Appendix A: List of specimen details of 27 freshwater fish species used in this study. SLM-HWE(PH)
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1 Appendix: Appendix A: List of specimen details of 27 freshwater fish species used in this study. 1 Barbonymus schwanenfeldii SLM-BS(PH)-01 Department of Fisheries, Lubuk Paku SLM-BS(PH)-02 Freshwater Fisheries (Pahang) SLM-BS(PH) SLM-BS(PH) SLM-BS(PH) Amblyrhynchicthys truncatus SLM-AT(PH)-04 Department of Fisheries, Lubuk Paku SLM-AT(PH)-05 Freshwater Fisheries (Pahang) SLM-AT(PH) SLM-AT(PH) SLM-AT(PH) Hypsibarbus wetmorei SLM-HWE(PH)-01 Department of Fisheries, Lubuk Paku SLM-HWE(PH)-02 Freshwater Fisheries (Pahang) SLM-HWE(PH) SLM-HWE(PH) SLM-HWE(PH) Pseudolais micronemus SLM-PM(PH)-01 Department of Fisheries, Kuala Mai SLM-PM(PH)-02 Freshwater Fisheries (Pahang) SLM-PM(PH) SLM-PM(PH) SLM-PM(PH) Osteochilus vittatus SLM-OH(PH)-01 Department of Fisheries, Lubuk Paku SLM-OH(PH)-02 Freshwater Fisheries (Pahang) SLM-OH(PH) SLM-OH(PH) Barbichthys laevis SLM-BL(PH)-01 Department of Fisheries, Lubuk Paku SLM-BL(PH)-02 Freshwater Fisheries (Pahang) SLM-BL(PH) SLM-BL(PH) Bagarius yarrelli SLM-BY(PH)-01 # Department of Fisheries, Kuala Mai SLM-BY(PH)-02 Freshwater Fisheries (Pahang) SLM-BY(PH) SLM-BY(PH) xvii
2 Appendix A continued' 8 Puntioplites proctozystron SLM-PP(PH)-01 Department of Fisheries, Lubuk Paku SLM-PP(PH)-02 Freshwater Fisheries (Pahang) SLM-PP(PH) SLM-PP(PH) Rasbora sumatrana SLM-RS(PH)-02 Department of Fisheries, Lubuk Paku, SLM-RS(PH)-04 Freshwater Fisheries Kuala Mai SLM-RS(PH)-05 (Pahang) Luciosoma setigerum SLM-LS(PH)-01 # Department of Fisheries, Kuala Mai SLM-LS(PH)-02 Freshwater Fisheries (Pahang) Hampala macrolepidota SLM-HM(PH)-10 (1) Department of Fisheries, Lubuk Paku SLM-HM(PR)-02 Freshwater Fisheries (Pahang) SLM-HM(PR)-03 Temenggor (Perak) (2) University of Malaya 12 Probarbus jullieni SLM-PJ(PH)-01 Department of Fisheries, Lubuk Paku Freshwater Fisheries (Pahang) 13 Pangasius nasutus SLM-PN(PH)-01 Department of Fisheries, Kuala Mai SLM-PN(PH)-02 Freshwater Fisheries (Pahang) SLM-PN(PH) SLM-PN(PH) Thynnichthys thynnoides SLM-TT(PH)-01 Department of Fisheries, Kuala Mai SLM-TT(PH)-02 Freshwater Fisheries (Pahang) xviii
3 Appendix A continued' 15 Hemibagrus wyckii SLM-HW(PH)-01 # Department of Fisheries, Kuala Mai SLM-HW(PH)-02 Freshwater Fisheries (Pahang) Cirrhinus Caudimaculatus SLM-CC(PH)-01 Department of Fisheries, Kuala Mai SLM-CC(PH)-02 Freshwater Fisheries (Pahang) Cyclocheilichthys apogon SLM-CA(PH)-02 Department of Fisheries, Kuala Mai SLM-CA(PH)-03 Freshwater Fisheries (Pahang) SLM-CA(PH) Anabas testudineus SLM-ATE(PH)-01 # Department of Fisheries, Kuala Mai Freshwater Fisheries (Pahang) 19 Belodontichthys dinema SLM-BD(PH)-01 Department of Fisheries, Kuala Mai SLM-BD(PH)-02 Freshwater Fisheries (Pahang) Puntioplites bulu SLM-PB(PH)-01 Department of Fisheries, Kuala Mai SLM-PB(PH)-02 Freshwater Fisheries (Pahang) SLM-PB(PH) SLM-PB(PH) SLM-PB(PH) Channa striata SLM-CS(JH)-08 (1) Department of Fisheries, Batu Pahat dam SLM-CS(JH)-11 Freshwater Fisheries (Johor) SLM-CS(JH) SLM-CS(JH) SLM-CS(JH)-30 (2) University of Malaya xix
4 Appendix A continued' SLM-CS(PP)-01 Permatang Kuang, SLM-CS(PP)-02 Paddy Fields SLM-CS(PP)-03 (Pulau Pinang) SLM-CS(PP) SLM-CS(PP) SLM-CS(TG)-05 Terengganu River SLM-CS(TG)-06 (Terengganu) SLM-CS(TG) SLM-CS(TG) SLM-CS(PH)-06 Pahang River SLM-CS(PH)-07 (Pahang) SLM-CS(PH) SLM-CS(PH) SLM-CS(PH) SLM-CS(KD)-01 Kubur Panjang, SLM-CS(KD)-02 Paddy Fields SLM-CS(KD)-03 (Kedah) SLM-CS(KD) SLM-CS(NS)-06 Sikamat, SLM-CS(NS)-11 Paddy Fields SLM-CS(NS)-13 (Negeri Sembilan) SLM-CS(NS) SLM-CS(NS) SLM-CS(SL)-16 Tanjung Karang, SLM-CS(SL)-18 small stream SLM-CS(SL)-20 (Selangor) SLM-CS(SL) SLM-CS(SL) CS(SRW)-1,2 Miri River CS(SRW)-3,4 (Sarawak) CS(SRW)-5, CS(SRW)-7, CS(SRW)-9, Rasbora paviana SLM-RP(PR)-01 University of Malaya Temenggor SLM-RP(SL)-02 (Perak) Kepong (Selangor) xx
5 Appendix A continued' 23 Hemibagrus nemurus SLM-HN(PR)-01 University of Malaya Temenggor SLM-HN(PR)-02 (Perak) SLM-HN(PR) Devario regina SLM-DR(PR)-01 University of Malaya Temenggor SLM-DR(PR)-02 (Perak) SLM-DR(PR) Channa lucius SLM-CL(PR)-01 University of Malaya Temenggor (Perak) 26 Puntius banksi SLM-PBA(SL)-01 University of Malaya Kepong SLM-PBA(SL)-02 (Selangor) SLM-PBA(SL) Poropuntius smedleyi SLM-PS(SL)-02 University of Malaya Kepong SLM-PS(SL)-03 (Selangor) SLM-PS(SL) SLM-PS(SL) SLM-PS(SL) # Image data obtained from FishBase Sample sequence not submitted to BOLD * Refer BOLD ( project code: FWFM for more details on specimens. xxi
6 Appendix B: A list of optimum PCR mixture used in this study. Component Stock concentration Working concentration Working volume (µl) 5X Buffer 5X 1X 6.0 (Promega) MgCl 2 25mM 2.5mM 2.0 (Promega) dntps 10mM 0.25mM 0.5 (each) F. Primer (Fish F1) 10µM 0.25µM 0.5 R. Primer (Fish R1) 10µM 0.25µM 0.5 Taq Polymerase 5U/µl 3U 0.6 (Promega) Autoclaved dh 2 O 6.8 DNA Template 50ng/µl 80ng 1.6 Total volume/tube 20.0 xxii
7 Appendix C: A list of optimum thermal cycling profile used in this study. Thermal Cycle Temperature ( C) Time (min) Initial denaturation 96 5 Denaturation 94 45sec Annealing 45 45sec Extension 72 30sec Final extension Hold 4 xxiii
8 Appendix D: List of components of 10X TBE buffer Component Tris Base Boric Acid EDTA dh 2 O Volume 108 g 55 g 40 ml Top up to 1 litre xxiv
9 100bp Clear single band ~ 700bp amplicon Appendix E: Banding pattern of COI amplicon with optimized annealing temperature in the range of and optimum amount of 25mM MgCl 2 of 2µl per reaction volume. Note: (1) Lane 1: 45 C, Lane 2: 46.1 C, Lane 3: 47.4 C, Lane 4: 48.9 C, Lane 5: 51.0 C, Lane 6: 53.4 C and Lane 7: 54.7 C. (2) Marker: 100bp DNA Ladder (Vivantis). (3) PCR products run on 1% agarose gel at 80 volt for 35 minutes. xxv
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