Genetic studies on the swimming crab (portunus pelagicus linnaeus, 1758) with implications for fisheries management

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY _ MUHAMMAD ARIFUR RAHMAN GENETIC STUDIES ON THE SWIMMING CRAB (Portunus pelagicus LINNAEUS, 1758) WITH IMPLICATIONS FOR FISHERIES MANAGEMENT MASTER THESIS KHANH HOA – 2018 i MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY _ MUHAMMAD ARIFUR RAHMAN GENETIC STUDIES ON THE SWIMMING CRAB (Portunus pelagicus LINNAEUS, 1758) WITH IMPLICATIONS FOR FISHERIES MANAGEMENT MASTER THESIS Marine Ecosystem based Management and Climate Change 58CH153 Major: Code: Topic allocation Decision Decision on establishing the Committee: Defense date: Supervisors: 6th June, 2018 Prof Dr Henrik Glenner Dr Dang Thuy Binh Chairman: Assoc Prof Ngo Dang Nghia Faculty of Graduate Studies: KHANH HOA – 2018 ii UNDERTAKING I undertake that the thesis entitled: “Genetic Studies on the Swimming Crab (Portunus pelagicus Linnaeus, 1758) with Implications for Fisheries Management” is my own work The work has not been presented elsewhere for assessment until the time this thesis is submitted All the given information is true to best of my knowledge 14.06.2018 Muhammad Arifur Rahman iii ACKNOWLEDGMENT I would like to express my deepest gratitude to the Department of Graduate Studies Nha Trang University for giving me the opportunity to study here and finish my thesis My heartfelt appreciations to Dr Dang Thuy Binh for her continuous support during my master Study and accept me to conduct my research in her Molecular Biology Lab Her outstanding supervision helped me in all the time of research and writing of this thesis I would like to express my heartiest thanks to my supervisor Prof Dr Henrik Glenner from University of Bergen, Norway My gratitude is always there with all the Lecturers and the coordinators of the Norhed Master‟s Program Moreover, I would like to thank all the members of Biodiversity and Conservation research group at Nha Trang University especially Mr Thinh, Ms Oanh and Mr Sang for their continuous support during my research Last but not least, I would like to thank to all of my family members for supporting me spiritually throughout the program Thank you! 14.06.2018 Muhammad Arifur Rahman iv TABLE OF CONTENTS UNDERTAKING iii ACKNOWLEDGMENT iv TABLE OF CONTENTS ……………………… ……………………………………………v LIST OF ABBREVIATIONS vii LIST OF TABLES viii LIST OF FIGURES ix LIST OF APPENDICES x ABSTRACT xi CHAPTER 1: INTRODUCTION 1.1 Background 1.2 Environmental change and human impact on marine population 1.3 Applications of advance genomic techniques in fisheries management 1.4 Rationale of the research 1.5 Objectives of the study 1.6 Thesis contents CHAPTER 2: LITERATURE REVIEW 2.1 Overview of the study area 2.1.1 Overview of Vietnam climate 2.1.2 Overview of Vietnamese coastline 2.2 Study on swimming crab (Portunus pelagicus Linnaeus, 1758) 2.2.1 Classification 2.2.2 Habitat and distribution 2.2.3 Life cycle 10 2.3 Overview of the research methodology 11 2.3.1 Molecular markers for studying population genetics 11 2.3.2 Restriction site-associated DNA sequencing (RADseq) 14 2.3.3 Next generation sequencing (NGS) 15 2.4 Overview on population genetics 16 2.4.1 Population genetics of marine organisms 16 2.4.2 Review on Portunus pelagicus 16 2.5 Population genetic study and fisheries management 18 CHAPTER 3: MATERIALS AND METHODS 20 3.1 Sampling sites and tissue collection 20 3.2 Research outline 23 v 3.3 Research methodology 24 3.3.1 DNA extraction and digestion 24 3.3.2 EzRAD library preparation 24 3.3.3 SNP discovery and filtering 25 3.3.4 Outlier loci detection 26 3.3.5 Genetic diversity and effective population size 26 3.3.6 Analyses of population structure 26 3.3.7 Historic migration pattern 27 CHAPTER 4: RESULTS AND DISCUSSION 28 4.1 Results 28 4.1.1 Reference genome and SNP detection 28 4.1.2 Outlier loci detection 28 4.1.3 Genetic diversity of Portunus pelagicus 28 4.1.4 Effective population size (Ne) 29 4.1.5 Genetic differentiation of Portunus pelagicus 29 4.1.6 Population structure 30 4.1.7 Historic migration pattern 32 4.2 Discussion 33 4.3 Implications for fisheries management 36 4.3.1 Record keeping 36 4.3.2 Gear regulation and habitat monitoring 37 4.3.3 Sanctuary establishment 37 4.3.4 Implementation of ban season 38 4.3.5 Control of pollution 38 4.3.6 Implementation of management action plan 39 4.3.7 Creating awareness 39 CONCLUSION AND RECOMMENDATIONS 40 REFERENCES 41 APPENDICES a vi LIST OF ABBREVIATIONS µL Microliter µM Micromol AB Allele Balance AF Allele Frequency AFLP Amplified Fragment Length Polymorphisms A, T, C, G Adenine, Thymine, Guanine, Cytosine BI Bayesian inference bp Base pairs DNA Deoxyribonucleic acid RAD Restriction site Associated DNA Sequencing RAPD Random Amplification of Polymorphic DNA RFLP Restriction Fragment Length Polymorphisms RNA Ribonucleic acid SNPs Single nucleotide polymorphisms 16S rRNA 16S ribosomal Ribonucleic acid PCR Polymerase Chain Reaction MVP Minimum Viable Population COI Cytochrome Oxidase I Cm Centimeter GENO Genotype Km Kilometer vii LIST OF TABLES Table 3.1 Portunus pelagicus sample site information and genetic diversity based on neutral SNPs 21 Table 4.1 Ne for Portunus pelagicus in Vietnam 29 Table 4.2 Pairwise values of Fst (below the diagonal) and their respective P-values (above the diagonal) of P pelagicus 29 Table 4.3 Log probabilities of the data given the model (*marginal likelihood, based on the Bezier approximation score) and Δ values (difference from largest 1Lm value) and rank according to largest likelihood value 33 viii LIST OF FIGURES Figure 2.1 The economical zones and activities in Vietnamese coastal area (Thanh et al., 2004) Figure 2.2 Blue swimming crab Figure 2.3 Ventral view of male and female P pelagicus Figure 2.4 Distribution of P pelagicus 10 Figure 2.5 Life cycle of P pelagicus 11 Figure 2.6 SNPs 14 Figure 2.7 DNA library construction using ezRAD technique 15 Figure 3.1 Dorsal view of P pelagicus (A Male and B Female) 20 Figure 3.2 Map of sampling stations for genetic study of P pelagicus along the coastal line in Vietnam 22 Figure 3.3 Flow diagram of the research work 23 Figure 4.1 Population structure results for K=3 amongst P pelagicus populations obtained using STRUCTURE on a data set of 80 individuals and 721 polymorphic SNPs (A) Optimal K value, (B) Bar plot of P pelagicus populations, (C) Individual group distribution percentage (%) among sampling sitesError! Bookmark not defined Figure 4.2 (A) Principal Component Analysis (PCA) of P pelagicus based on neutral polymorphic SNP markers (B) Scatter plot from PCoA of P pelagicus 31 Figure 4.3 Principal Component Analysis (PCA) of P pelagicus based on under selection (divergent) loci 32 Figure 4.4 Berried female blue swimming crab in the landing center 38 ix LIST OF APPENDICES Appendix No.1 Purification of total DNA a Appendix No.2 Calculation of concentration of the samples a Appendix No.3 Collection of DNA Libraries b Appendix No.4 Quality scores and base calling accuracy (Phred scale) d Appendix No.5 Individual and SNPs filtering step by step e Appendix No.6 Detection of outlier SNPs using multiple algorithms f x Appendix 3: Collection of DNA Libraries ezRAD method, a simplified method developed by Toonen et al., 2013 for genomic genotyping in non-model organisms, was used to collect DNA libraries from the samples and the enriched DNA libraries were stored at -400C Fragmentation Before beginning the work the bench top and all equipments were wiped with 70% ethanol and the tips were sterilized 100 ng DNA was pipetted and put in the PCR tubes strip (8 tubes per strip) To clean salts and enzymes in the DNA elution AmpureXP Beads was used MboI and Sau3AI cut-off enzymes together with 10X SmartCut buffer was added in each tube to fragment DNA at 5´-GATC-3´ position The reaction was carried out in BIO-RAD DNAEngine (Peltier Thermal cycler, Serial Number: EN031947) for hours at 37ºC and 20 at 65ºC to stop fragmenting Finally, the samples were hold at 4ºC until next step The samples were cleaned up again with Ampure PEG solution Component Volume (µl) 10X SmartCut Buffer 2.5 MboI 5U/ µl (NEB) 0.5 Sau3AI 5U/ µl (NEB) 0.5 DNA template 21.5 Total volume 25 Blunt Ends repair with Illumina Kit In this step, overhangs resulted from fragmentation were converted into blunt ends using End Repair Mix2 The 3´ to 5´ exonuclease activity of the mix removes the 3´ overhangs and the 5´ to 3´ polymerase activity fills in the 5´ overhangs Fragment selection (350-550 bp) with Illumina Kit In this step 350 to 550 bp DNA fragment was selected using sample purification beads (SPB) Bead particles in SPB are designed to prioritize prior linkages with large size DNA fragments in the solution Bead particles attached DNA fragments larger than 550 bp Therefore, the bead particles were removed and the solution that contains target DNA fragments was kept for next steps After that SPB was used to bind fragments 350 to 550 bp Smaller fragments were washed away during ethanol washing b A-tailing Illumina A-tailing Mix was added in each sample to add a single “A” nucleotide to the 3´ end of each DNA fragment to bind the adapter during the ligation step The reaction was carried out for 30 at 37ºC, at70ºC and at 4ºC in DNAEngine As early as the incubation was finished, directly proceeded to next step without delay in the thermal cycler holding at 4ºC Adaptor ligation The adapter has three parts: sequences that make the fragment bind to the flowcell, sequences that are identified by the primer during sequencing and unique sequences that identify individuals Appropriate indexed adaptor was added to each sample and labeled as which index is applied The samples were incubated at 30ºC for 10 and while reached at 4ºC, immediately 1.7 µl of Stop Ligation Buffer was added to each sample Remaining adapter was removed through double washing with SP beads, resuspension buffer and ethanol After washing the DNA is in µl of supernatant PCR amplification of libraries The reaction incorporates dilution of 75% sample and 25% H 2O (3:1) The amplification of DNA with PCR was contained the following components Table Component of PCR to amplify the gene library of P pelagicus Component Volume (µl) Illumina PCR Primer Cocktail 1.5 Illumina Enhanced PCR Mix H2O 1.875 DNA Library Template 5.625 Total 15 c The strip labeled as PCR was run in the DNAEngine (Peltier thermal cycler) with the following parameters: (i) Pre-heat lid to 100ºC (ii) 95ºC for min, (iii) cycles of a 98ºC for 20 Sec b 60ºC for 15 Sec c 72ºC for 30 Sec (iv) 72ºC for (v) Hold at 4ºC Visualize Libraries Finally, µl of PCR product was run on 1.5% agarose gel (Invitrogen, USA) dyed with ethidium bromide to visualize libraries and to check for presence of adaptor dimer and the 350-500 bp regions The DNA library was stored at -40ºC Appendix 4: Quality scores and base calling accuracy (Phred scale) Phred quality score Probability of incorrect base call Base call accuracy Q10 in 10 90% Q20 in 100 99% Q30 in 1000 99.9% Q40 in 10000 99.99% Q50 in 100000 99.999% (Source: https://www.illumina.com/documents/products/technotes/technote_Q-Scores.pdf) d Appendix 5: Individual and SNPs filtering step by step Filtering Conditions Number of Individuals SNPs 86 11547 MAF 0.01 (minor allele frequency) 86 11547 DP (read depth) 86 10049 Remove indel 86 6946 Number of alternate observations on the forward and reverse strand 86 2900 Mean mapping quality of observed alternate and reference alleles 86 2123 Remove individuals have missing at least 10% 80 2097 PAIRED/PAIREDR 80 2021 Minor Allele Frequency 80 1651 Max-missing 0.95 80 1646 Remove loci which at least 10% missing data 80 1646 HWE 0.001 (Hardy-Weinberg Equilibrium p-value) 80 761 Significant SNPs (final filtering) 80 731 Max-missing 0.9 (genotype missing) MinQ 30 (mean quality) Mac (minor allele count) Max-non-ref-af 0.9999 Number of reference observations on the forward and reverse strand e Appendix 6: Detection of outlier SNPs using multiple algorithms Each dot represents an SNP marker The outlier SNPs potentially under divergent selection are marked in the figure: (A) Lositan - Fst values are plotted as a function of expected heterozygosity (He) for inter-population comparisons Loci under positive selection (red area), neutral selection (light shaded area and markers under balancing selection (yellow area) (B) BayeScan – Fst is plotted against the log10 of the posterior odds (PO) f ... Graduate Studies: KHANH HOA – 2018 ii UNDERTAKING I undertake that the thesis entitled: ? ?Genetic Studies on the Swimming Crab (Portunus pelagicus Linnaeus, 1758) with Implications for Fisheries Management? ??... Detection of outlier SNPs using multiple algorithms f x Genetic Studies on the Swimming Crab (Portunus pelagicus Linnaeus, 1758) with Implications for Fisheries Management ABSTRACT The blue swimming. .. EDUCATION AND TRAINING NHA TRANG UNIVERSITY _ MUHAMMAD ARIFUR RAHMAN GENETIC STUDIES ON THE SWIMMING CRAB (Portunus pelagicus LINNAEUS, 1758) WITH IMPLICATIONS FOR FISHERIES MANAGEMENT