ГЕНЕТИКА, 2015, том 51, № 10, с. 1171-1183


УДК 575.17:597.593.6


© 2015 V. S. Baisvara, R. Kumara, M. Singha, A. K. Singha, U. K. Chauhanb, N. S. Nagpurea, and B. Kushwahaa

aMolecular Biology and Biotechnology Division, ICAR-National Bureau of Fish Genetic Resources,

Lucknow-226002, Uttar Pradesh, India e-mail: ravindra.scientist@gmail.com bSchool of Environmental Biology, Awadhesh Pratap Singh University, Rewa-486003, Madhya Pradesh, India

Received November 8, 2014

The mitochondrial DNA (mtDNA) ATPase 8/6 gene has been used in phylogenetic as well as in phylogeo-graphic studies along with other mtDNA markers. In this study, ATPase gene sequences were used to assess the genetic structuring and phylogeographic patterns in Channa striata. Out of 884 nucleotide positions generated in ATPase 8/6 genes, 76 were polymorphic. The study suggested 23 unique haplotypes from 67 individuals of nine populations collected from different riverine systems of India. The ATPase 8/6 sequence revealed highest haplotype as well as nucleotide diversities in Imphal River population and lowest diversities in Tapti River population. The pattern of genetic diversity and haplotype network indicated distinct mitochondrial lineages for Chaliyar population, whereas mismatch distribution strongly suggested a population expansion in mid pleistocene epoch (0.4 Mya) with distinct genetic structuring in C. striata. The baseline information on genetic variation and the population sub-structuring would facilitate conservation and management of this important snakehead murrel.

Keywords: ATPase 8/6, C. striata, genetic variation, mtDNA polymorphism, phylogeography. DOI: 10.7868/S0016675815100045

Channa striata (Bloch, 1793), commonly known as striped snakehead murrel, is a tropical freshwater food fish in the Asia-Pacific region [1] and has also been used for medicinal and pharmaceutical purposes [2]. It is a carnivorous, air-breathing species found across the North part of Asia, southern China, Indochina and the Sunda Islands in ponds, swamps, rivers, small streams, canals, drains, reservoirs, rice fields, mining pools, and lakes. It is abundant in nature and may survive for a longer time without water. Culture of snakehead is important in certain countries like India, Pakistan, Tibet, Nepal, Bhutan, Bangladesh, and Sri Lanka [3]. In this region, the aquaculture potential of the species has not been fully exploited despite its high market price and tolerance to adverse climatic conditions including air breathing capability [1].

Being an important food-fish, indiscriminate harvesting has resulted in decline in the stocks of the species [4]. An earlier study of this species has focused on reproductive biology [5], breeding [6], medicinal properties [2—7], biochemical composition [8], dietary intake [9], ecology [10], and morphological characters [11]. In C. striata, genetic variation in natural populations of Malaysia was described using mitochondrial COI and nuclear microsatellites [12, 13]

markers. For conservation and management of this species, information on relevant population genetics is essential, specifically through assessment of its genetic diversity and structuring for potential brood-stock identification. As information on population structure of this species in Indian region is not available, so this study was undertaken to assess the genetic diversity of the natural populations for utilization in management of the species in culture systems.

The mitochondrial ATPase subunits 8/6 were sequenced to examine the genetic diversity and structure of C. striata stocks within India and particularly to infer the mechanisms or forces most likely to have been involved in distribution of snakehead populations; thus, providing genetic information for broodstock management and species conservation in nature.


Sample collection

A total of 67 individuals, representing nine populations of C. striata from India, were sampled during the years 2010 to 2013. Details of sample collection, i.e., river, sample size, sampling locations with latitude/longitude and time of collection are shown in



Fig. 1. Sampling locations of C. striata.

Fig. 1 and Table 1. These populations, classified into nine categories (i.e., Betwa, Brahmaputra, Chaliyar, Ganga, Gomti, Imphal, Krishna, Narmada, and Tapti rivers), were located in different geographical areas in India. These riverine locations were selected so as to cover geographically distant populations. Gomti and Betwa (Vindhya Range) belongs to Ganges River basin in Northern India, Narmada (Amarkantak Hill) and Tapti (Satpura Range) in Central India. Brahmaputra River and Imphal River are major rivers in North East part of India. Chaliyar and Krishna rivers originated from Western Ghat in Southern India, but flowing west-ward and east-ward, respectively. Fish muscle tissue samples were collected, fixed in 95% ethanol and stored at 4°C for further use. Voucher specimens were preserved in formalin.

DNA isolation, amplification, and sequencing

Genomic DNA was isolated from ethanol preserved muscle tissue using standard phenol-chloroform method. The DNA pellet was dissolved in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) and the DNA quality as well as concentration was determined on 0.7% agarose gel and with Nanodrop 2000 (Thermo Scientific, USA). The genomic DNA was diluted to 50 ng/^L and stored at 4°C. ATPase 8/6 regions of mitochondrial DNA (mtDNA) was amplified in 50 ^L reaction volumes. The PCR reaction mix contained 10x buffer (with 15 mM MgCl2), 10 mM dNTPs mix, 10 pmole of each primer (ATP.2L8331 and COIII.2H 9236), 2.5 U Taq DNA polymerase and 200 ng genomic DNA. Amplifications were performed in thermal cycler (Eppendorf AG 22331, Hamburg, Germany) with initial denaturation at 94°C for 3 min,

ATPase 8/6 GENE BASED GENETIC DIVERSITY ASSESSMENT Table 1. Sampling locations of C. striata used in the present study

River N

Gomti 9

Narmada 8

Betwa 10

Tapti 4

Chaliyar 8

Krishna 9

Ganga 10

Brahmaputra 6

Imphal 3

Total 67

Note: N = number of individuals.

Sampling locations (latitude and longitude)

Sampling time

Lucknow, Uttar Pradesh (26°52' N; 80°55' E) Indore, Madhya Pradesh (23°10' N; 75°45' E) Jhansi, Uttar Pradesh (25°33' N; 79°24' E) Surat, Gujarat (21°14' N; 72°24' E) Nilambur, Kerala (11°15' N; 76° 11' E) Vijaywada, Andhra Pradesh (16°36' N; 80°17' E) Roorkee, Uttarakhand (29°51' N; 77°18' E) Guwahati, Assam (26°10' N; 91°41' E) Imphal, Manipur (24°50' N; 93°58' E)

March, 2010 November, 2012 November, 2012 November, 2012 February, 2013 February, 2013 March, 2013 March, 2013 March, 2013

followed by 33 cycles of denaturation at 94°C for 45 s, annealing at 54°C temperature for 30 s and elongation at 72°C for 1 min, with a final elongation at 72°C for 10 min. The amplified PCR products were checked on 1% agarose gel and sequenced from both side by the dideoxynucleotide chain-termination method [14] using ABI sequencer 3730. The quality of sequencing results was checked by viewing the electropherograms in Finch TV (Geospiza, Inc., Seattle, WA 98119) software.

Sequence alignment

The total 67 sequences were aligned using Clustal W [15] in MEGA 5.05 software [16]. The mismatches were referred against the electropherograms and the sequences submitted to GenBank (Table 2). All parameters were used as default with gap opening penalty 15, gap extension penalty 6.66 and multiple alignment parameters set as gap opening penalty of15, gap extension 6.66, DNA weight matrix IUB and Transition weight 0.5.

Genetic diversity analysis

Intra-population diversity was analyzed by haplo-type diversity and nucleotide diversity. The overall genetic differentiation between each sampled population was tested using pairwise /-statistics. For deriving the correlation between geographical distribution and genetic structure, genetic differentiation of all the nine populations was calculated in the framework of a predefined geographical locations and analysis of molecular variance (AMOVA) based on pairwise squared Euclidean distances matrix amongst populations and between individuals [17]. All populations analyses were performed using Arlequin version 3.5 [18]. The measurement of contribution of diversity (CSR), differentiation (CDR), and allelic richness (CTR) of

each population were computed by Contrib (version 1.02) software [19].

Phylogeographic analysis

Sequence data were analyzed through neighbor-joining (NJ) and maximum-likelihood (ML) methods with bootstraps of 1,000 replicates. All phylogenetic analyses were carried out using MEGA (version 5.05) software. The UPGMA cladogram was computed on the basis of pairwise genetic relatedness (i.e., F& scores). To find out whether ancestral haplotypes are extinct, dataset was analyzed using phylo-geographi-cal techniques based on haplotype networks using NETWORK (version 4.612) software [20]. The p-dis-tance was computed using between group mean distances option in MEGA to find the proportions of nucleotides at particular positions are different.

Test of population patterns, isolation by distance (IBD) and relationships among geographical distance of the rivers and genetic distance among stocks was done using Mantel test [21, 22] in XLSTAT-2010 software with 10,000 permutations. Pairwise FsL value were genetic distance matrix between populations, while geographical distances (in kilometer) between populations were sampling site of the river.

Neutrality and demographic history

Arlequin computer software version 3.5 [18] was used for pairwise mismatch distribution analyses and selective neutrality tests. Raggedness index was included in mismatch analysis to determine goodness of fit for unimodal distribution [23]. Two tests, viz. Taji-ma's D-test [24] and Fu's Fs-test [25], were applied under the

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