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).

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).  

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).

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).

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.

 

References