PHYLOGENETIC RELATIONSHIP OF GENUS Staurois (ANURA: RANIDAE) VIA PARTIAL 16S RIBOSOMAL SUBUNIT GENE IN SARAWAK Nur Athirah Binti Amirrudin (27589) Bachelor of Science with Honours (Animal Resources Science and Management) 2013 Faculty of Resources Science and Technology
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PHYLOGENETIC RELATIONSHIP OF GENUS Staurois (ANURA: RANIDAE) VIA
PARTIAL 16S RIBOSOMAL SUBUNIT GENE IN SARAWAK
Nur Athirah Binti Amirrudin
(27589)
Bachelor of Science with Honours
(Animal Resources Science and Management)
2013
Faculty of Resources Science and Technology
PHYLOGENETIC RELATIONSHIP OF GENUS Staurois (ANURA:
RANIDAE) VIA PARTIAL 16S RIBOSOMAL SUBUNIT GENE IN
SARAWAK
Nur Athirah Binti Amirrudin
This project is submitted in partial fulfilment of the requirement for the degree of
Bachelor of Science with Honours
(Animal Resource Science and Management)
Department of Zoology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
2013
Declaration
I hereby declare that the thesis is based on my original work except for the citation
which has been duly acknowledged. I also declare it has not been previously or
concurrently submitted for any degree for any other degree at UNIMAS or any other
institution of higher learning.
_____________________
Nur Athirah Amirrudin
Animal Resource Science and Management Programme
Department of Zoology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
i
ACKNOWLEDGMENT
First of all, Alhamdulillah praise to Allah for giving me the strength in completing my
final year project. God granted me so much pleasure while doing this project. I would
like to express my thank and sincere appreciation to my supervisor, Mr. Mohd Zacaery
Khalik for his helpful advice, guidance, and suggestion in the fulfilment of this project.
Thanks also to Dr. Ramlah Zainudin for her suggestions and support during the
completion of this project.
Most thanks to my beloved parents, Amirrudin Bin Haji Asmuni and Suhana Binti Haji
Wahab for their support and encouragement. Thank you very much for the financial
support. To all my family members, thanks a lot for the endless encouragement while
accomplishing this project.
Last but not least, my gratitude goes to all lecturers and staffs of the Department of
Zoology. Kind thanks all the laboratory assistants Isa Sait, Huzal Irwan Husin, Nasron
Ahmad and Trevor Allen Ak Nyaseng, for providing guidance, facilities and materials.
Not forgetting the postgraduates students and my colleagues for providing me help in the
fulfilment of this project. Thanks to all of you.
ii
Table of Contents
CHAPTERS TITLES PAGES
Acknowledgements
Table of Content
i
ii
List of Tables iii
List of Figures iv
List of Abbreviation
List of Appendices
vi
vii
1 Introduction
1.1 Background of Study
1.2 Objectives
2
4
2 Literature Review
2.1 The study of Staurois
2.2 Mitochondrial DNA
2.3 16S Ribosomal Sub-unit gene
5
7
8
3 Material and Method
3.1 Study Site
3.2 Field Sampling
3.3 Identification and Measurement
3.4 Processing Specimens
3.4.1 Sample Collections
9
10
11
11
iii
3.4.2 Isolation of Mitochondrial DNA from
Muscle Tissue Specimen
3.4.3 Amplification of Targeted Sequences by
using PCR
3.4.4 Agarose Gel Electrophoresis
3.4.5 Purification and Sequencing
12
13
15
15
4 Results and Discussion
4.1 Nucleotide frequencies
4.2 Pairwise Genetic Distance
4.3 Phylogenetic Analysis
4.4 Gene Variation of Eastern and Western Sarawak
samples.
4.5 The Tectonic Event
17
18
24
29
30
5 Conclusions and Recommendation
5.1 Conclusion
5.2 Recommendation
32
32
References
Appendix
33
iv
List of Tables
Table No. Title Page
Table 1: List of primers for 16S gene used in this study. 14
Table 2: Master mix preparation for one time reaction cocktail. 14
Table 3: Amplification profile. 14
Table 4: Pairwise genetic distance of 48 individuals analyzed
based on Kimura 2-parameter.
19
Table 5: Pairwise genetic distance samples from Western part
of Sarawak only analyzed based on Kimura 2
parameter.
22
v
List of Figure
Figure No. Title Page
Figure 1: Map of Study Site in Sarawak. 10
Figure 2: The position of 16S gene in mtDNA genome. 14
Figure 3: The relationships among the genus Staurois as
estimated using Neighbour Joining tree (Western part
of Sarawak).
26
Figure 4: The relationships among the genus Staurois as
estimated using Maximum Parsimony tree (Western
part of Sarawak).
27
Figure 5: The relationships among the genus Staurois as
estimated using Maximum Parsimony (Western and
Eastern part of Sarawak).
28
vi
List of Abbreviation
bp base pair
CTAB Cetyltrimethylammonium bromide
MgCl2 Magnesium chloride
NaCl Sodium Chloride
TAE Tris-acetate-EDTA
CIA Chloroform-Isomyl Alcohol
DNA Deoxyribonucleic acid
mtDNA Mitochondrial Deoxyribonucleic Acid
PCR Polymerase Chain Reaction
SVL Snout – vent length
UV Ultra Violet
MEGA Molecular Evolutionary Genetic Analysis
oC Degree celcius
vii
List of Appendices
Appendix 1: List of tissue samples used in this study.
Appendix 2: The sequence alignments.
Appendix 3: Nucleotide compositions of genus Staurois in this study.
1
Phylogenetic Relationship of Genus Staurois (Anura: Ranidae) via Partial 16S
Ribosomal Mitochondrial DNA in Sarawak
Nur Athirah Binti Amirrudin
Animal Resources Science and Management Programme
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
The partial 16S ribosomal mitochondrial DNA gene was used to infer the phylogenetic relationship of
selected species of genus Staurois and elucidated the extent of genetic variation within the genus Staurois.
The three from six species that were sequenced and used in phylogenetic analysis are Staurois guttatus, S.
latopalmatus, and S. tuberilinguis thar are endemic to Sarawak. Samples are collected from Western part
of Sarawak including Kubah National Park, Sebangkoi Recreational Park, dan Batang Ai National Park.
A total of 498 bp of 16S subunit gene from 48 samples were analysed using genetic analysis softwares
such as Molecular Evolutionary Genetic Analysis 4.1 (MEGA 4.1). The phylogenetic trees were
constructed using Neighbour Joining (NJ), Maximum Parsimony (MP), and Maximum Likelihood (ML)
analyses. All trees produced similar tree topology showing three major clades that distinguish haplotypes
from different species and the sister clades distinguishing haplotypes from Eastern and Western part of
Sarawak. It also proven that 16S is the useful genetic marker for the genetic study within the genus level.