CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES GENETIC DIVERSITY OF CLIMBING PERCH (Anabas testudineus BLOCH, 1792) POPULATIONS BASED ON RAPD AND ISSR MARKERS By PHAM THI TRANG NHUNG A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Can Tho, December 2013 CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES GENETIC DIVERSITY OF CLIMBING PERCH (Anabas testudineus BLOCH, 1792) POPULATIONS BASED ON RAPD AND ISSR MARKERS By PHAM THI TRANG NHUNG A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Supervisor Dr. DUONG THUY YEN Can Tho, December 2013 ACKNOWLEDGEMENT First of all, I want to thank to Can Tho university and college of aquaculture and fishery for giving me a chance to study and complete this course. Also, I want to express my special thanks to my supervisor, Dr. Duong Thuy Yen for her invaluable guidance, advice, caring and encouragement. Many thanks are also given to all professors who taught our class, and other teachers of the College of Aquaculture and Fisheries, and especially to those of the Department of Freshwater Aquaculture for providing me with great working and learning conditions. I would love to express my sincere appreciation to many of my friends and class Advance Aquaculture Program course 35 as well as my family for supporting me. i ABSTRACT Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) techniques were used to evaluate genetic diversity of four climbing perch (Anabas testudineus) populations including square-head and wild populations. Total 65 specimens were amplified using primers (1 RAPD and ISSR primers). The percentage of polymorphism and heterozygosity ranged from 66.15% and 0.166 (Dong Thap) to 81.54% and 0.237 (Ca Mau), respectively. Therein, Ca Mau population had the highest genetic diversity. Results also revealed that a high portion of total genetic variation existed within populations (91%), while genetic differentiation among populations was low (Gst value 0.0859), accounting for 9% of total genetic variation. Based on Nei’s genetic distance, the highest dissimilarity was observed between Dong Thap and each of the other populations. This study indicated that RAPD and ISSR techniques can be useful for genetic diversity studies in fish. ii TABLE OF CONTENTS ACKNOWLEDGEMENT i ABSTRACT . ii TABLE OF CONTENTS iii LIST OF FIGURES vi LIST OF ABBREVIATIONS vii INTRODUCTION . 1.1 General introduction 1.2 Research Objectives: 1.3 Research Contents: CHAPTER LITERATURE REVIEW 2.1 Distribution of climbing perch . 2.2 Biological characteristics of climbing perch . 2.2.1 Classification and taxonomy . 2.2.2 Morphological characteristics of climbing perch 2.2.3 Feeding behavior of climbing perch 2.2.4 Growth characteristics . 2.2.5 Spawning . 2.3 Genetic diversity 2.3.1 Definition of genetic diversity . 2.3.2 Importance of genetic diversity . iii 2.4 Genetic markers commonly used to study genetic diversity . 2.5 Studies of genetic diversity of fish and shrimp populations using RAPD and ISSR 2.5.1 Studies of genetic diversity of fish and shrimp populations using RAPD . 2.5.2 Studies of genetic diversity of species using ISSR . 10 2.6 PCR reaction method . 11 2.6.1 Principle of PCR reaction 11 2.6.2 Some main factors affect PCR reaction 11 2.6.3 Application of PCR method 12 CHAPTER 13 MATERIALS AND METHODS 13 3.1 Time and sites of study 13 3.2 Materials 13 3.3 Methods . 14 3.3.1 Fish sampling 14 3.3.2 DNA extraction . 14 3.3.3 Electrophoresis 15 3.3.4 Screening primers and optimize PCR conditions 15 3.3.4 PCR reaction 19 3.3.4 Data scoring . 20 3.3.5. Data analysis . 20 RESULTS AND DISCUSSION 21 4.1. Result 21 4.1.2. Genetic diversity of climbing perch populations . 26 4.1.3. Genetic differentiation among climbing perch population 27 CHAPTER 32 CONCLUSION AND RECOMMENDATION 32 iv LIST OF TABLES Table3.1. Selected primers with sequence, GC content, melting temperature for RAPD and ISSR analysis in climbing perch. .16 Table 3.2 Optimize MgCl2 and primer (OPA08) concentrations .17 Table 3.3 Gradient temperatures used to optimize PCR conditions of ISSR primers .18 Table 3.5 PCR components .19 Table 4.1.The size and number of PCR products for all primers .26 Table 4.2 Genetic diversity parameters (Mean ± SE) of climbing perch populations based on primers (1 RAPD + ISSR) 27 Table4.3. Pairwise population matrix of Nei genetic identity (under the diagonal line) and genetic distance among populations 28 Table4.4. Analysis of genetic variance (AMOVA) of climbing perch populations .28 v LIST OF FIGURES Figure 2. External appearance of climbing perch Figure 4.1. Test OPA 09 for different samples of populations. 21 Figure 4.2. Test OPA 07 with gradient temperature. . 22 Figure 4.3. Testing primer OPAH 08 with different concentrations of MgCl2 and primer for two samples CM 3p and HG4 23 Figure 4.4. Testing ISSR 06 with different concentrations of MgCl2 and temperatures for two samples CM42 and CM43 . . 24 Figure 4.5.Test primers ISSR 01, ISSR 05, ISSR 11, ISSR 14, ISSR 15 with samples DV 55D, DT 41, and CM 21. . 25 Figure 4.6. Test ISSR 15 at different annealing temperature for samples DV 60C, HG12, DT 31, CM 18. 25 Figure 4.8. UPGMA consensus tree of Nei’s (1978) genetic distances between populations from four A.testudineus populations. . 28 vi LIST OF ABBREVIATIONS µg Microgram µl Microlitter AFLP Amplified fragment length polymorphism bp Base pair DArT Diversity Arrays Technology DNA Deoxyribonucleic acid dNTP Dinucleotid tripgotphate EDTA Ethylenediamine ISSR Inter-Simple Sequence Repeat MgCl2 Magenium Chloride mM milimolar mtDNA Mitochrondrial DNA NaCl Potassium chloride ng Nanogram PCR Polymerase Chain Reaction RAD marker Restriction site associated DNA markers RAPD Random Amplified Polymorphic DNA RFLP Restriction fragment length polymorphism SDS Sodium dodecyl sunphate SNP Single nucleotide polymorphism SSLP Simple sequence length polymorphism TBE Tris borate Ethylenediamine TE Tris Ultrapure and Ethylenediamine VNTR Variable number tandem repeat vii CHAPTER INTRODUCTION 1.1 General introduction Climbing perch (Anabas testudineus) is one of the most common freshwater fish in tropical and subtropical Asia, widely distributing in Pakistan, Bangladesh, Nepal, Bhutan (probable), Sri Lanka, India, Indonesia, southern China, Myanmar, Thailand, Cambodia, Laos, Vietnam, Malaysia, Brunei Darussalam and Singapore (www.fishbase.org). This species is also one of main aquaculture species, popularly cultured over the Mekong Delta of Viet Nam (Truong Thu Khoa and Tran Thi Thu Huong, 1993) because of its high nutrition value, fast growth, easy cultivation, high market demands, and high tolerance to adverse environmental conditions (Pethiyagoda, 1991). In 2009, a new strain of climbing perch was found in cultured conditions. It has square head (therefore it is called square head climbing perch), faster growth, and bigger size than the normal one. It was reported that the first batch of square head broodstock consisted of only 70 individuals. If so, genetic diversity of this strain could be low. Nowadays, many species have trended to increase potential of threatens due to environmental changes, competition for water resources, unregulated fishing, and high population growth (Sverdrup-Jensen, 2002). In addition, aquaculture practices are more growing, which probably inadvertently reduces genetic variability of species due to inappropriate selection and inbreeding (Wasco et al., 2004). And climbing perch is not an exception, neither. Fish population has declined more and more rapidly. Therefore, genetic diversity is very important for a species or population to adapt to continuous changes of the environment. Conservation of genetic variability is essential for the wellbeing of present and future generations (Akram and Kianoosh, 2012). Investigating genetic diversity of wild and cultured climbing perch is very important not only for domestication, conservation and sustainable development as well as for further breeding program of the species. Recently, advances in molecular biology techniques have provided numerous DNA markers and methods for studying genetic diversity of Table 4.2 Genetic diversity parameters (Mean ± SE) of climbing perch populations based on primers (1 RAPD + ISSR) Population No. No. population private band Ca Mau 15 Dong Thap 16 Hau Giang 18 Dau Vuong 16 65 TOTAL Polymorphism (% P) 81.54 Observed Expected Heterozygosialleles allele ty (He)* (na)* (ne)* 1.692 1.389 0.237 (0.085) (0.043) (0.022) 1.385 1.266 0.166 (0.111) (0.041) (0.022) 1.554 1.374 0.225 (0.103) (0.045) (0.023) 1.508 1.363 0.215 (0.103) (0.046) (0.024) 74.23 1.535 1.348 0.211 (3.29) (0.051) (0.022) (0.011) 66.15 76.92 72.31 * Mean and SE of polymorphism, observed and expected allele and heterozygosity for all genes 0.300 40 0.200 20 0.100 0.000 Camau Heterozygosity Number Band patterns across populations 60 Dongthap Haugiang Square head Populations No. Bands No. Bands Freq. >= 5% No. Private Bands No. LComm Bands ([...]... breeding and conservation programs of the species 1.2 Research Objectives: To evaluate genetic diversity based on RAPD and ISSR makers of climbing perch population distributed in the MeKong Delta, providing basic information for further breeding and conservation program 1.3 Research Contents: - Optimize PCR conditions for 10 universal RAPD and 10 ISSR primers - Genetic diversity of climbing perch populations. .. excitement and 5 spawning of climbing perch 0.3-0.4 m water level is compatible for climbing perch to spawn 2.3 Genetic diversity 2.3.1 Definition of genetic diversity Genetic diversity is the diversity of gene segments between individuals of the same species and among species Genetic diversity can be inherited in a population or among populations (www.wiseGeek.com) 2.3.2 Importance of genetic diversity Genetic. .. 60 0.000 Camau Dongthap Haugiang Square head Populations No Bands No Bands Freq >= 5% No Private Bands No LComm Bands ( . samples CM42 and CM43 . 24 Figure 4 .5. Test 5 primers ISSR 01, ISSR 05, ISSR 11, ISSR 14, ISSR 15 with 3 samples DV 55 D, DT 41, and CM 21. 25 Figure 4.6. Test ISSR 15 at different annealing temperature. DNA extraction 14 3.3.3 Electrophoresis 15 3.3.4 Screening primers and optimize PCR conditions 15 3.3.4 PCR reaction 19 3.3.4 Data scoring 20 3.3 .5. Data analysis 20 RESULTS AND DISCUSSION. populations. Total 65 specimens were amplified using 7 primers (1 RAPD and 6 ISSR primers). The percentage of polymorphism and heterozygosity ranged from 66. 15% and 0.166 (Dong Thap) to 81 .54 % and 0.237