Assessment of Mahseer Population Structure and Genetic Variation Using Atpase6

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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

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. for Mahseer (Sample Voucher and Collection Site)

S.No. Sample Voucher Collection Site Accession No. 1 CR1 Chambal River MT010726 2 CR2 Chambal River MT010727 3 CR3 Chambal River MT010728 4 CR4 Chambal River MT010729 5 CR5 Chambal River MT010730 6 CR6 Chambal River MT010731 7 CR7 Chambal River MT010732 8 CR8 Chambal River MT010733 9 CR9 Chambal River MT010734 10 CR10 Chambal River MT010735 11 CR11 Chambal River MT010736 12 RPS1 Rana Pratap Sagar MT010737 13 RPS2 Rana Pratap Sagar MT010738 14 RPS3 Rana Pratap Sagar MT010739 15 RPS4 Rana Pratap Sagar MT010740 16 RPS5 Rana Pratap Sagar MT010741 17 RPS6 Rana Pratap Sagar MT010742 18 RPS7 Rana Pratap Sagar MT010743 19 RPS8 Rana Pratap Sagar MT010744 20 RPS9 Rana Pratap Sagar MT010745 21 RPS10 Rana Pratap Sagar MT010746 22 BD1 Bassi Dam MT010747 23 BD2 Bassi Dam MT010748 24 BD3 Bassi Dam MT010749 25 BD4 Bassi Dam MT010750 26 KSR1 Kali Sindh River MT010751 27 KSR2 Kali Sindh River MT010752 28 KSR3 Kali Sindh River MT010753 29 KSR4 Kali Sindh River MT010754 30 KSR5 Kali Sindh River MT010755 31 KSR6 Kali Sindh River MT010756 32 KSR7 Kali Sindh River MT010757 33 KSR8 Kali Sindh River MT010758 34 KSR9 Kali Sindh River MT010759 35 KSR10 Kali Sindh River MT010760 36 KSR11 Kali Sindh River MT010761 37 KSR12 Kali Sindh River MT010762 38 KSR13 Kali Sindh River MT010763 39 KSR14 Kali Sindh River MT010764 40 KSR15 Kali Sindh River MT010765 41 PR1 Parvan River MT010766 42 PR2 Parvan River MT010767 43 PR3 Parvan River MT010768 44 PR4 Parvan River MT010769 45 BL1 Bari Lake MT010770 46 BL2 Bari Lake MT010771 47 BL3 Bari Lake MT010772 48 BL4 Bari Lake MT010773 49 BL5 Bari Lake MT010774 50 BL6 Bari Lake MT010775 51 BL7 Bari Lake MT010776 52 BL8 Bari Lake MT010777 53 BL9 Bari Lake MT010778 54 BL10 Bari Lake MT010779 55 BL11 Bari Lake MT010780 56 BL12 Bari Lake MT010781 57 BL13 Bari Lake MT010782 58 DD1 Daya Dam MT010783 59 DD2 Daya Dam MT010784 60 DD3 Daya Dam MT010785 61 DD4 Daya Dam MT010786 62 MT1 Madar Tank MT010787

Journal of Wildlife Research | October-December, 2020 | Volume 08 | Issue 04 | Pages 74-80 © 2020 Jakraya 76 Summarwar et al…Assessment of Mahseer population structure and Genetic variation using ATPase6

63 MT2 Madar Tank MT010788 64 MT3 Madar Tank MT010789 65 MT4 Madar Tank MT010790 66 MT5 Madar Tank MT010791

Table 2: Nucleotide composition of ATPase6 gene sequences T C A G Total CR1 27.53 27.85 32.44 12.18 460 CR2 26.95 28.39 31.56 13.11 508 CR3 26.96 28.39 31.81 12.84 520 CR4 26.94 28.91 31.41 12.75 589 CR5 27.12 28.63 31.51 12.74 558 CR6 27.11 27.84 32.07 12.97 496 CR7 27.11 27.66 32.97 12.27 438 CR8 26.83 28.05 32.72 12.40 389 CR9 26.36 28.34 32.13 13.18 503 CR10 27.63 27.41 33.11 11.84 353 CR11 27.40 27.84 31.74 13.02 484 RPS1 27.40 28.01 31.96 12.63 485 RPS2 27.44 27.60 32.73 12.23 447 RPS3 27.16 28.12 32.59 12.14 448 RPS4 27.70 27.54 32.24 12.52 454 RPS5 26.63 28.18 32.65 12.54 401 RPS6 26.27 29.14 31.05 13.54 552 RPS7 26.77 27.99 32.86 12.37 390 RPS8 27.53 27.10 33.33 12.04 357 RPS9 27.73 27.07 33.41 11.79 350 RPS10 26.61 28.23 32.86 12.30 392 BD1 27.40 26.58 33.70 12.33 207 BD2 27.46 27.62 32.54 12.38 445 BD3 27.83 27.61 33.04 11.52 344 BD4 26.71 27.04 34.20 12.05 445 KSR1 27.46 27.62 32.54 12.38 445 KSR2 27.08 28.22 31.52 13.18 511 KSR3 27.51 27.51 32.37 12.61 467 KSR4 27.58 27.73 32.05 12.63 461 KSR5 27.57 27.57 32.31 12.56 463 KSR6 27.63 27.48 32.21 12.67 466 KSR7 27.66 27.50 32.59 12.24 462 KSR8 26.71 28.57 31.68 13.04 540 KSR9 27.01 28.30 31.47 13.22 512 KSR10 26.69 28.94 31.48 12.89 563 KSR11 27.21 28.09 31.76 12.94 493 KSR12 27.11 28.35 31.40 13.14 539 KSR13 27.06 28.09 31.91 12.94 496 KSR14 27.09 28.71 31.40 12.80 584 KSR15 27.66 27.66 32.19 12.50 456 PR1 27.23 29.03 31.10 12.65 599 PR2 27.48 27.48 33.01 12.03 445 PR3 27.77 27.46 32.29 12.48 454 PR4 27.14 28.14 31.57 13.14 516 BL1 27.34 27.66 32.59 12.40 442 BL2 26.56 28.59 31.72 13.13 536 BL3 26.91 28.89 31.40 12.80 582 BL4 26.88 28.83 31.17 13.12 586 BL5 27.57 27.73 32.24 12.46 472 BL6 27.39 28.23 31.46 12.92 545 BL7 27.19 28.55 31.56 12.70 562 BL8 27.19 27.55 32.92 12.34 451 BL9 27.10 28.59 31.57 12.74 567 BL10 27.10 28.67 31.38 12.84 593 BL11 26.90 28.89 31.42 12.78 575 BL12 26.53 29.17 30.69 13.61 651 BL13 27.71 28.54 31.03 12.72 582 DD1 27.52 26.90 33.06 12.53 376

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DD2 27.52 26.90 33.06 12.53 376 DD3 27.47 28.07 30.96 13.51 548 DD4 27.29 28.02 31.27 13.42 567 MT1 27.78 27.26 32.12 12.85 464 MT2 27.53 27.84 31.26 13.37 527 MT3 27.52 27.35 31.71 13.42 484 MT4 27.52 27.35 31.71 13.42 484 MT5 27.52 27.35 31.71 13.42 484

Table 3: The haplotype codes and corresponding number of individuals

Haplotype Frequency Sequence Population Hap_1 57 1-57 CR1 CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11 RPS1 RPS2 RPS3 RPS4 RPS5 RPS6 RPS7 RPS8 RPS9 RPS10 BD1 BD2 BD3 BD4 KSR1 KSR2 KSR3 KSR4 KSR5 KSR6 KSR7 KSR8 KSR9 KSR10 KSR11 KSR12 KSR13 KSR14 KSR15 PR1 PR2 PR3 PR4 BL1 BL2 BL3 BL4 BL5 BL6 BL7 BL8 BL9 BL10 BL11 BL12 & BL13 Hap_2 9 58-66 DD1 DD2 DD3 DD4 MT1 MT2 MT3 MT4 & MT5

Table 4: Population pair wise FST Values (Distance method: pair wise difference) Population CR RPS BD KSR PR BL DD MT CR ─ ─ ─ ─ ─ ─ ─ ─ RPS 0.000 ─ ─ ─ ─ ─ ─ ─ BD 0.000 1.000 ─ ─ ─ ─ ─ ─ KSR 0.000 0.000 0.000 ─ ─ ─ ─ ─ PR 0.000 0.000 0.000 0.000 ─ ─ ─ ─ BL 0.000 0.000 0.000 0.000 0.000 ─ ─ ─ DD 1.000 1000 1.000 1.000 1.000 1.000 ─ ─ MT 1.000 1.000 1.000 1.000 1.000 1.000 0.000 ─

Table 5: AMOVA result for natural population of Tor tor Source of variation d.f. Sum of Variance Percentage of Fixation indices P values squares components variation

Among groups 7 0.0000 0.0000 0.000 FCT=0.000 1.00±0.00 Among populations 58 -0.0000 -0.08889Vb -20.00 FSC=-0.2000 1.00±0.00 within groups Within Populations 330 176.00 0.53333Vc 120.00 FST=-0.200 1.00±0.00 Total 395 176.00 0.4444

Table 6: Results from Tajima's Neutrality Test m S Ps Θ π D 66 6 0.013245033 0.002782994 0.00314917 0.317531964 Abbreviations: m = number of sequences, n = total number of sites, S = Number of segregating sites, ps = S/n, Θ = ps/a1, π = nucleotide diversity, and D is the Tajima test

statistic.

Tajima’s neutrality test (Table 6) was done. The relationships of taxa are presented in the form of a likelihood of the maximum composite estimate of the graph. Using UPGMA, evolutionary history has been pattern of nucleotide substitution with the genetic inferred. The optimal tree with the sum of branch distance between different populations were length 0.01706665 was shown (Fig 1). The tree is determined. drawn to a scale, with the branch lengths in the same The highest distance was observed between units as the evolutionary distances used to infer the Madar Tank and River Parwan and then in Madar Tank phylogenetic tree. and River Kali Sindh. Lowest was observed between The Dendrogram showed a big difference River Kali Sindh and River Chambal, and River Kali between the populations collected from the Daya Dam Sindh and Rana Pratap Sagar Dam. Evolutionary and Madar tank as one unit and the populations –

Journal of Wildlife Research | October-December, 2020 | Volume 08 | Issue 04 | Pages 74-80 © 2020 Jakraya 78 Summarwar et al…Assessment of Mahseer population structure and Genetic variation using ATPase6

Fig 1: Evolutionary relationships of taxa (The evolutionary history was inferred using the UPGMA the optimal tree with the sum of branch length = 0.01706665 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

collected from the rest of the other sites. On the other relationships among haplotypes found in a sample of hand, it can be stated that the populations collected homologous DNA. A haplotype is a set of markers on a from other sites (BL, PR, KSR, BD, RPS, and CR) single chromosome that tended to be inherited together. were found to have a close association. However, It was observed that out of 66 fishes, only 9 among these similar populations, differences were fishes from two sites denoted close association, and rest found. It can be instituted that few samples from CR, other 57 fishes from six sites exhibited similarities for RPS, BD, and KSR denoted differences. It is reiterated mitochondrial DNA, ATPase 6 gene sequences which that the sequence of the ATPase 6 region of were analysed for species identification and mitochondrial DNA acted as a potential marker for phylogenetic relationship. Sequence analysis of this studying genetic variations. The high rate of evolution gene very lucidly exhibited that all the 66 fish species in the ATPase 6 gene, distinguished haplotypes in a fell into two distinct groups. These were genetically small sample size. Haplotype is a collection of genes distant from each other and divulged identical within an organism that are inherited together from a phylogenetic reservations. All the ATPase 6 gene single parent. They displayed the evolutionary sequences from 66 fishes offered enough phylogenetic

Journal of Wildlife Research | October-December, 2020 | Volume 08 | Issue 04 | Pages 74-80 © 2020 Jakraya 79 Summarwar et al…Assessment of Mahseer population structure and Genetic variation using ATPase6 information and evolutionary relationship to Oxidase I and ATPase 6, the populations from Madar differentiate the Tor tor . This study attempted to tank and Daya dam exhibited genetic differences from provide the utility of mitochondrial DNA ATPase 6 the fish samples collected from other sites. It is gene sequence-based gambit in identifying and important to appreciate that the dendrogram is a recognizing Tor tor at a rapid rate. summary of the distance matrix. Further studies are needed to find out the clear differences. 4. Conclusion Present investigation lucidly showed that there Conflict of interest is definite genetic differentiation in Tor tor populations The authors declare that they have no conflict of along the water division in Southern Rajasthan. It can interests. be concluded that for both the cases, Cytochrome

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