Research Article

Locus Specific Genetic Diversity and Population Differentiation Analyses of Badis badis (Hamilton-Buchanan, 1822) in Sub-Himalayan West Bengal, India  

Mukhopadhyay T. , Bhattacharjee S.
Cell and Molecular Biology Laboratory, Department of Zoology, University of North Bengal, P.O. North Bengal University, India
Author    Correspondence author
International Journal of Aquaculture, 2016, Vol. 6, No. 4   doi: 10.5376/ija.2016.06.0004
Received: 20 Feb., 2016    Accepted: 12 Apr., 2016    Published: 26 Apr., 2016
© 2016 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Mukhopadhyay T. and Bhattacharjee S., 2016, Locus Specific Genetic Diversity and Population Differentiation Analysis of Badis badis (Hamilton-Buchanan, 1822) in Sub-Himalayan West Bengal, India., International Journal of Aquaculture, 6(4): 1-9 (doi: 10.5376/ija.2016.06.0004)

Abstract

Badis badis is a threatened ornamental freshwater fish in the Indian scenario. We have studied RAPD based locus specific population genetic structure of this species in the eastern sub-Himalayan hotspot region of West Bengal state of India, known as the Terai and Dooars. We have studied six populations of Badis badis from the Terai region riverine streams and seven populations from the Dooars region riverine streams. We have used twenty different RAPD decamer primers which allowed amplification of 184 loci in the Terai populations and 134 loci in the Dooars region Badis populations. Observed number of locus specific alleles, effective number of alleles, Shannon’s diversity index and Nei’s genetic diversity were analyzed. The population differentiation of different Badle is badis populations was measured through PhiPT analyses. 

Keywords
Badis badis; RAPD fingerprinting; Locus specific diversity; Population differentiation

(The advance publishing of the abstract of this manuscript does not mean final published, the end result whether or not published will depend on the comments of peer reviewers and decision of our editorial board.)

1 Introduction
Dwarf Chameleon fish Badis badis (Hamilton-Buchanan 1822) (Family Badidae) is a freshwater fish species found in the hill streams of the eastern sub-Himalayan region of West Bengal, India, known as the Terai and Dooars. It has an important ornamental and commercial value and has been reported as a Vulnerable (VU) category by National Bureau of Fish Genetic Resources (NBFGR), India (Lakra et al., 2010). However, the molecular characterization with regard to the genetic background of this fish species has not been carried out in this eastern sub-Himalayan hotspot region. Therefore, it is essential to ascertain the genetic architecture of any threatened species having economic value for its management, conservation and most importantly proper reestablishment in natural population to prevent its extinction from the wild. Since scanty of genomic data is available in Badis badis species till date, we have used RAPD-PCR fingerprinting (Welsh and McClelland, 1990; Williams et al., 1990), an efficient and inexpensive molecular tool, to investigate and characterize the genetic background of Badis sp through the study of DNA polymorphisms. We have studied the locus specific genetic diversity and population differentiation between different populations of Badis badis from terai and dooars region of West Bengal, India. This locus specific diversity analysis will help to ascertain further calculation of diversity indices of this fish species in the study region.
 
2 Results
The present study revealed that a significant number of loci were amplified using twenty decamer arbitrary RAPD primers. Six populations (consisting of thirty individuals) of Badis badis from the Terai region generated total 184 numbers of amplified fragments correspond to 184 numbers of amplified loci (Table 1). Whereas, seven populations (consisting of seventy individuals) of Badis badis from the Dooars region generated total 134 numbers of amplified fragments correspond to 134 numbers of amplified loci (Table 2). The observed number of alleles, effective number of alleles, Nei’s genetic diversity and Shannon’s diversity index in Terai region populations were 1.9598±0.1969, 1.4846±0.3008, 0.2983± 0.1425 and 0.4589±0.1834 respectively (Table 1). The observed number of alleles, effective number of alleles, Nei’s genetic diversity and Shannon’s diversity index in the Dooars region populations were 1.8657±0.3423, 1.5311±0.3486, 0.3082±0.1740 and 0.4589±0.2366 respectively (Table 2). Locus-specific Nei’s genetic diversity was found to be highest in OPB03-8, OPA16-12 and OPA2-9 loci (Terai rivers) and that in OPA19-5, OPB12-3 loci (Dooars rivers) (Table 1 and Table 2).

 

Table 1 Locus specific diversity indices of Terai population of Badis badis

 

 

Table 2 Locus specific diversity indices of Dooars population of Badis badis

 

The PhiPT values showed the degree of population differentiation. The highest level of differentiation was found between TR-1 and TR-5 populations (0.719) in the the Terai region (Table 3) and between DR-3 and DR-7 populations (0.810) in the Dooars region (Table 4).  

 

 

Table 3 Pair-wise PhiPT values between six populations of Badis badis collected from the Terai region

 

 

Table 4 Pair-wise PhiPT values between six populations of Badis badis collected from the Dooars region

 

3 Discussion
RAPD technique has been widely used to ascertain the genetic architecture of different subdivided populations of a species. Due to its cheap, inexpensive and rapid use, RAPD is the best alternative to other more sophisticated molecular tools in population genetic analyses. The ornamental fish Badis badis has been considered to be a vulnerable fish species in the Indian context, demanding proper conservation, management and stock enhancement in the sub-Himalayan Terai and Dooars regions of West Bengal, India. We have previously studied the genetic architecture of this species in detail in the Terai region through RAPD fingerprinting (Mukhopadhyay and Bhattacharjee, 2015). In the present study we have ascertained locus specific genetic diversity analyses and population differentiation of the Terai and Dooars region Badis badis populations. We have focused only the locus specific diversity of this species in the Dooars region of West Bengal, India. The study has identified few more diverse RAPD bands or loci which could be further used to study similar populations in the nearby regions to study changes in diversity patterns. The detailed study of this species in the study region should be done to analyze its genetic diversity in a robust way. 
 
We have also carried out population differentiation study in these two regions to show how much genetic differentiation has occurred in this species in the particular study area. We have found that a significant level of population differentiation has occurred between some populations (Table 3 and Table 5). This differentiation might have happened due to lower level of gene flow between these populations or lower level of intermixing between these populations due to some allopatric or sympatric processes. The region is within a biodiversity hotspot therefore, acquiring information regarding the population genetic structure of this species might be helpful to the development of suitable conservation strategies and management practices. 
 
4 Materials and Methods
4.1 Survey and sample collection
A total thirty Badis badis individuals were collected from six spots of the major streams of the Terai Region of West Bengal, India (five samples from each spot) and geographic coordinates of the collection spots were recorded. The collection spots were as follows: TR1=Mahananda Barrage, Fulbari, TR2=Mahananda-Panchanoi River Junction, TR3=Balason River at Palpara, TR4=Panchanoi River, TR5=Mahananda River at Champasari and TR6=Balason River at Tarabari (Figure 1). A total seventy fish samples of Badis badis were collected from seven different spots of Dooars Region of West Bengal, India (ten samples from each spot) and geographic co-ordinates were recorded. The collection spots were as follows: DR-1= Sevoke (Teesta River), DR-2=Ghish river, DR-3=Gajoldoba (Teesta Barrage), DR-4=Chel river, DR-5= Neora river, DR-6=Dharla river and DR-7=Jalpaiguri (Teesta river). The collection spots are depicted in Figure 1. Fishes were identified according to Talwar and Jhingran (Talwar and Jhingran, 1991).

 

 

Figure 1 Map showing the collection spots of Badis badis from different riverine streams of sub-Himalayan West Bengal, India. (see text for detail)

 

4.2 Genomic DNA isolation
High molecular weight genomic DNA was extracted and quantified using the protocol followed by Mukhopadhyay and Bhattacharjee (2014).
 
4.3 RAPD–PCR amplification, documentation and analysis
Twenty RAPD primers from Kit-A and B (Imperial Life Science Pvt. Ltd., India) were selected for further analyses on the basis of the numbers, variability and reproducibility of the bands obtained (Table 5). RAPD analyses were performed in a 96 well Peltier Thermal Cycler (Applied Biosystems 2720, Life Technologies, USA) following Mukhopadhyay and Bhattacharjee (2014). The amplified products were electrophoresed in an ethidium bromide (0.5µg/ml) pre-stained 1.4 % agarose gel (Lonza, Basel, Switzerland) in TAE buffer using BenchTop Labsystems BT-MS-300, Taiwan electrophoretic apparatus. The standard 100 base pair ladder (NEB, USA) were used to approximate molecular weight of each band. The RAPD data was analysed using three software viz., Popgene ver. 1.32 (Yeh et al., 1999) and GenAlEx 6.5 (Peakall and Smouse, 2006; Peakall and Smouse, 2012). The data matrix was used to estimate locus specific different diversity indices i.e.,  the observed number of alleles (Na), effective number of alleles (Ne), percentage of polymorphic loci (P), Shannon Diversity Index (I) and Nei’s genetic diversity (H). Inter-population genetic differentiation was estimated by pairwise PhiPT values, where PhiPT = AP / (WP + AP) = AP / TOT (AP = Estimated variance Among Populations, WP = Estimated variance Within Population, TOT= Estimated variance) (Excoffier et al., 1992).

 

 

Table 5 The primer name, its sequences and GC content

 

Acknowledgments
The work was supported by a research grant from University Grants Commission (UGC), India [MRP Sanction No. 40-289/2011 (SR)] awarded to corresponding author.
 
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