Low depth sequencing reveals the critically endangered Batagur kachuga (Red-crowned roofed turtle) mitochondrial genome and its evolutionary implications.

The Batagur kachuga (B. kachuga), commonly known as the Red-crowned roofed turtle, is a critically endangered species native to India and its neighboring countries like Bangladesh, and Nepal. The present study is the first report of the complete mitochondrial genome of B. kachuga (16,517 bp) construed via the next-generation sequencing (NGS) approach from eggshell DNA. There are 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), 13 protein-coding genes (PCGs), and one putative control region (CR/D-loop) in the mitogenome. The CR region from the current study reveals conserved TAS, CD, and CSB domains and two AT-rich tandem repeat regions. Most genes are encoded in the heavy strand except the NADH dehydrogenase subunit 6 (ND6) gene and seven tRNA genes. Most PCGs start with the initiation codon ATG, except the COI (Cytochrome Oxidase Subunit-I) gene, which starts with the GTG codon. The present investigation also predicts the distinctive cloverleaf structures of tRNAs except for tRNA-Ser1 and tRNA-Ser-2, which lack a DHU arm. The comparative analysis of Ka/Ks with other 33 species from Order Testudines, in relation to B. kachuga, revealed negative selection in most PCGs, indicating a process of preservation and purification that aids in eliminating undesirable or detrimental substitutes. Phylogenetic analysis of this species has been analysed using the complete mitogenome of 33 turtle species. The maximum likelihood phylogenetic tree strongly supports each family in different clades and also reveals a close relationship between the Pangashura and Batagur genera. Our study suggests the generation of genome-wide molecular data, in terms of mitogenomes, SNPs, and SSRs, is needed to improve the understanding of this species and their phylogenetics and evolutionary relationships, which will help to improve the conservation efforts of this species.

An In Vitro System Mimics the Intestinal Microbiota of Striped Beakfish (Oplegnathus fasciatus) and Inhibits Vibrio alginolyticus by Limosilactobacillus reuteri-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are functional substances secreted by microbes and host cells, and it has been discovered that they participate in the interactions between different microorganisms. Our recent findings indicate that Limosilactobacillus reuteri-derived EVs have the potential to improve the intestinal microbiota of Oplegnathus fasciatus fish and inhibit pathogenic bacteria. Previous research has reported that the host intestinal cells play a regulatory role in the intestinal microbiota. This suggested that to investigate the mechanisms through which L. reuteri-derived EVs regulate the intestinal microbiota, a system that excludes interference from host intestinal cells should be established. In this study, an in vitro cultured intestinal bacteria system, without host factors, was used to simulate the intestinal microbiota of O. fasciatus fish. After adding L. reuteri-derived EVs to the system, the changes in the microbiota were analyzed. The results showed that L. reuteri-derived EVs effectively reduced the abundance of Vibrio spp. In the results of the in vitro experiments, it was also observed that L. reuteri-derived EVs have the ability to inhibit Vibrio alginolyticus. We further sequenced the small RNA contained in L. reuteri-derived EVs and found that these small RNAs can interfere with genes (LysR, pirin, MIpA/OmpV, CatB, and aspartate-semialdehyde dehydrogenase) related to the growth of V. alginolyticus. Taken together, the results indicate that in the absence of host involvement, the small RNAs present in L. reuteri-derived EVs have the function of inhibiting pathogenic bacteria and exhibit the potential to regulate the intestinal microbiota.

Complete mitogenome sequence analysis of an Indian Black Bengal goat, Capra hircus, and its phylogenetic consequences.

Capra hircus (Black Bengal goat) is a prized goat breed reared across the Southeast Asian region. Known for its excellent chevon quality and adaptability to hot and humid climates, it supports the livelihood of millions of farmers. The present study reveals the complete mitogenome of a Black Bengal goat of Indian origin utilizing a next-generation sequencing approach (GenBank acc. no.-MZ073671). This newly assembled whole mitochondrial genome (16,637 bp) is composed of a non-coding control region (D-loop region), two ribosomal RNA (rRNA genes), 13 protein-coding genes, and 22 transfer RNA (tRNA). Overall, the current mitogenome shares 99.77% sequence homology with previously reported mitogenomes of Capra hircus. The phylogenetic analysis inferred that the current mitogenome shares a close relationship with the Teressa goat breed of the Andaman and Nicobar Islands.

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.

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.

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.

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.

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.

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.

Population divergence and structure of Cirrhinus mrigala from peninsular rivers of India, revealed by mitochondrial cytochrome b gene and truss morphometric analysis.

Genetic diversity and population structure of Cirrhinus mrigala from peninsular riverine systems of India was studied using mitochondrial DNA gene, cytochrome b and truss morphometric analysis. Analysis of 982 bp of the cytochrome b gene from 182 samples collected from six rivers revealed the presence of 28 haplotypes with overall high haplotype diversity value of 0.78981 and nucleotide diversity value of 0.00215. Analysis of molecular variance (AMOVA) showed that genetic variation is mainly harbored within populations rather than among populations. The pairwise Fst values (-0.009 to 0.084) was indicative of a low genetic structure among mrigal populations. Morphometric examination of 243 samples using 10 truss landmarks and principal component analysis showed a similar trend when compared with genetic data. Overall, low genetic and morphometric differences were observed despite those populations from different geographic locations. The results of this study would provide essential information to resource recovery and help in delineating populations for fishery management. Besides, the data will provide a valuable baseline for further investigations on the geographic distribution of this commercially important fish species.