CHAPTER-IV Results and Discussion 4.1 Standardization of genomic DNA extraction from fish tissue The fishes under study viz., Badis badis and Amblyceps mangois have been classified as threatened in category as reported by NBFGR. Therefore it would be unethical to standardize the genomic DNA (gDNA) isolation and RAPD PCR experiments with these fish samples. For this reason closely related fish samples (Labeo bata related to Badis badis and Heteropneustes fossilis related to Amblyceps mangois) were chosen for the standardization of sufficient amount of genomic DNA extractraction from minute quantity of tissue samples from these fishes. The DNA isolation method employed in this study showed that sufficient amounts of high molecular weight gDNAs could be purified from fish scales/fins (Figure 12). While the integrity of the isolated gDNAs appeared satisfactory ample amount of RNAs were present in the isolated gDNA samples (Figure 12 A–C). The figure showed that the yields of high molecular weight gDNAs from live scale and fin samples were substantially higher than that from frozen samples. However the gDNAs isolated from different quantities of fin clips of both live and frozen Heteropneustes (scaleless species) were more degraded than that from scales or fins of Labeo (Figure 12 C). Live Frozen Live Frozen Live Frozen A. Labeo bata (Scale) B. Labeo bata (Fin) C. Heteropneustes fossilis (Fin) Figure 12. 0.7 % agarose gel electrophoreses of genomic DNAs (gDNAs) isolated from Live and Frozen scale and fin samples of L. bata and H. fossilis. A. Lane 1 Lambda DNA/Hind III Digest DNA size marker (kb), lanes 2–6: 5, 10, 20, 30, 50 pieces of scale gDNA preparations from live L. bata, lanes 8–12: 5, 10, 20, 30, 50 pieces of scale gDNA preparations from frozen L. bata. B. Lane 1 Lambda DNA/Hind III Digest DNA size marker (kb), lanes 2–6: 1, 5, 10, 20, 30 mg of fin gDNA preparations from live L. bata, lanes 8–12: 1, 5, 10, 20, 30 mg of fin gDNA preparations from frozen L. bata. C. Lane 1 lambda DNA/Hind III Digest DNA size marker (kb), lanes 2–6: 1 mg, 5, 10, 20, 30 mg of fin gDNA preparations from live H. fossilis, lanes 8–12: 1, 5, 10, 20, 30 mg of fin gDNA preparations from frozen H. fossilis. Quantification of gDNA yields was done after treating each isolate with RNase A. The total yield of gDNAs from 5 and 50 pieces of scales were 8.40 and 80.0 µg respectively Page | 131 and from 1 and 30 mg fin were 4.65 and 123.50 µg respectively in live L. bata. In live H. fossilis the total yield were 14.60 and 132.60 µg from 1 and 30 mg fin respectively. However, the yield of gDNAs from frozen samples of both L. bata and H. fossilis were relatively low in comparison to that from the live ones (Table 10). Table 10. Spectrophotometric assessment of gDNA yields and purity in both live and frozen tissues from a carp (Labeo bata) and a catfish (Heteropneustes fossilis) after treatment with RNase A. Specimen Tissue Sample OD260 OD280 OD260/ Concentration Total type OD280 (µg/ll) yield (µg) 5 pieces 0.0056 0.0032 1.75 0.084 8.40 Scale Labeo bata 50 pieces 0.0530 0.0243 2.18 0.800 80.0 (live) 1 mg 0.0031 0.0016 1.94 0.0465 4.65 Fin 30 mg 0.0823 0.0446 1.85 1.235 123.50 5 pieces 0.0021 0.0011 1.91 0.032 3.20 Scale Labeo bata 50 pieces 0.0190 0.0102 1.86 0.285 28.5 (frozen) 1 mg 0.0009 0.0005 1.80 0.0135 1.35 Fin 30 mg 0.0240 0.0137 1.75 0.36 36.0 Heteropneustes 1 mg 0.0097 0.0049 1.98 0.146 14.60 Fin fossilis (live) 30 mg 0.0884 0.0463 1.86 1.326 132.60 Heteropneustes 1 mg 0.0011 0.0006 1.83 0.0165 1.65 Fin fossilis (frozen) 30 mg 0.0165 0.0093 1.77 0.2475 24.75 The gDNAs isolated from lowest (5 pieces) and highest (50 pieces) numbers of scales of L. bata and that from lowest (1 mg) and highest (30 mg) amounts of fin tissues of the fishes were used as templates in RAPD-PCR analyses (Figure. 13). In live L. bata scale and fin, OPA02 primer produced nine bands and OPA 07 produced seven bands respectively [Figure. 13 A (lanes 2, 3, 6, 7), 2B (lanes 2, 3, 6, 7)]. But in frozen L. bata scale and fin the result was different, the bands appeared faint, OPA02 primer produced three bands and OPA07 primer produced four bands [Figure 13 A (lanes 4, 5, 8, 9), b (lanes 4, 5, 8, 9)]. In live H. fossilis fin OPA 02 gives eight bands and OPA07 gives four bands (Figure 13 C lanes 2, 3, 6, 7) whereas in frozen sample the numbers of bands were relatively less, both OPA 02 produced six bands and OPA07 produced three bands (Figure 13 C lanes 4, 5, 8, 9). Page | 132 A. Labeo bata (Scale) B. Labeo bata (Fin) C. Heteropneustes fossilis (Fin) Figure 13. RAPD banding patterns in 1.4 % agarose gels from L. bata and H. fossilis using decamer primers OPA02 and OPA07. A Lane 1 100 bp DNA ladder (kb), lanes 2–3, 6–7 RAPD profile from 5 and 50 pieces of scale respectively from live L. bata, lanes 4–5, 8–9 RAPD profile from 5 and 50 pieces of scale respectively from frozen L. bata. B Lane 1 100 bp DNA ladder (kb), lanes 2–3, 6–7 RAPD profile from 1 and 30 mg of fins respectively from live L. bata, lanes 4–5, 8–9 RAPD profile from 1 and 30 mg of fin respectively from frozen L. bata. C Lane 1 100 bp DNA ladder (kb), lanes 2– 3, 6–7 RAPD profile from 1 and 30 mg of fin respectively from live H. fossilis, lanes 4–5, 8–9 RAPD profile from 1 and 30 mg of fin respectively from frozen H. fossilis. Page | 133 4.2 Genetic diversity study of Badis badis A detailed survey was carried out in the Mahananda (Terai region), Teesta and Jaldhaka (Dooars region) river systems for the collection of Badis badis samples. Total seventeen spots were selected for collection purpose (Figure 14) and samples were collected from the selected sites with the help of scoop net. The fourteen collection spots were as follows, viz., Mahananda Barrage, Fulbari (BTR-1), Mahananda-Panchanoi River Junction (BTR-2), Balason River, Palpara, Matigara (BTR-3), Panchanoi River (BTR-4), Mahananda River, Champasari (BTR-5), Balason River, Tarabari (BTR-6) from Mahananda river system; Sevoke (BDR-1), Ghish river (BDR-2), Gajoldoba-Teesta Barrage (BDR-3), Chel river (BDR-4), Neora river (BDR-5), Dharla river (BDR-6), Jalpaiguri (BDR-7) from Teesta river system; and Jaldhaka River (BDR-8), Murti River (BDR-9), Ghotia River (BDR-10), Diana River (BDR-11) from Jaldhaka river system. See the map below for detail: BDR-2 BDR-5 BDR-1 BDR-4 BDR-11 BDR-10 BDR-9 BTR-5 BTR-6 BTR-4 Teesta River System Jaldhaka River System Mahananda River System BDR-3 BTR-3 BTR-2 Water channel BDR-6 BTR-1 N sampling site BDR-8 BDR-7 10 Km Figure 14. Badis badis collection sites. Also refer to Table 2 for the geographic coordinates of the collection spots for Badis badis. Page | 134 4.2.1 Findings based on RAPD based analyses 4.2.1.1 Intra-Population Genetic Diversity Study 4.2.1.1.1 Mahananda River System Table 11. Intra-population genetic diversity indices based on RAPD analyses of Badis badis of Mahananda-Balason river system. Populations RAPD Markers Diversity Indices H´ H´ Np Nper S H H´ or I E= e /S EHeip=(e - 1/S-1) Mahananda Barrage, 40 28.37% 1.2837± 0.1061± 0.1562± 0.910696 0.595911 Fulbari (BTR-1) 0.4524 0.1841 0.2637 Mahananda-Panchanoi 35 24.82% 1.2482± 0.0898± 0.1330± 0.915118 0.573127 River Junction (BTR-2) 0.4335 0.1718 0.2471 Balason River, Palpara, 34 24.11% 1.2411± 0.0865± 0.1283± 0.916037 0.567789 Matigara 0.4293 0.1690 0.2433 (BTR-3) Panchanoi River 36 25.53% 1.2553± 0.0946± 0.1395± 0.915876 0.586364 (BTR-4) 0.4376 0.1766 0.2533 Mahananda River, 37 26.24% 1.2624± 0.0953± 0.1409± 0.912001 0.576637 Champasari 0.4415 0.1764 0.2531 (BTR-5) Balason River, Tarabari 44 31.21% 1.3121± 0.1166± 0.1717± 0.904902 0.600197 (BTR-6) 0.4650 0.1890 0.2709 Mahananda river 53 37.59% 1.3759± 0.1207± 0.1836± 0.873272 0.53614 system 0.4861 0.1789 0.2600 Note: Np=number of polymorphic loci, Nper=percentage of polymorphic loci, S=observed number of alleles, H= Nei's gene diversity, H´ or I= Shannon's Information index, E= measure of evenness, EHeip= Heip’s evenness index. Based on the RAPD profile of Badis badis from Mahananda-Balason river the number of polymorphic loci and the percentage of polymorphic loci vary across six populations (BTR-1 to BTR-6) (Table 11 and Figure 15 A). The highest number of polymorphic loci was observed in BTR-6 population (44 numbers) and the percentage of polymorphism was 31.21.