Tài liệu Genetic diversity of climbing perch (anabas testudineus bloch, 1792) populations based on rapd and issr markers

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 3 wild 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 (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 1.1 General introduction ................................................................................ 1 1.2 Research Objectives:.................................................................................... 2 1.3 Research Contents: ...................................................................................... 2 CHAPTER 2 ...................................................................................................... 3 LITERATURE REVIEW .................................................................................. 3 2.1 Distribution of climbing perch ..................................................................... 3 2.2 Biological characteristics of climbing perch ............................................... 3 2.2.1 Classification and taxonomy..................................................................... 4 2.2.2 Morphological characteristics of climbing perch .................................. 4 2.2.3 Feeding behavior of climbing perch ...................................................... 4 2.2.4 Growth characteristics ........................................................................... 5 2.2.5 Spawning ............................................................................................... 5 2.3 Genetic diversity .......................................................................................... 6 2.3.1 Definition of genetic diversity............................................................... 6 2.3.2 Importance of genetic diversity ............................................................. 6 iii 2.4 Genetic markers commonly used to study genetic diversity ....................... 6 2.5 Studies of genetic diversity of fish and shrimp populations using RAPD and ISSR ............................................................................................................ 9 2.5.1 Studies of genetic diversity of fish and shrimp populations using RAPD ............................................................................................................. 9 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 3 .................................................................................................... 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 5 .................................................................................................... 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 4 climbing perch populations based on 7 primers (1 RAPD + 6 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 4 climbing perch populations...............................................................................................................................28 v LIST OF FIGURES Figure 2. External appearance of climbing perch .............................................. 3 Figure 4.1. Test OPA 09 for different samples of 4 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 5 primers ISSR 01, ISSR 05, ISSR 11, ISSR 14, ISSR 15 with 3 samples DV 55D, DT 41, and CM 21. ......................................................... 25 Figure 4.6. Test ISSR 15 at different annealing temperature for 4 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 1 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 1 different species (Akram and Kianoosh, 2012). Over the last decade, polymerase chain reaction (PCR) technique has become widespread and popular due to simplicity and high profitability of success (Bardakci, 2001). Over other techniques, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) techniques have been attracted many scientists’ interest. These techniques are simple and cost-effective, create high reproducible and polymorphic bands, require small amount of DNA without requiring flanking sequence characteristics as other methods. RAPD and newly applicable ISSR markers have been successfully used in genetic diversity analysis of some aquatic species (Chen and Leibenguth, 1995; Partis et al. 1996; Ali et al. 2004; Nie et al. 2012; Saad et al. 2012; Zhigileva et al. 2013; Dexiang et al. 2007). Despite the importance of the species in aquaculture and genetic conservation, few researches have been focused on genetic diversity of A.testudineus in their naturally distributed areas, especially, in the Mekong delta of Viet Nam. Therefore, this study aimed to evaluate genetic diversity of climbing perch populations distributed in the Mekong Delta using RAPD and ISSR techniques. The study would provide basic information for further 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 - Genetic difference among climbing perch populations 2 CHAPTER 2 LITERATURE REVIEW 2.1 Distribution of climbing perch Climbing perch is a freshwater species, distributed wildly in tropical regions, mainly in islands between Indian and Australia, Africa, and in Southeast Asia, climbing perch is distributed in Thailand, Lao, Cambodia, Myanmar and Viet Nam where average temperature is very compatible for grow of climbing perch (22-300C) . They are likely to live in shallow water with lots of plankton and organic matter (Kottelat, 2001). In Viet Nam, climbing perch is popular around the country. Climbing perch live in rice fields, lakes, rivers; they can also live in estuarine. Square head climbing perch has been found since 2009 in Hau Giang province. 2.2 Biological characteristics of climbing perch Figure 2. External appearance of climbing perch 3 2.2.1 Classification and taxonomy Class: Osteichthves Subclass: Neoptervgii Superordo: Acanthoptergii Ordo: Perciformes Farmilia: Anabantidae Genus: Anabas Species: Anabas. testudineus (Bloch, 1792) There are two phenotypes of climbing perch: normal climbing perch and square head climbing perch. Square head climbing perch was just found in 2009. It looks similar to the normal climbing perch except its square head. However, square head has some advantages compared to the normal one such as bigger than the normal climbing perch, high growth rate, male and female has similar growth rate; thus its production and market have developed very fast, bring more income for fish farmers. 2.2.2 Morphological characteristics of climbing perch Climbing perch has a well-balanced oval body which is covered by scales with black or grey spots on the outer edge. They have big eyes in front of their head. Their odd and even fins all have hard spikes. Operculum bone has incisors and rounded caudal fin which is not divided into lobes. Among the caudal peduncle has a bunch of black pigments which are lighter when climbing perch is mature (Truong Thu Khoa and Tran Thi Thu Huong, 1993). Furthermore, climbing perch has air breath organ that helps them tolerance with the adverse environmental conditions. Square head climbing perch looks like the normal climbing perch when it is young. When it grows up, it has big and square head, dark yellow scales, pink and spread tail, pretty curve and long body with two black spots near the gills and tail. 2.2.3 Feeding behavior of climbing perch Climbing perch start eating at the 3rd day. It is an omnivorous species who consumes a variety of food items such as fish, shrimp, crustaceans, 4 worms, mollusks, detritus, aquatic plants, rice, and insects (Duong Nhut Long 2003). When it grows up, it can eat many types of food, in which animal feeds are dominant. In addition, it’s possible to eat processed food and agriculture by-product very well. 2.2.4 Growth characteristics The size of normal climbing perch is quite small and growth rate is pretty slow compared with other species. In the Mekong Delta, the average size of climbing perch is about 60-120 g/individual. Normally for many fish, the male is bigger than the female. In contrast for climbing perch, the male is smaller than the female. In culture ponds, if it is fed enough food, after 6 months, it can reach 60-80 g/individual. Common length of climbing perch is about 12 cm and can reach maximum 25 cm (Talwar and Jhingran, 1991). Square head climbing perch grows very fast compare to the normal climbing perch. The male and female has similar growth rate. This makes it different from normal climbing perch. Female has growth rate as twice as male. After 4-5 months, it can reach 0.1-0.2 kg/individual and 0.4-0.6 kg/individual in 7 months. In contrast to the normal climbing perch, the longer we culture square head climbing perch, the bigger it is (Phuong Thanh, 2010). 2.2.5 Spawning Climbing perch has intermittent spawning behavior. It does not build a nest or prepare a spawning substrate, and does not provide parental care to its offspring, neither. Mating system of the climbing perch is either polygamy or promiscuity. Sexual dimorphism in the climbing perch was not found. The eggs of the climbing perch have positive buoyancy and floating, which is rare for freshwater fish (Zworikin, 2012). Climbing perch has high fecundity, about 300,000-700,000 eggs/kg female. The mature eggs often have ivory or yellowish white color. Water swollen eggs have diameter about 1.2-1.3 mm (Marimuthu et al., 2009). Climbing perch is one of fish species that are mature early. In nature, the smallest one that gets maturity is about 25 g. Square head climbing perch gets mature after 8 months. In the Mekong Delta, climbing perch spawn in rainy season, mainly from June to July. Normally after heavy rains, it all spawns. When spawning, it usually finds places where water is cool and has slow flows. Water current is the main factor stimulates the 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 diversity plays an important role in evolution by allowing species to adapt to a new environment. Analysis of genetic diversity is a key element for the study of biodiversity, ecosystem functioning, and the consequences of man-made impact on natural systems such as climate change, habitat fragmentation, and biological invasions. It’s very important for the ecology and has great effects on ecological processes such primary productivity, population recovery from disturbance, interspecific competition, community structure, and fluxes of energy and nutrients (Hughes et al., 2008). Decreased population genetic diversity can lead to reduce fitness of the population. Furthermore, in low genetic diversity population, the combination of declined fitness and increase variability can increase extinction rate of that population (Markert et al., 2010). 2.4 Genetic markers commonly used to study genetic diversity There are some commonly used genetic markers: Random amplification of polymorphic DNA (RAPD): RAPD markers are decamer (10 nucleotide length) DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence and which are able to differentiate between genetically distinct individuals. The ease and simplicity of RAPD make it ideal for individual and pedigree identification, pathogenic diagnostics, and trait improvement in genetics and breeding programs (Yoon and Kim, 2001; Holsinger et al., 2002). RAPD technique has further advantages compared to other techniques because it has a universal set of primers and does not require preliminary work 6 such as probe isolation, filter preparation, or nucleotide sequencing (Williams et al., 1990). There are many researches use RAPD for various purposes such as discrimination of species, identification of endangered species, genetic diversity, gene mapping, for breeding program…. In many cases, only a small amount of primers are required for detecting polymorphism (Williams et al., 1990). In some cases, a single primer may often be sufficient to distinguish all of the sampled varieties (Mulcahy et al., 1995). In addition, RAPD fingerprinting offers a rapid and efficient method for generating a new series of DNA markers in fishes (Foo et al., 1995). Especially, this method is suitable when finance is limited. However, like other techniques, RAPD also has some limitations we should concern about. It’s a dominant marker; hence we cannot distinguish between homozygous and heterozygous individuals because of the presence of bands. Besides, RAPD is not high repeatable due to random primer and depend much on PCR conditions. Microsatellite polymorphism, Inter-Simple Sequence Repeat (ISSR): is RAPD- like approach that assesses variation in the microsatellite regions dispersed throughout the various genomes of eukaryotes (Schulz, 2004). Microsatellites are very short stretches of DNA (usually 10-20 basepairs) that are very hypervariable, showed as different variants within as well as among populations. These short microsatellites are mono-, di-, or trinucleotide repeats. However, the most popular repeats used are di- or trinucleotides. ISSR technique has similar protocol as RAPDs, except that ISSR primers anneal to terminal parts of microsatellite regions. In addition, ISSR annealing temperatures is higher than RAPD technique and vary among different primers with different base compositions (Pharmawati et al., 2005). Normally, the optimal annealing temperatures range 45-50oC (Jabbarzadeh at al., 2010). These are many advantages of ISSR that are definitely suitable for detecting genetic diversity. First of all, this method requires small quantity of DNA. Besides, it provides dominant, reproducible and large number of markers. Additionally, this method allows to investigate in large scale genetic mapping and population studies. Furthermore, this method has extremely high variability and high mapping density compare to RAPD and RFLP. Also, ISSR doesn’t require flanking sequence characteristics as other methods. 7 Of course, nothing is perfect for everything in whatever case and so does ISSR. The limitation of this method is that it’s dominant marker, we can’t distinguish individuals that are heterozygous or homozygous of DNA bands at specific position (Kosman and Leonard, 2005) and hence not as informative as SSRs (Genet, 1983). Restriction fragment length polymorphism (RFLP): is a technique that exploits variations in homologous DNA sequences. RFLP analysis is the first DNA profiling technique inexpensive enough to see widespread application. RFLP is an important tool in genome mapping, localization of genes for genetic disorders, determination of risk for disease, and paternity testing (Welker et al., 1986). Amplified fragment length polymorphism (AFLP): is a PCR-based tool used in genetic research, DNA fingerprinting, and in the practice of genetic engineering. AFLP uses restriction enzymes to digest genomic DNA, followed by ligation of adaptors to the sticky ends of the restriction fragments. AFLP-PCR is a highly sensitive method for detecting polymorphism in DNA (Vos et al., 1995). Variable number tandem repeat (VNTR): is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. It’s often referred to as micro- or minisatellite DNA, are ubiquitous in eukaryotes and humans.VNTR typing generates portable digit-based data. VNTR-based PCR analysis is easy, rapid, and highly specific and can be conducted worldwide for genetics and biology research, forensics, and DNA fingerprinting (Smittipat et al., 2005). Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. Single nucleotide polymorphism (SNP): is a DNA sequence variation occurring when a single nucleotide — A, T, Cor G — in the genome (e.g. in exons, introns, intergenic regions, in promoters or enhancers, etc) differs between members of a biological species or paired chromosomes in a human. Common SNPs has only 2 alleles. The genomic distribution of SNPs is not homogenous. SNPs usually occur in noncoding regions more frequently than in coding regions or, in general, where natural selection is acting and fixating the allele of the SNP that constitutes the most favorable genetic adaptation (Schork et al., 2000). 8 Restriction site associated DNA markers (RAD marker): useful for association mapping, QTL-mapping, population genetics, ecological genetics and evolution. Isolating RAD tags is an important part in RAD marker and mapping. Isolated RAD tags can be used to identify and genotype DNA sequence polymorphisms mainly in form of SNPs (Chutimanitsakun et al., 2011). 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 RADP technique was invented and used as soon as PCR technique has been introduced. Even though RAPD technique doesn’t give a high reliable consequence, it is used due to low cost and easy way to use. RAPD is used to evaluate genetic diversity. In fisheries and aquaculture, RAPD is used to estimate genetic diversity and genetic difference among populations. For instance, five randomly primer pairs are used to compare genetic difference between freshwater prawn strains in Viet Nam and China. . The result showed that there are genetic different between those strain. There are 14.9 alleles of fresh water prawn in China, and 12.9 alleles for Viet Nam strain. Genetic diversity value of Chinese prawn strain is 0.156; 0.84-0.88 and 0.179; 0.86-0.88 for Viet Nam strain (Nguyen Thanh Tam and Pham Thanh Liem, 2012). The authors concluded that fresh water prawn in China in this experiment may be of the same species of Macrobrachium rosenbergii that has had long domesticated process. In addition, genetic variation of grass carp and common carp populations could be evaluated by using RAPD (Huaiyun et al., 1998). As consequently, RAPD patterns of grass were significant different from those of common carp. Genetic distance and band sharing between them were 0.2583 and 0.2394. In contrast, RAPD patterns of red common carp and common carp were pretty similar to each other. Genetic distance and band sharing between them were 0.7612 and 0.0947. The genetic variability of grass carp, red common, and common carp were 0.18, 0.29, 0.26, respectively. Besides, Bardakci and Skibinski (1994) used RAPD to identify three species of tilapia genus Oreochromis and four subspecies of O. niloticus. They 9 manipulated thirteen random primers for detecting polymorphism within as well as between populations. Different RAPD patterns were inspected for varies species. The result indicated that RAPD marker was possibly subservient for investigation of species as well as subspecies. Also, RAPD was used to evaluate genetic diversity and genetic distance among redbreast sunfish that lived in reference and contaminated streams (Nadig et al., 1998). They used thirteen primers which produced 45 polymorphic bands among all population. Consequently, fish that lived in contaminated streams had little different result to the others. They had less genetically distant from each other than they lived in reference sites. In sum, application of RAPD could be useful for analysis differences in genetic distance between populations of sunfish. 2.5.2 Studies of genetic diversity of species using ISSR ISSR is not as popular to use as RAPD. In the past, there were also many researchers used ISSR in genetic diversity study or other purposes. However, they have been applied more in plants, and less for animals including fish. Some studies in fish using this type of marker have been published recently. Schulz (2004) with his assistants used ISSR to assess DNA variations of the noble crayfish (Astacus Astacus L.) in Germany and Poland. In this research, total 22 unambiguous and polymorphic markers were detected. The result showed that the number of polymorphic loci in one population ranged from 4 to 19. The author concluded that ISSR proved suitable for DNA variation and establishing separation of the stock. The study also showed the correlation of the result on the local, regional and supra-regional levels. In addition, Varela (2007) with his assistants applied ISSR to assess genetic differentiation in six sampling localities distributed along the European Atlantic coast to expose the potential of these markers in genetic studies and as a source of sequences for developing microsatellite markers. As the result, 51 ISSR markers were found 148 microsatellites and 2 polymorphic microsatellites were developed. The author found that mussels of a sampling locality in the Baltic Sea were not significantly different from a pure M. edulis locality supporting an extensive introgression of M. edulis in these individuals. In another case, ISSR was used to analyze genetic variation of the bivale G.gema between marine and Virginia USA, among 10-m-diameter 10 patches within localities, and within patches (Casu et al., 2005). They investigated 30 individuals/patch and 3 patches/locality. Five primers ISSR were used and found out 67 polymorphic loci. The result revealed significant differentiation at individual and patch levels while genetic variation between localities was low. 2.6 PCR reaction method Polymerase chain reaction (PCR) enables researchers to produce millions of copies of a specific DNA sequence in approximately two hours. This automated process bypasses the need to use bacteria for amplifying DNA. 2.6.1 Principle of PCR reaction PCR method (polymerase chain reaction) was invented in 1985 by Kary Mullis with his assistances. This method uses pairs of primer to synthesize a large amount of transcriptions from a special DNA sequence base on activity of enzyme polymerase. PCR method base on activity of DNA polymerase in proceed of synthesizing a new DNA from primary DNA. We need short DNA segments as primer for taq DNA polymerase to anneal to the complementary sequences. After that, taq polymerase attaches to priming sites and extend to synthesize the new perfect strand. PCR reaction is a chain of continuous sequences, each sequence include 3 stages: Denature DNA: separate DNA into single strands. Sample is heated to 94-960C in one to several minutes. Anneal primer: temperature is lower to 50-650C for one to several minutes. This step allows the left and right primers to anneal to their complementary sequences. This stage decides the specificity of reaction. Extension: temperature is again raised to 720C for one to several minutes. This allows taq polymerase to attach at each priming site and extend (synthesize) a new DNA strand. 2.6.2 Some main factors affect PCR reaction There are many factors affect PCR reaction as template DNA, enzymes (taq polymerase), type and concentration of primers, temperature and time, nucleotide concentration, buffer, Mg2+ concentration, dNTP, number of 11