USE OF EPIC INTRONS AS NUCLEAR DNA MARKERS FOR THE

CHAPTER II USE OF EPIC INTRONS AS NUCLEAR DNA MARKERS FOR THE

CHARACTERIZATION OF SPECIES IN THE Himantura uarnak SPECIES COMPLEX Abstract Nuclear-DNA markers DNA have been widely used to characterize the species and populations. Intron used as an alternative of molecular marker to know the capable of providing extensive information related to nuclear DNA polymorphism even just using a universal primer. Polymorphism alleles at the species or population level causing the genetic variability that can occur as a result of gene flow inter-oceanic. The purpose of this study is to determine the genetic information of allele’s polymorphism on each individual in Himantura uarnak species complex. The research method used intron length polymorphism markers in a nuclear DNA using PCR-Exon Primed Intron Crossing EPIC. The study showed two marker EPIC has produced polymorphism alleles in individuals and formed 4 clusters different species. Two clusters I and IV are cryptic species currently under H. leoparda census Manjaji-Matsumoto and Last. Keywords: EPICs, Intron, Cryptic spesies, Himantura uarnak species complex, Stingray Introduction Microsatellite and introns are molecular markers that are currently being developed and in demand by many researchers in the field of population genetics Wainwright et al. 2012; Quattro et al. 2006; Berrebi et al. 2006; Atarhouch et al. 2003. Excess from microsatellite are widely used and can provide high-quality data, but requires a primer with a very specific design and require considerable time to the development of microsatellite marker Atarhouch et al. 2003; Wainwright et al. 2012. In addition to the transcribed part of the genome, many other parts that are not transcribed used as a genetic marker, such as introns Quattro et al. 2006. The development of the current science showed that introns are known to have their own meaning, which have been found in the introns of genes in eukaryotic organisms, and there are variety of genes, including genes that produce proteins and thought to have role in the evolution from an organism Penny et al. 2009. Application of DNA molecular markers from the core have been widely used in the characterization of species, populations, and intraspecific genetic structure. Introns are not part of gene involved in the process of mRNA translation to form a protein. Intron as an alternative molecular marker has a system that can provide extensive information related to nuclear DNA of polymorphism even just using universal primers. The molecular markers do not require knowledge of the genome to be analyzed, however, generally are polymorphic and sometimes hipervariabel. Introns can be easily amplified by PCR and visualized on agarose gel or gel akrilamide. This marker can generate data at a cost that is much less expensive than other core DNA molecular marker Atarhouch et al. 2003. The use of the marker PCR primer pair exon primed intron-crossing EPIC can amplify multiple loci simultaneously. The amplification results may reflect the condition polyploidization and tandem duplications that happened before, or other phenomena that might occur during the process of evolutionary divergence of these organisms Atarhouch et al. 2003; Berrebi et al. 2006. EPIC-PCR are molecular marker from introns that have several advantages, such as can be used to investigate the genetic of polymorphism in the the genome core. Utilization of conserved sequence of bases from exon can be done as PCR primers were attached. The advantage is the primary location to be sure and not going misprime and region of polymorphism in intron pair from loci can be explored properly. The same primer pairs can function in different species, where the introns appear to accumulate the level of genetic of polymorphism that is much larger than the exons on inter and intraspecies Chow 1998. EPIC from Actin and Calmodulin genes has successfully demonstrated the existence of polymorphism alleles is very diverse in fish species from 19 different families Atarhouch et al. 2003. Primers from EPIC markers are universal so it can be used to analyze variety of species and only use the primer pair was able to amplify several different loci from these genes, such as primer pair EPIC actin 2 have successfully amplify loci Actin2 Actin2-A and-B. Similarly, the primary EPIC Calmodulin 3 has successfully amplified loci Calmodulin3-A, Calmodulin3-B and Calmodulin3-C. The resulting fragment sizes varied accross species Berrebi et al. 2006. Some information about EPIC has been available in the literature Palumbi et al. 1991; 2002, and can be designed by using cDNA database Janech et al. 2006; Zhu et al. 2006. Genes that have been isolated from skeletal muscle from Oryzias latipes medaka fish with access code EMBL AB015886 produce skeletal muscle actin gene, known as actin or gene OlMA1 Kusakabe et al. 1999. Actin intron portion from genes that have been used as markers of EPIC is intron 2 Atarhouch et al. 2003; Berrebi et al. 2006. The position of intron 2 was initiated in 1494 to the base sequence of up to 2147 bases all, which has been isolated fragment length of 654 bp Kusakabe et al. 1998. Calmodulin genes known to encode a calcium binding protein that is highly conserved regulate cell function. Several studies indicate that genes Calmodulin literature are one from minority of genes that encode proteins that are highly conserved, phylogenetically widespread Corte-Real et al. 1994. Intron 3 from calmodulin gene has been isolated from fish Xiphias gladius Swordfish SW19A strains derived from the Mediterranian Sea, the size of 359 bp. This gene is also known by another name ie CAM gene with access code EMBL GeneBank AF069912 and AF069913 Chow and Takeyama 2000. Primers were used to amplify intron 3 in the CAM gene 359 bp is located in exon 3 and exon 4. The purpose of this study was to characterize species in the Himantura uarnak species complex using size polymorphic Exon Primed Intron Crossing EPIC intron markers. Materials and Methods Time and place of study Data collection was conducted in August 2006 Arlyza 2006-2008, unpublished to October 2011, covering 8 location in western and eastern Indonesia Figure 2.1. Sample collection for the western region, include: 1 Sunda Strait Banten, 2 Batang Central Java and 3 Baron, south coast of Yogyakarta, while the eastern region, include: 4 Singaraja Bali, 5 Labuan Bajo NTT, 6 Jampea and 7 Makassar south Sulawesi, and 8 Kendari Southeast Sulawesi. Data collection of Himantura uarnak species complex in Indonesian waters presented in Appendix 1. Specimen collection Collection of samples and specimens performed at fish landing sites production center and ordering directly through fishing of stingray collector. Any information relating directly to the documented of samples, such as: sex, length, weight, width, location of catchment, fishing gear, local name, date of sampling, specimen labels, museum code from each species one individual has had a code of museums number, MZB 20875 and still there two more are yet coded. Specimen’s collection only for small to medium size of individual, large size specimens were not collected. After the information and photograph recorded, then the tissue of each individual cutted and preserved in alcohol 95. Whole specimen was immersed in 4 formalin solution in a period of 2-3 weeks and then re-immersed in a solution of 70 alcohol, and deposited to the Museum of Zoology Bogoriense MZB, Cibinong. Figure 2.1 Sampling locations of Himantura uarnak species complex Genetic sample analysis a. DNA extraction The main sample and sub-samples are stored in a solution of 95 alcohol; the alcohol solution was changed every day for three consecutive days at the beginning of sample storage. The tissue samples of each individual were taken as much as 10-25 mg for DNA extraction. In this study, DNA extraction was done by using the Qiagen DNeasy ® Tissue Kit Qiagen GmbH, Hilden, Germany. Stages of processing carried out in accordance with the instructions from the kit manufacturer. b. DNA Amplification with PCR Nuclear DNA used as a template to see fragment length polymorphism from intron using Primed Exon Intron Crossing EPIC was amplified using PCR. In this study, we used two genes i.e. Actin and Cam. Amplification of intron 2 in Actin gene was using universal primers actinatf Act-2F fluo and actinatr Act-2R, whereas intron 3 in gene Cam used primers camf Cam fluo-3F and camr Cam-3R Table 2.1. Table 2.1 Target genes, primer names, and sequences of each primers of EPIC marker Gene, family gene Primer name Sequences primer Actin Act-2-F 5’-GCTATAACCCTCGTAGATGGGCAC-3’ Intron 2 Act-2 Act-2-R 5’-ATCTGGCACCACACCTTCTACAA-3’ Calmodulin Cam-3-F 5’-TGACGGAGCTCTGCAGCACTGAC-3’ Intron 3 Cam-3 Cam-3-R 5’-GTGAGGAGGAGCTCCGTGAGGC-3’ Source: Atarhouch et al. 2003 The second design primer pairs were obtained from results of research conducted by Kusakabe et al. 1998, 1999 for the actin gene and Chow and Takeyama 2000 for Calmodulin genes. The position and length of the both introns from the gene can be seen in Figure 2.2 and Figure 2.3. Figure 2.2 The position of intron-2 in Actin gene from Oryzias latipes AB015886 Kusakabe et al. 1998, 1999; Atarhouch et al. 2003 Figure 2.3 The position of intron-3 in Cam gene from Xiphias gladius AF069913 Chow and Takeyama 2000; Atarhouch et al. 2003 Both the primers have been applied by Atarhouch et al. 2003 on several species of fish from the sea and fish from freshwater Table 2.2 with PCR products are very diverse. Table 2.2 The application of primers Act-2-FAct-2-R and Cam-3-FCam-3-R on several fish that have been investigated by Atarhouch et al. 2003 Fish species Primers Act-2FAct-2R Cam-3FCam-3R Ta = 58°C c Ta = 54°C Ta = 52°C Sardina pilchardus 380M, 400P 470M, 580P Sardinops melanosticus a 380M, 400M 550P Engraulis encrasicolus 400P - Scomber scombrus 350P, 680P 450P Merluccius merluccius 700P - Lichia amia 340M, 700M, 800P 380P Salmo marmorata 330M, 400P, 510M, 540M, 730M 450P Barbus carpathicus 4n 370M, 600M - Zacco platypus 350M, 450M, 550M - Opsariichthys bidens 350M, 450M, 550M - Cichla monoculus 390M, 680P, 700M, 850M c 190M, 210M Cyprichromis leptosomab 350M, 650P 110P, 180M, 420M Mauligobius maderensis b 320, 600 360M Pomatoschistus microps 280M, 350M, 550M, 950P 540M Pomatoschistus minutus 520M, 1100M - Pomatoschistus marmoratus 110P, 350M, 550M, 1000P 540M Note: c Ta = 54°C; independent loci = 320, 600; M Monomorphic, P Polymorphic PCR amplification to generate polymorphic alleles using a total reaction volume of 10μl master mix with the following composition: 4.8 µl ddH2O, 10xPCR buffer Exon 2 Exon 3 nt 1494 nt 2147 Intron 2 654 bp 2913 bp 2913 1 Exon 3 464 1 Exon 4 nt 58 nt 416 Intron 3 359 bp 464 bp Promega 1 μl, 2mM dNTPs Invitrogen 1 μl, 25mM MgCl 2 1 μl, each primer 0,5 μl MWG-Biotech AG, labeled with Cy5 or fluorescein, Taq polymerase Sigma 0.2 µl and 1 μl DNA extract. PCR run for 35 cycles with initial conditions of denaturation 95°C for 2 min, denaturation 95°C for 1 min, annealing 54 o C for Actin-2 loci and 55 o C for CAM-3 loci for 1 min, extension 72 o C for 1.5 minutes and 10 minutes for the final extension 72 o C. Results in the form of amplification of polymorphism alleles were visualized with a mixture consisting of 2.5 µ l PCR amplification product plus 2.5 µ l loading dye were loaded into the gel wells akrilamide 8 denatured Biorad. Amplification result of polymorphism alleles contained in the gel were visualized by scanning akrilamide FMBIOs using fluorescent imaging system Hitachi. Allele sizes were determined by fluorescent labeled ladder Promega using 8.0 ANALYSIS FMBIO image analyzer program Berrebi et al. 2006. c. Genotyping for loci Actin and Calmodulin Allele size of PCR products from both genes were compared with fluorescent labeled ladder Promega by using a scanning FMBIO ANALYSIS 8.0 image analyzer program. The result can be read manually or use a program analyzer through a computer connected to the device. Genotyping is done by observing the alleles that exist in each individual and determine the pair of alleles at each of these individuals to obtain genotype individuals homozygous and heterozygous genotype. Homozygous genotype is shown with a thick ribbon on the size of single basepair bp specific, whereas the heterozygous genotype will feature two bands at different base sizes, where the tape of the bottom tended to be thicker than the top of the ribbon. d. Data Analysis A total of 115 individuals of Himantura uarnak species complex consist of 113 from Indonesia, one individual from Zanzibar and one from Taiwan. Data analysis was performed by observing the nuclear DNA of polymorphism alleles from each individual. Variation in genotypic frequencies will be analyzed to infer genetic differentiation. Opportunity for cryptic species will be detected from individual genotypes based on nuclear DNA Act-2A, Act-2B, Cam-3A, Cam-3B and Cam-3C. To estimate the level of genetic differentiation within populations, were analyzed using a Correspondent Analysis CA based on the data matrix allelic frequency Benzecri 1982 with the program Genetix 4.02 Belkhir et al. 2000 based on each individual genotype at 5 size- polymorphic intron loci. Bayesian analysis of the structure using the software STRUCTURE 2.3.1 Falush et al. 2007 to describe the grouping of individuals based on genetic similarity character from genotype results EPIC maker. This program using Markov Chain Monte Carlo MCMC algorithms for cluster formation in individuals in the population based on multilocus genotype data Pritchard et al. 2000; Falush et al. 2003. The program working based on a mixed model that assumes the allele frequencies in the two populations are correlated. Dimensions of the K value for the parent cluster formation on the number of individuals assigned ingroup ranged from grades 2-6. The STRUCTURE program at an early stage using 50,000 repetitions later increased to 100,000 repetitions. Results and Discussion Alleles produced from two molecular markers of EPIC of nuclear DNA showed high polymorphism Appendix 2. Genotyping results against several individuals in the Himantura uarnak species complex from intron at Cam-3 loci Figure 2.4 showed heterozygous individuals column no. 2 and 7. It shown with different sized alleles are 129 bp and 125 bp, while the individuals homozygous column no. 1, 3 and 6 have the same size of the 129 bp allele. The homozygous individual in column no. 4, 5 and 8 has a size of allele of 125 bp. Figure 2.4 The alleles polymorphism of Cam-3 intron loci Usage of each universal primer of Actin and Cam genes from individuals of Himantura uarnak species complex have been identified to produce 2 loci on the gene of Act-2 i.e. Act-2A and Act -2B, while the Cam-3 gene has been identified that consist of 3 loci i.e. Cam-3A, Cam-3B and Cam-3C. Act-2A loci produces 4 different alleles, they are 186 bp, 187 bp, 190 bp and 192 bp. Act-2A loci produces 4 genotypes Table 2.3 which consist of 2 pairs of homozygous 186186 bp and 190190 bp and 2 pairs of heterozygous 190187 bp and 192190 bp. Act-2B loci produces 3 different alleles i.e. 97 bp, 98 bp and 100 bp, and at this loci there are three genotypes Table 2.3 which consist of one pair of homozygous 100100 bp and two pairs of heterozygous 10097 bp and 10098 bp. Table 2.3 The application of primers Act-2-FAct-2-R and Cam-3-FCam-3-R on Himantura uarnak species complex Family gene Gene Intron Primer name Loci Genotype producing Actin Act-2 2 Act-2-F Act-2A 186186 bp, 190190 bp, Act-2-R 190187 bp, 192190 bp Act-2B 100100 bp, 10098 bp, 10097 bp Calmodulin Cam-3 3 Cam-3-F Cam-3-R Cam-3A 175175 bp, 178178 bp, 178175 bp Cam-3B 163163 bp, 165165 bp, 165163 bp Cam-3C 125125 bp, 129129 bp, 144144 bp, 129125 bp, 144129 bp Cam-3A loci produces 2 different alleles, they are 175 bp and 178 bp and produces 3 genotypes Table 2.3, which consist of two pairs of homozygous 175175 bp and 178178 bp and one pair of heterozygous 178175 bp. Cam-3B loci consists of 2 different alleles, they are 163 bp and 165 bp, and produce 3 genotype consist of two pairs of homozygous 163163 bp and 165165 bp and one pair of heterozygous 165163 bp. Cam-3C produces 3 different alleles 125 bp, of 129 bp and 144 bp, there are as many as the number of genotypes 5 consisting of 3 pairs of homozygous 125125 bp, 129129 bp and 144144 bp and 2 pairs of heterozygous 129125 bp and 144129 bp. Results of this - 129 bp - 125 bp study showed that the alleles from Himantura uarnak species complex different alleles produced by Atarhouch et al. 2003 despite using the same type of primer. Genotypes generated by 5 size-polymorphic intron loci from each marker EPIC Appendix 3, then analyzed using a Correspondence Analysis CA that from each individual in the population from each Himantura uarnak species complex each defined by their genotype at 5 intron loci. Oval-shaped circles depict individual group-rated 90 to one group from three groups 1, 3, and 4 is obtained from the structure of Bayesian analysis. Cluster 2 according to one individual, which alienated Group II = Himantura undulata. Individuals geometrically as intermediates between Cluster 1 with one of the Cluster 3 or Cluster 4 allele interpreted as jointly owned sharing or forms of the same genotype results from a combination of the allele pair Figure 2.5. The four cluster of nuclear DNA in each individual is shown with different colors, namely Claster 1 whitish gray, Claster 2 light gray, Claster 3 dark gray and Claster 4 blackish gray. Figure 2.5 Clustering formation based on Correspondent Analysis CA using genotype data Genotype of Himantura uarnak species complex comprising 113 individuals have been determined based on size of the alleles at 5 size-polymorphic intron loci Act-2A, Act-2B, Cam-3A, Cam-3B and Cam-3C in the nuclear DNA. Determination of criteria based on the order of the sampling locations Batang, Java Sea; Strait of Sunda, Banten; Makassar Strait; Jampea-Selayar islands, Flores Sea; Kendari, Banda Sea; Labuanbajo, Flores Sea; Bali Sea; south of Java, Yogyakarta; Zanzibar, Western Indian Ocean; Taiwan, the Pacific northwest. Individuals that come from Batang are individuals who have allele frequency and highest varied Appendix 3 and 4. Individuals which came from Makassar Strait, Selayar, Kendari, and Labuanbajo have almost 100 frequency of each allele at each loci and clustered in one cluster is Cluster 3 Group III = Himantura uarnak. This signifies that individuals in these clusters have experienced reproductive isolation within the geographic range. Group I have the same allele with Group II, III and IV or the opposite occurs in Act-2B loci, Cam-3A and Cam-3B. Similarity allele that occurs in loci Act-2B consist of alleles 100 bp and 98 bp. Loci of Cam-3A its allele similarities occur at 178 bp and 175 bp, while the Cam-3B loci occurs at 165 bp allele. Differences in allele of Group I with the other group occurred in Group II and IV. Differences in Group II consisted of alleles 190 bp, 187 bp, 144 bp and 97 bp. Differences in Group IV occurs in loci Act-2A with alleles 192 bp and 190 bp, for Cam-3C allele consist of 144 bp and of 129 bp. Two intron loci are Act-2A with genotype 192190 bp and Cam-3C with allele genotype 144129 bp may be appropriate as intron polymorphic markers to separate each species Table 2.4. Table 2.4 The allele frequency of five polymorphic intron loci on Himantura uarnak species complex Locus Act-2A Alleles Alleles frequency Alleles frequency per species H. leoparda var.1 Group I H. undulata Group II H. uarnak Group III H. leoparda Group IV 186 bp 98 1 1 187 bp 1 0,5 190 bp 114 0,5 0,99 192 bp 1 0,01 Total 2n 214 52 2 46 114 Loci: Act-2B Alleles Alleles frequency Alleles frequency per species H. leoparda var.1 Group I H. undulata Group II H. uarnak Group III H. leoparda Group IV 97 bp 1 0,5 98 bp 7 0,06 0,02 0,03 100 bp 108 0,94 0,5 0,98 0,97 Total 2n 216 54 2 46 114 Loci: Cam-3A Alleles Alleles frequency Alleles frequency per species H. leoparda var.1 Group I H. undulata Group II H. uarnak Group III H. leoparda Group IV 175 bp 167 0,1 1 1 0,99 178 bp 49 0,9 0,01 Total 2n 216 54 2 46 114 Loci: Cam-3B Alleles Alleles frequency Alleles frequency per species H. leoparda var.1 Group I H. undulata Group II H. uarnak Group III H. leoparda Group IV 163 bp 47 0,92 0,01 165 bp 159 0,08 1 1 0,99 Total 2n 206 50 2 46 108 Loci: Cam-3C Alleles Alleles frequency Alleles frequency per species H. leoparda var.1 Group I H. undulata Group II H. uarnak Group III H. leoparda Group IV 125 bp 91 0,94 1 129 bp 11 0,06 0,5 0,08 144 bp 82 0,5 0,92 Total 2n 184 48 2 46 88 Allele from each group or cluster is seen as a couple of different alleles heterozygous genotype are interpreted as a hybrid, which is considered a hybrid offspring with allele sharing. There were 43 individuals known to have heterozygous genotype, after the calculated results indicate that there are as many as 3.8 is a hybrid. Group I and IV showed highly variable pattern of genotypes seen with many combinations of alleles, while Group III had genotype patterns with combination of alleles that tend to be consistent and dominant against a particular genotype one pattern homozygous except one individual with the allele pair 100098 bp on Act-2B loci. Frequency of alleles per loci were observed Table 2.4 looks stable on the species H. undulata Group II and H. uarnak Group III, and more varied for H. leoparda var. 1 Group I and H. leoparda Group IV. Two intron polymorphic data from Group I and 3 intron polymorphic data from Group IV were not included in the analysis because the data is only represented by 1- 3 loci, whereas for the analysis process must be represented by at least 4 polymorphic loci Appendix 3. Individual probability assessment on one of the groups 1, 3, 4 are determined by the Bayesian approach of nuclear DNA intron multilocus genotypes Appendix 4. Burn-in is done without the individual sampling location information in estimating the proportion of a mixture of individual and assign individuals to populations. Five independent loci carried on the parent cluster formation K by using the limit value from 2 to 6 to get the informative diagrams. The resulting diagram are most informative on the value of the K = 3. The results from diagram based on Bayesian analysis of population structure aligned with the results of Correspondent Analysis CA Figure 2.6. Figure 2.6 The result of Bayesian population structure analysis, where it was match to Correspondence Analysis CA According to the diagram above, Clade II is in accordance with the individual that alienated the IR007. Individual probabilities from each assessment are shown on the y- axis, the individual is represented by a vertical bar and sorted according to the type clade Clade I-IV displayed on the horizontal bar. Individuals from different groups adjusted to the Correspondent Analysis CA, IR007 marked with an asterisk Cluster 2. White horizontal lines underscore individual with valuation probability 90 that occurred in Cluster 1, 3 and 4. Two EPIC markers were used in this study similar to Atarhouch et al. 2003, namely Actin 2 and Calmodulin 3. Two markers produce different alleles of polymorphism with results obtained by Atarhouch et al. 2003. Polymorphism alleles generated in this study is less than 200 bp. There were as many as 14 total alleles obtained in this study only two alleles have a similar size to the results of research that has been conducted by Atarhouch et al. 2003, they were 100 bp alleles at loci Act-2B and 178 bp allele Cam-3A. Two loci of EPIC marker are Act-2A loci with genotype 192190 bp and P roba bi li ta s ra te Cluster 1 4 3 0.2 0.4 0.6 0.8 1 Clade I IV III II Cam-3C loci with genotype 144129 bp can be used as intron polymorphic markers appropriate to separate each species. The two genotypes have specific characteristics that is only found in two loci and observed only owned by two separate groups of individuals, namely Group II and IV. It shows that have found the sharing of alleles between two groups of individuals. Four different groups of individuals have been defined through the multilocus genotype complex species of H. uarnak. Hybridization has occurred between the clusters were deduced through observation that heterozygous genotype is interpreted as a hybrid. It has been detected with a very low proportion 4 but still showed hybridization has occurred in H. uarnak species complex. Chance of this happening if it really happened will likely be very rare. Incidence of genetic homogeneity across large geographic distances was maintained in each cluster, although there may be a cross between clusters. This is reflected by the formation of 4 species consisting of H. uarnak and H. undulata, while the others were cryptic species that have been released as H. leoparda according to the current definition Manjaji 2004; Manjaji-Matsumoto Last 2008. If there is a shortage of individuals by ancestral mix-species with H. uarnak species complex will support the general view that the hybridization is a rare phenomenon in the group of elasmobranch Ward et al. 2008; Dudgeon et al. 2012. The existence of cryptic species was found in H. leoparda showed that the species is sympatric with large geographic areas Futuyma 2009. Members of H. uarnak species complex have undergone a process of overlap in the broad geographical range and habitat sharing. Based on observation that know geographical distribution of reproductive isolation has occurred in Group III. Individuals in Group III had done isolation mechanism, i.e. individuals which come from Flores Sea Labuan Bajo and Selayar Island, the sea around Kendari to Makassar Strait and the Banda Sea. Individuals were grouped in a single cluster that is separate namely Cluster 3 has a pattern of alleles were detected almost uniform. Conclusions 1. Actin and Calmodulin genes from EPIC markers have demonstrated the existence of polymorphism alleles at each locus were analyzed with the highest number of 4 alleles in Act-2A loci. 2. Heterozygous genotypes of two loci Act-2A and Cam-3C can be used as intron polymorphic markers were correctly for Himantura uarnak species complex. 3. Obtained 4 clusters that show the differences for each species in the complex species of H. uarnak.

CHAPTER III MARKER CYTOCHROME C OXIDASE SUBUNIT I COI OF