 22 tRNAs. The WANCY region (a cluster of five tRNA genes) contains the 50 bp OL light strand origin of replication. Phylogenetic analysis of Syngnathidae revealed M. deocata to cluster with Oostethus manadensis, forming a sister group with Doryrhamphus japonicas and Dunckerocampus dactyliophorus. The mitochondrial genome sequence data generated in the present study will play an important role in population genetic analysis and developing conservation strategies for this species.

The complete mitochondrial genome of Labeo catla (Hamilton, 1822) using long read sequencing.

Labeo catla is a widely cultured species in monoculture and polyculture systems of the Indian subcontinent. In this study, the complete mitochondrial genome sequence of catla was reconstructed from Oxford Nanopore sequence data. The mitochondrial genome is 16,600 bp in length (accession no. is MN830943) which is larger than the previously reported catla mitogenomes. Like other vertebrate mitochondrial genomes, it has 13 protein-coding genes, 22 tRNAs, 2 rRNAs and a putative control region. Most of the mitogenes are encoded on H-strand. Phylogenetic analysis showed that Labeo catla is more closely related to Labeo rohita than other labeo species. The catla mtgenome reported here will facilitate population genetics, phylogenetics and molecular taxonomy of Indian major carps.

The genome of walking catfish Clarias magur (Hamilton, 1822) unveils the genetic basis that may have facilitated the development of environmental and terrestrial adaptation systems in air-breathing catfishes.

The walking catfish Clarias magur (Hamilton, 1822) (magur) is an important catfish species inhabiting the Indian subcontinent. It is considered as a highly nutritious food fish and has the capability to walk to some distance, and survive a considerable period without water. Assembly, scaffolding and several rounds of iterations resulted in 3,484 scaffolds covering ∼94% of estimated genome with 9.88 Mb largest scaffold, and N50 1.31 Mb. The genome possessed 23,748 predicted protein encoding genes with annotation of 19,279 orthologous genes. A total of 166 orthologous groups represented by 222 genes were found to be unique for this species. The Computational Analysis of gene Family Evolution (CAFE) analysis revealed expansion of 207 gene families and 100 gene families have rapidly evolved. Genes specific to important environmental and terrestrial adaptation, viz. urea cycle, vision, locomotion, olfactory and vomeronasal receptors, immune system, anti-microbial properties, mucus, thermoregulation, osmoregulation, air-breathing, detoxification, etc. were identified and critically analysed. The analysis clearly indicated that C. magur genome possessed several unique and duplicate genes similar to that of terrestrial or amphibians' counterparts in comparison to other teleostean species. The genome information will be useful in conservation genetics, not only for this species but will also be very helpful in such studies in other catfishes.

The draft genome of Labeo catla.

OBJECTIVE: Labeo catla (catla), one of the three Indian major carps, is native to the Indo-Gangetic riverine system of India as well as the rivers of Pakistan, Bangladesh, Nepal and Myanmar. Its higher growth rate and compatibility with other major carps, specific surface feeding habit, and consumer preference have increased its popularity in carp polyculture systems among the fish farmers in Indian subcontinent. Recent advancement in sequencing technology coupled with massive parallel sequencing platforms has facilitated accelerated genetic improvement in aquaculture species through integration of genomics tools. A draft genome and allied resources are lacking in catla. Therefore, in the present study, we have performed de-novo assembly of Labeo catla for the first time. DATA DESCRIPTION: A male farm reared catla was used for extracting high molecular weight genomic DNA followed by sequencing in Oxford Nanopore and Illumina platforms. Approximately, 80× coverage of sequence data was assembled adopting the hybrid assembly strategy. The assembled genome size of catla was 1.01 Gb containing 5345 scaffolds with N50 value 0.7 Mb and more than 92% BUSCO completeness. Gene annotation resulted in 25,812 predicted genes.

Establishment of base population for selective breeding of catla (Catla catla) depending on phenotypic and microsatellite marker information.

The phenotypic and microsatellite marker information of nine strains of catla (Catla catla) for growth trait was used to infer relationship within and between strains. This information helped in optimizing the proportion of individuals to be used from each strain while creating a base population for selective breeding. For this purpose, nine strains were collected from different sources and places of India namely West Bengal, Bihar, Odisha, Andhra Pradesh and Uttar Pradesh. Two riverine sources i.e. Ganga and Subarnarekha were also represented among the nine strains collected for base population. They were brought to Indian Council of Agricultural Research-Central Institute of Freshwater Aquaculture (ICAR-CIFA) at fry stage and reared separately till fingerlings. After passive integrated transponder tagging fingerlings were stocked in three communal ponds for one year culture. Live body weights were then measured and least square means were obtained after pond effect correction. A wide range of variation was observed among and between strains. Microsatellite markers were used to estimate genetic differences of different strains of catla using pair wise F ST estimates. Overall multi locus F ST, including all loci was estimated to be 0.4137 (P < 0.05), indicating genetic heterogeneity among them. Analysis of molecular variance revealed that, 58.63% of variation was due to within individual variation, 3.45% of variation was due to among individuals within strain and 37.92% was due to among strain variations. Both phenotypic as well as microsatellite data will be used to form a base population of catla with individuals from the stock having broad genetic variation for selective breeding programme.

Genetic diversity and population structure of Cirrhinus mrigala revealed by mitochondrial ATPase 6 gene.

The genetic diversity and population structure of Cirrhinus mrigala were studied using partial mitochondrial ATPase 6 gene. 155 individuals were collected from six distant rivers of peninsular India. A total of 11 haplotypes were found with haplotype diversity value of 0.46711 and nucleotide diversity 0.00154 indicating a low degree of genetic divergence among populations. The result of AMOVA analysis also revealed that 97.53% of the genetic variation contained within populations and 2.47% occurred among populations. An overall fixation index was found to be 0.02466 (p < .05). Pairwise Fst analysis showed significant genetic differentiation between the Kaveri and rest of the populations. The minimum spanning tree of the haplotype data demonstrated a common ancestral origin of the mrigal populations in peninsular rivers. From the overall results of the present study it may be inferred that mrigal population in peninsular India have low genetic differentiation. This information would be very essential for setting up of genetic improvement programmes, effective management, and conservation of natural genetic resources of mrigal.

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-ß (TGF-ß) 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.

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

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.

Genetic variation in Labeo fimbriatus (Cypriniformes: Cyprinidae) populations as revealed by partial cytochrome b sequences of mitochondrial DNA.

Labeo fimbriatus, a medium sized carp is assessed as a commercially important aquaculture species in Indian subcontinent. In the present study, the genetic diversity and population structure of four Indian riverine populations of L. fimbriatus have been evaluated using partial cytochrome b sequences of mitochondrial DNA. Sequencing and analysis of this gene from 108 individuals defined 7 distinct haplotypes. Haplotype diversity (Hd) and nucleotide diversity (π) ranged from 0.067 to 0.405 and 0.00023 to 0.03231, respectively. The Mahanadi population had the highest π level. Analysis of molecular variance (AMOVA) indicated that 47.36% of genetic variation contained within population and 53.76% of genetic variation among groups. Pairwise FST analysis indicated that there was little or no genetic differentiation among populations (-0.0018 to 04572) from different geographical regions except Mahanadi population. The Mahanadi population can be considered as a separate stock from rest three riverine populations. Accordingly, the genetic information generated from this study can be implemented while taking decision in formulating base population for the sustainable selective breeding programs of this species.

Complete mitochondrial genome sequence of E. suratensis revealed by next generation sequencing.

The complete mitochondrial genome of Etroplus suratensis, the Green chromide cichlid, was determined for the first time through NGS method. The genome is 16,467 bp (Accession no. KU301747) 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 plus strand was A: 28.3%, G: 15.2%, C: 30.9%, T: 25.6%, and the A + T content 53.9%. The control region contains 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 cichlid fish of India.

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

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.