JOURNAL OF WILDLIFE RESEARCH Journal homepage: www.jakraya.com/journal/jwr ORIGINAL ARTICLE Assessment of Mahseer Population Structure and Genetic Variation using ATPase6 Sudha Summarwar¹*, K.P. Yadav ² and S.P. Tailor³ 1Reseach Associate, Department of Zoology, 2Vice Chancellor, 3Dean, School of Agriculture Science, Sangam University, Bhilwara, Rajasthan 311001, India, Abstract This study depicts research on molecular characterization of 66 Mahseer ( tor sp.) collected from 8 waterbodies situated in Southern Rajasthan based on ATPase 6 mitochondrial genes with the help of DNA barcoding technique. Study areas included Rivers Chambal, Kali Sindh and Parwan, Dams Rana Pratap Sagar, Bassi, Daya, Lake Badi, and Madar Tank. Non-invasive techniques were used to collect Fin tissues. Total DNA extracted (isolation, amplification, and sequence analysis of DNA) were isolated from the finclip tissue of collected fishes. ATPase 6 gene of Fish F1 and Fish R1 was amplified using a balanced solution as per guidelines. The raw DNA sequences were edited using Bio Edit sequence alignment. *Corresponding Authors: BLASTN program was used for comparing the sequences from the two Dr. Sudha Summarwar chromatograms, the fragment showing 100% alignment with no gap or Email: [email protected] insertion/deletions were selected. A total of 66 ATPase sequences for Tor tor generated were submitted to the NCBI Gen Bank. The Pairwise Received: 07/11/2020 nucleotide composition was measured. The respective values of T, C, A, Accepted: 29/11/2020 and G were 27.45, 28.20, 32.56, and 12.65 %. The pairwise FST comparison of the sample using the ATPase 6 gene sequence showed significant genetic differences among the population. The nucleotide diversity index was 0.003149% which is considered low suggesting that effective plans should be made to avert the extinction of the population. Hierarchical AMOVA was performed to test the significance of the partitioning of genetic variances resulting from different groupings of the populations into geographical groups. The FST results showed no sharing of genetic material between the fish population collected from Daya Dam with others except Madar Tank and likewise between fishes of Madar Tank and the others except Daya Dam. It can be concluded that the fish population of the Madar Tank and Daya Dam were genetically similar, more studies are needed to confirm the same. The Dendrogram showed a big difference between the populations collected from the Daya Dam and Madar tank as one unit from the other sites. Only 9 fishes out of 66 fishes from two sites shoed close association, rest other 57 fishes from six sites exhibited similarities for mitochondrial DNA. ATPase 6 gene sequences were analysed for species identification and phylogenetic relationship. Keywords : ATPase 6, Mahseer, Tor to r , DNA sequence, Dendogram. 1. Introduction molecular characterization of Mahseer ( tor sp.) from India has a rich natural heritage and nurtures a southern Rajasthan based on ATPase 6 mitochondrial unique bio-diversity, placing it among the 12 most genes. The molecular genetic diversity of Mahseer from biodiverse countries. Of the 31,100 fish species the natural reservoir of Rajasthan was studied with the currently in nature, 2,438 are known from the Indian help of the DNA barcoding technique. subcontinent (Froese and Pauly, 2018). According to Leveque et al. (2008), the know-how of the fish fauna 2. Materials and Methods of tropical Asia remains in its exploratory phase, The southern part of Rajasthan is illustrious for especially in India. It is important to understand that its water bodies. Various samples of Mahseer were despite the imperative role that biodiversity plays, all collected from eight sites of water bodies situated in species that together make biodiversity, have been Southern Rajasthan along with water divisions. Study largely encountering vast perils owing to several areas included River Chambal, Rana Pratap Sagar Dam, anthropogenic factors (Jena and Gopalakrishnan, 2012). Bassi Dam, River Kali Sindh, River Parwan, Badi The present study attempted to furnish research on Journal of Wildlife Research | October-December, 2020 | Volume 08 | Issue 04 | Pages 74-80 © 2020 Jakraya Summarwar et al…Assessment of Mahseer population structure and Genetic variation using ATPase6 Lake, Daya Dam, and Madar Tank from Rajasthan, mean amount of nucleotide T was highest in Bari Lake India. and lowest in River Kali Sindh. Similarly, Nucleotide Fin tissues were collected using non-invasive G was having a high presence in Madar Tank and techniques (Wasko et al., 2003) followed by proper lowest in Bassi Dam. Nucleotide C shows the highest antiseptic treatment of organisms before they were value in Bari Lake and lowest in Bassi Dam and reverted to their habitat. All the fishes were handled as Nucleotide A was highest in River Parvan and lowest in per the approved guidelines of Agricultural Laboratory, Madar Tank. Aligned sequences of ATPase 6 gene Udaipur, Rajasthan, India. Total DNA extracted (DNA sequences in the present study identified 2 haplotypes isolation, amplification, and sequence analysis of DNA) (Table 3). Haplotypes were revealed in all eight of Mahseer were isolated from the fin clip tissue of populations. The frequency for Haplotype_ 1 was 57 randomly collected fishes. The sample was stored in and Haplotype_2 was 9. Genetic differentiation 90% ethanol and DNA was isolated using the Phenol between the Tor tor populations was assessed using Chloroform method. FST pairwise comparison (Table 4). The pairwise FST ATPase 6 gene was amplified using Fish F1 and comparison of the population sample using the ATPase Fish R1. To amplify 50 ng template DNA a reaction 6 gene sequence showed genetic differences mixture of 25 μl was prepared to contain 1X PCR significantly among the population. buffer, 2 mM MgCl2, 10 picomoles of each primer, In the present study nucleotide diversity index 0.25 mM of dNTP mix, and 0.25 U Taq polymerase. was 0.003149%. It can be considered low and The cycle parameters consisted of initial suggested that plans should be made effectively to denaturation (94ºC, 180seconds) followed by 35 cycles conserve the population so that extinction can be of denaturation (94ºC, 30 seconds), annealing (54ºC, 30 averted efficiently. Further research on the genetic seconds), and extension (72ºC, 60seconds). The PCR diversity of species and populations at remote locations product was directly sequenced with the Big Dye is needed to measure if there are differences in Terminator v3.1 Cycle Sequencing kit using only Fish geographic patterns of genetic diversity between F1 primer. An amplified product was purified with the members of the population. Determining patterns of sodium acetate purification method. Lyophilized PCR genetic diversity is important because if geographically product was sequenced with a 310 automated genetic remote populations have high genetic diversity than this analyzer (Applied Biosystems). should increase their evolutionary potential to adapt to The raw DNA sequences were updated using the the increasing impacts. Bio Edit sequence alignment editor version 7.0.5.2. Hierarchical AMOVA (Table 5) was performed BLASTN program was used to compare the sequences to test the significance of the partitioning of genetic retrieved from the two chromatograms, and the variances resulting from different groupings of the fragment showing 100% alignment with no gap or indel populations into geographical groups. A Further (insertion/deletions) were selected. The selected conducted AMOVA test also found it among fragments of the sequence aligned using ClustalX population difference (% of the variation was 20) software. Finally, each of the sequences was compared within the population it was 120. A hierarchical in NCBI through BLASTN to examine the complete analysis of molecular variance (AMOVA) was alignment with the partial coding sequence of the fish performed to test for genetic differentiation between mitochondrial COI gene. The sequences were translated regions. The total nucleotide diversity was 0.0031. It using the online software ORF finder can be inferred based on FST results that there was no (http://www.ncbi.nlm.nih.gov/gorf/ gorf.html) and sharing of genetic material between the fish population aligned through BLASTP. In this way, the generated collected from Daya Dam with others except Madar sequences were confirmed to be the fragments of the Tank and likewise between fishes of Madar Tank and mitochondrial COI gene. All sequences from claimed the rest other population except Daya Dam. Further, it specimens were submitted to the GenBank database can be clarified that the fish population of the Madar with accession numbers (AN). Further, the Tank and Daya Dam were found genetically similar, identification of the specimen was mainly done using a though more studies are needed to confirm the same. A DNA barcode sequence using a similarity match in the decrease in population size can have a major impact on BOLD species identification system (BOLD-IDS, genetic variation due to the relationship between www.barcodinglife.org). genetic drift and the size of the population. Phylogenetic analysis of the mitochondrial 3. Results and Discussion sequences using the Neighbour-Joining Tree (NJ) A total of 66 fishes were collected from the 8 method strongly supported the reciprocally sites and a total of 66 ATPase sequences for Tor tor monophyletic status between Tor tor populations. The were generated. All sequences representing the ATPase Neighbour-joining tree of Mahseer using ATPase 6 6 gene were submitted to the NCBI Gen Bank (Table gene data showed that the sequences were clustered 1). The Pairwise nucleotide composition was measured into two major groups. Phylogenetic analysis of (Table 2). The respective values of T, C, A, & G were ATPase 6 using the UPGMA method strongly 27.45, 28.20, 32.56, and 12.65 percent. In general, the supported the monophyletic status between Tor tor .– Journal of Wildlife Research | October-December, 2020 | Volume 08 | Issue 04 | Pages 74-80 © 2020 Jakraya 75 Summarwar et al…Assessment of Mahseer population structure and Genetic variation using ATPase6 Table 1: Accession No.