POPULATION of Malaysia (Chee, 1996). Longtail tuna may


2 Defined and important of the genetic population study 2.3 Mitochondrial DNA of D-Loop markerCHAPTER 3: MATERIALS AND METHOD 3.1 Sample sites and sample collection3.2 DNA salt extraction3.3 DNA purification3.4 Polymerase Chain Reaction (PCR) Optimization3.

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5 Gel electrophoresis3.6 DNA Sequencing3.7 Data analysisCHAPTER 4: EXPECTED RESULT 4.1 Expected results REFERENCESGANTT CHART CHAPTER 1INTRODUCTION 1.1 Background of the study Tuna species such as Thunnus tonggol, Euthynnus affinis and Auxis thazard has been caught in Malaysia waters by the fisherman by using a variety of fishing gear. In addition, due to Malaysia has joined Exclusive Economic Zone (EEZ) and lead to exploring the sea when the fishing instrument and fishing vessel become advanced. The fishery for tuna harvest increases from artisanal to the commercial industries.

Because of the introduction of new gear like purse seine and trawl, the catch of pelagic species such as tuna has drastically increased and contributed to the commercial fishery of Malaysia (Chee, 1996).Longtail tuna may be caught by using a variety of fishing gear. For example, by using gillnet, trolling or purse seines. Purse seines highly used by fishermen in Malaysia to capture tuna species. Purse seines are widely used because it is more effective from another fishing gear for pelagic species. The tuna species that catch in a bulk form for the commercial fish industry is Euthynnus affinis, Auxis thazard and Thunnus tonggol (Chee, 1996).Management for longtail tuna are limited or absent, and assessments for longtail tuna are managed by certain states only and in progress by SEAFDEC (Southeast Asian Fisheries Development Centre). Longtail tuna species are complicated to assess when these species are accidentally caught in multispecies and the landing report for longtail tuna are insufficient or incomplete.

The tuna catches need to be reported so the tuna harvest may be able to maintain at the optimum levels and able to manage tuna species from exploitation. Since tuna is migrated fish, a regulation needs to be implemented for tuna species because tuna are likely to be shared with other countries.One of the important of longtail tuna is in the recreational fishing sport such as in Oman. This sport becomes widely known in recreational fishing years by years. In addition, the recreational fishing increase due to increases of human population. When the human population increases, the participant of anglers or tourism also increase and lead to sports fishing (Cooke and Schramm, 2007). Besides, modern fishing equipment such as electric fishing and sonar, or also the sophistication of internet spread the news between the anglers such as fishing club, causes highly increases the recreational fishing sport (Griffiths et al.

, 2010, McPhee et al., 2002). Besides, longtail tuna is widely used in fisheries industry markets. Longtail tuna’s meat is sold in canned form, or sell fresh in markets to make dishes or sashimi.   Population genetic is a study about the composition of biological genetic and changes in the genetic pattern.

The genetic population also relate with natural selection in the genetic composition. Natural selection of genes may occur in variants when another variant is reproduced for fit in the surrounding. When there are any changes occur in the population genetic, commonly it called ‘ evolution’. The change (evolution) may happen due to mutation, natural selection or genetic drift. Besides, to study about a natural population in the genetic, it depends on whether the polymorphic neutral available or not.

However, based on the previous study that have been done, DNA markers are highly used to obtain the information in fish population, such as frequencies of allele or gene flow (Neigel, 1997), compared to the electrophoresis although it also one of the way to study about the genetic variation of the fish population. 1.2 Problem Statement The problem statement for longtail tuna species is tuna are widely used as food sources and also sports fishing. This problem could lead to the over-exploitation of the longtail tuna species and may threaten this habitat. Unfortunately, the current data for this population status is lacking. The data is important to ensure the conservation and management strategy can be done well.1.

3 Significance of Study The data obtained from this study will be helpful in the strategic plan which is able to manage and ensure the conservation of this longtail tuna population is continuous in the east coast of Peninsular Malaysia. 1.4 Objectives The objectives of this study are to study the diversity of Thunnus Tonggol in the east coast of Peninsular Malaysia based on D-loop marker and to investigate the genetic structure of longtail tuna in Malaysia waters.CHAPTER 2LITERATURE REVIEW 2.1 Introduction of longtail Tuna (Thunnus tonggol) Tuna species is sleek, powerful and impressive wild animals. Tuna from family Scombridae. It has an elongated and streamlined body shape.

Because of their body structure, they are able to swim in fast motion and some tuna has the ability to swim as fast as 43 miles per hour. Tuna becoming high demand year by year because of their meat value, easy to catch, and precious in the industry such as canned tuna, sushi or sashimi. Tuna has a various type and each type has them on different valuable depends on each species. Tuna is species that migrate through the ocean for thousand miles. They usually migrate for spawning and food resources. They will migrate when spawning seasons begin, and they will migrate to the place where they are born.

Longtail tuna is neritic species that distribute from tropical to temperate oceans water in Indo-west specific and Red-Sea. Also, longtail tuna may be able to locate the tuna from East Africa, East New Guinea, north to southern Japan and south to Australia (Bleeker 1851). Even though tuna prefer inshore waters, but these species usually avoiding estuaries and turbid waters (Bleeker 1851). Longtail tuna is pelagic species which is commonly live near the surface of the water column and avoiding bottom column or sea. Usually, species that live in the pelagic area are more to migratory and mobile or fast-moving fish. They tend to migrate according to the food supply, seasonal or for spawning. Besides, longtail tuna is small in size compared to others types of tuna, restricted coastal distribution and fast-moving schools form (Yesaki, 1993) cause these species are hard to catch. The certain underdeveloped country such as Iran, Taiwan or Indonesia using gillnet, purse seine or troll to catch the longtail tuna, and unfortunately, it becomes heavily exploited.

During 1985, the global catches of longtail tuna were increased rapidly around 100,000 tonnes and continued throughout the year until in 2007, it reached about 248000 tonnes (Yonemori, Yanagawa, Pong, 1996). Researched show that over the past decade, Malaysia and other countries like Iran have higher global landings of longtail tuna. However, the statistics catch of longtail tuna are undercalculate because some of the landings were not reported especially targeted species in artisanal fisheries (Yonemori, Yanagawa, Pong, 1996).

Artisanal fisheries mean small scale of fisheries industries that involve small markets or by using traditional fishing gear and fishing technique and use small boats.Longtail tuna species are not valuable compared to the others species of tuna such as bluefin tuna (Jena, 2018). Even though the longtail tuna are frequently caught by fisherman but, the agencies management state that it is not included in major commercial species because it became smaller fishers’ earnings. Longtail tuna caught by using troll lines, gillnet or purse seines. Usually, the size target of tuna by using gillnet is in larger size while purse seines more to the smaller size. A gillnet is commonly used in Iran or India while purse seines use in Malaysia or Thailand, and for a troll, lines are usually used by Indonesia. Juveniles of longtail tuna also might be caught others species fishing target associated with FADs.

Fishery management effort supposedly continues to ensure the species and habitat are conserved and remained until the next generation. Unfortunately, very little information is available on fishery management of tuna and it is getting difficult due to overutilization of fish stocks, pollution and various human activities resulting in the reduction of genetic resources and variations ( Ciftci, 2003). Fishes not only provide us protein but also as the main contributor to food resources, fishery activities, providing jobs, and also an investment. The fishery management effort are including a closed zone for spawning grounds, reducing catches and others. These actions are ways to help the tuna populations to rebuild and ensure their long-term sustainability of the valuable species (Rooker, et al.

, 2007). When the times for the tuna spawning arrives, the spawning grounds area is prohibited for the fishermen. Thus, the tuna able to rebuild the depleted populations and protect their spawning fish (Rooker, et al., 2007), eggs and larvae from being destroyed.

2.2 Defined and important of a genetic population study A population is an organism that lives together in the same place with the same species. However, there are still many opinions from the researcher or biologists about the definition of population. A population is "a collection of organisms that we have lumped together because we believe they function together as a unit" (Roughgarden, 1979) or "a population as any group of organisms belonging to the same species at the same time and the same place" (E.O Wilson, 1992). Population genetic study is related to the natural population and population biology. These population genetic will successfully occur if it assisted by factors that contributed to the success of the genetic population.

These factors are a mutation, genetic drift, recombination, natural selection and gene flow. Besides, it also depends on the polymorphic neutral markers which are can proffer investigation of population study and provide scientific data for continues management, regulation and conservation to protect the fisheries population resources in long terms while the genetic variation is the study about the population changes and the frequencies of an allele in the population. The information about the gene flow or allele frequencies can be obtained by using DNA markers. These DNA markers are widely used and more popular compared to the electrophoresis, which is commonly used to study the genetic variations in fish populations (Neigel, 1997). One of the important of the genetic population studies is regulation can be applied to the specific species that need conservation based on the distributions. This is because fish harvest may cause the long-term effect on the population and surrounding if over-exploitation occurs. Next, population genetic analysis is important in planning, developing and implementing the strategy for the future of management and conservation.

The strategy should be not only provided for the economy but also for the natural population maintenance. The fisheries biologists must be the focus on the importance of the clarify the factor and conditions that allow communities and species to be sustained (Allendorf et al., 1987).Besides, the genetic population also method for studying about the electrophoresis which is it provides important ways how to measure and know the genetic discreteness of fish stock and also study about the relationship among the stocks available. Furthermore, electrophoresis contains data about the primary position among the method used for the identification of stock (Ihssen et al., 1981). 2.3 Mitochondrial DNA of D-Loop marker131445052705Figure 1.

Mitochondria DNA diagram.Mitochondria are part of the cell, which is always known as the powerhouse of the cell. In the animal cell, it is able to power all the processes and produce Adenosine triphosphate (ATP). The function of ATP provides energy throughout the cell so that the reaction will occur and function, regulate the cycle of a cell, heme production, calcium trafficking and uridine (Mineri, 2009). Mitochondria and free-living bacteria have similarities in their DNA and likely relative with the ?-proteobacteria which is phagocytosed by a heterotrophic cell. This hypothesis is known as the Endosymbiosis theory (Margulis, 1993), and it explains why the mitochondria have their own DNA, proteins and two membranes. The mitochondrial DNA (mtDNA) and prokaryotic genomes share some characteristics that present such as having a little non-coding sequence. loop marker is derived from the ‘displacement loop’, which is can be found in the mitochondrial DNA (mtDNA), where the genome of mitochondrial contains a fair amount of the non-coding sequence.

However, in some cases, D-loop was also known as a control region. DNA markers are highly used to obtain the information in the fish population such as gene flow, frequencies of allele and other parameters (Neigel, 1997), compared to the electrophoresis (Okasha, 2012). CHAPTER 3METHOD AND MATERIALS3.1 Sample sites and sample collectionThe random sample will be collected from three different sites consist of 20 to 30 of individuals throughout the east coast of Peninsular Malaysia at the Tok Bali(Kelantan), Terengganu and Kuantan (Pahang).

The size for each sample will be used approximately about 0.5cm- 0.9cm. The sample will be preserved in absolute ethanol.

DNA template will be isolated using salt extraction 3.2 DNA salt extractionFirstly, 300 µl of extraction buffer and 2 µl of proteinase K will be added to the sample. Then, the sample will be incubated for overnight at 65?. For this incubation process, it will incubate maximum 1200rpm for 24 hours. Next, for the extraction process, the sample will be added with 100 µl NaCl solution of 5M and spun for 5 minutes at 13000rpm.

The supernatant will be collect and transfer into the new tube. Next, 400 µl isopropanol will be added and mix by using the inverted tube and spun for 30 minutes at 14000rpm. The ethanol will be removed by using a pipette, and air dry for 30 minutes.

Next, the sample will be store at -20?. 3.3 DNA purificationUV spectrophotometer machine will be used to check the purity and concentration of the DNA sample extraction. Pipette the sample and insert into the Scan Drop (UV spectrophotometer machine) for identification of the sample for purity and concentration. 3.4 Polymerase Chain Reaction (PCR) OptimizationThe genome of DNA sample will take out from the freezer and will be amplified with mtDNA D-loop with primer pair forward and reverse primer. In PCR tube, will be mix with the 50-100ng of genomic extraction DNA, 0.

6 µM of each forward and reverse primer, 0.2 mM of dNTP, 1X PCR buffer, 4.2 mM of MgCl2 and 0.

08 U of Taq polymerase. The sample will be mix and spun for 5 seconds. After that, the sample will be amplified in a 25µl reaction volume in the MJ PTC-200 Thermal Cycle. The Thermal Cycle conditions will be set based on this steps: Step 1. Initial denaturation at 98°C (1 minute) Step 2. Denaturation at 95°C ( 15 seconds) Step 3. Annealing at 55°C (15 seconds to 1 minute)Step 4.

Extension at 72°C (2 minutes)Step 5. Repeat step number 2 for 35 cycles.Step 6. Final extension 72°C (1 minute)Step 7. Holding 12°C- ? Step 8.

End After the PCR amplification is done, the samples will be stored at 20°C. 3.5 Gel electrophoresisThe PCR samples will be visualised on 1.7% of an agarose gel stained by using the SyBr Safe. 3.6 DNA SequencingPCR products will be sent for DNA sequencing at First BASE Laboratories Sdn Bhd, Selangor by using forward primer only. 3.7 Data analysisMolecular Evolutionary Genetics Analysis (MEGA) is a software that will be used to determine the sequence alignment, inferring phylogenetic histories and also conduct the evolution of molecular analysis (Tamura et al.

, 2013). All the multiple sequences will be aligned and all the unambiguous of operational taxonomic units (OTUs) will be compiled and edited by using the ClustalW implemented in MEGA 6.0 (Tamura et al., 2013). The DNA sequences will be translated into protein to ensure the alignment will be accurate and detection the present of numts. By using the MEGA 6.

0, the complete aligned of the dataset will be analyzed. Arlequin 3.5 will be used to calculate the haplotype diversity and nucleotide diversity also used to describe the DNA polymorphism at sampling sites (Excoffier and Lischer, 2010).For the analysis of the molecular variance, SAMOVA 2.

0 (Dupanloup, 2016) will be used to identify the genetically of the similar group’s population and to evaluate the amount of the genetic variation among the partitions. The optimal number of groups will be determined based on the highest value of the variance among the groups, which is incorporating the information on haplotype divergence and the geographical proximity. In addition, based on the SAMOVA, the calculation for the relative genetic differentiation between the population structure and the population of the pairwise will be determined by using the Arlequin 3.5 which is will evaluate the significant differences among populations and spatial population structure. CHAPTER 4EXPECTED RESULT4.1 Expected resultBased on this study, the genetic diversity of the longtail tuna population will be expected to be low and the genetic structure of longtail tuna will be expected to be no structure in the east coast of Peninsular Malaysia.REFERENCESGANTT CHART-4000527940 YearTask 2018 2019Sep Oct Nov Dec Jan Feb Mar Apr May JuneProposal Writing                    Literature review Proposal presentation Proposal submission                    Preparing materials                    Sample collection                    DNA extraction                    PCR Optimization Data analysis                    Thesis writing Final Report preparation                    Final report presentation                    Final report correction                   Final report submission


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