International Journal of Aquatic Biology (2015) 2(4): 274-281 ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.NPAJournals.com © 2015 NPAJournals. All rights reserved Original Article Evaluation of 5.8S rRNA to identify Penaeus semisulcatus and its subspecies, Penaeus semisulcatus persicus (Penaeidae) and some Decapoda species Zahra Noroozi1, Seyed Javad Hosseini*1, 21 1Cell and Molecular Biology Department, Faculty of Science, Persian Gulf University, Bushehr, Iran. 2Biotechnology group of Persian Gulf Institute, Persian Gulf University, Bushehr, Iran. Article history: Received 1 July 2014 Accepted 2 June 2015 Available online 2 5 August 2015 Keywords: Genetic variation Nuclear marker Crustacean Decapoda Abstract: The green tiger prawn, Penaeus semisulcatus is one of the most important members of the family Penaeidae in the Persian Gulf. Based on the morphological characteristics, two groups, including P. semisulcatus and its subspecies viz. P. s. persicus are recognized. This study was conducted to investigate the genetic distance between P. semisulcatus and P. s. persicus by analyzing partial sequence of 5.8S rRNA. Another objective of this study is to evaluate the ability of 5.8S rRNA to identify the species of Decapoda. The results indicated that the 5.8S rRNA gene of both P. semisulcatus and P. s. persicus were exactly identical, and sequence variation was not observed. The results also indicated that 5.8S rRNA sequences between species of the same genus of analysed species of Decapoda are conserved, and no genetic distance was observed in species level. The low evolutionary rate and efficient conservation of the 5.8S rRNA can be attributed to its role in the translation process. Introduction Penaeid shrimps are the most important economic resource in the world’s crustacean fishery industry (Voloch et al., 2005; Holthuis, 1980; Dall et al., 1990). The genus Penaeus has 27 species (Holthuis, 1980) and among them, the green tiger shrimp (Penaeus semisulcatus) is included more than 90% of shrimp fishing in the Persian Gulf (Hosseini et al., 2004). Based on the morphological characteristics, two groups of P. semisulcatus are distinguished in the Persian Gulf. The first group (I) is characterized by a reddish body color with deep red or brown transverse bands, and cream and brown striped color of the whip antenna. Second group (II) i.e. subspecies of P. s. persicus, is characterized by a creamy pink body color without distinct transverse stripes, and its whip antenna has a cream color without stripes (Rahnama et al., 2010). The group I is the main species in the coast of Hormozgan * Corresponding author: Seyed Javad Hosseini E-mail address: sjhosseini@pgu.ac.ir Province but group II is found in the coast of Bushehr Province. The subspecies of P. s. persicus has been described based on the carapace morphology and protein electrophoresis patterns (Matinfar, 1999). The identification of shrimps traditionally are relied on morphometric analysis; however, it is well- known that such characteristics are influenced by environmental conditions (Bowman et al., 1982). To overcome this problem, molecular markers e.g. nuclear and mitochondrial DNA have been developed in the past two decades for study of the phylogenetic relationship and genetic diversity of such an aquatic organism (Ferguson and Danzmann, 1998; Liu and Cordes, 2004; Chauhan and Rajiv, 2010; Askary et al., 2013). Both nuclear and mitochondrial sequences are used for species identification and genetic diversity evaluation. The mitochondrial genes of 16S rRNA and subunit I of cytochrome oxidase (COI) were extensively used for 275 Noroozi and Hosseini/ Evaluation of 5.8S rRNA to identify Decapod species molecular study of the crustaceans especially shrimps of the family Penaeidae (Lavery et al., 2004; Chan et al., 2008; Nayak and Umadevi, 2012). Of the nuclear genes, 28S ribosomal RNA, 18S rRNA, 5.8S rRNA, phosphoenolpyruvate carboxy kinase and sodium-potassium ATPase α-subunit have been considered for the study of the phylogenetic relationships among shrimp (Porter et al., 2005; Calomata et al., 2009; Ma et al., 2009). The molecular comparison of P. semisulcatus in Persian Gulf with its subspecies, P. s. persicus using mitochondrial 16S rRNA showed a significant difference (Rahnema et al., 2010). The genetic distance between them, based on a 561 bp section of the mitochondrial 16S rRNA was calculated as 3.3% (Rahnema et al., 2010). The high mutation rate of the mitochondrial DNA limits its utility in the phylogenetics of deep divergences. Furthermore, the highly A/T-biased mitochondrial DNA, especially at the third codon position of the protein coding genes, suffers from high levels of homoplasy and thus exhibits strong negative effects in phylogenetic analyses (Chu et al., 2009). Therefore, it is necessary to evaluate the genetic distance between P. semisulcatus and the subspecies of P. s. persicus based on a proper and robust molecular marker. Of the nuclear marker, the 5.8S rRNA is considered for the study of the phylogenetic relationships among shrimps (Calomata et al., 2009) and other organisms (Gulling and Voglers, 1998). Hence, in the present study, we investigated the genetic distance between P. semisulcatus and P. s. persicus by analyzing partial sequence of 5.8S rRNA. Another objective of this study is to evaluate the ability of 5.8S rRNA to identify the different species of Decapoda. Materials and methods Sample collection and genomic DNA extraction: 10 and 8 specimens of P. semisulcatus and P. s. persicus were collected from Bushehr and Dayyer (Bushehr Province, South of Iran), respectively. Total genomic DNA was extracted from 100-150 mg muscle tissue from the ethanol-preserved samples according to Brandfass and Karlovsky (2008). DNA amplification: The partial sequence of 5.8S rRNA and complete sequence of internal transcribed spacer-II were amplified using ITS2F (5’ GATCACTTGGCTCGTGCGTC 3’) and ITS2R (5’ GCTCTTCCCGTTTCGGTCGC 3’) primers. These primers have been designed based on 5.8S rRNA sequence of P. merguiensis (AY331590) and P. vannamei (AF 124597) and 28S rRNA of P. semisulcatus (DQ079809) and P. vannamei (AF 124597), respectively. In fact, the ITS2F was designed to amplify of a 150 bp (93%) fragment of 3’ end of 5.8S rRNA. In the first stage, PCR conditions were optimized using DMSO (0, 2, 4 and 6%) and annealing temperature gradient. Polymerase chain reactions (PCRs) were performed in 50 μl volume, containing 5 μl Mg2+ free-PCR buffer (10X), 3 mM MgCl2, 0.4 mM of dNTP mix, 20 pico mole of each primer, 2.5 U Taq DNA polymerase (Fermentas), 200-400 nanogram DNA, and ddH2O. The PCR reaction was carried out according to the following thermal program: 4 min at 94°C for initial denaturation, followed by 30 cycle with 94°C for 1 min, 59°C for 45 sec and 72°C for 1.5 min. The final extension was at 72ºC for 5 min. The negative control reactions were also used. The size and quality of PCR products were visualized on 1% agarose gel. DNA sequencing and analysis: Three samples of P. semisulcatus (Sem3, Sem4 and Sem6) and three samples of P. s. persicus (Per3, Per4 and Per6) were selected for sequencing. PCR products were sequenced in both strands, using the same primer pairs for PCR. The sequencing was performed using ABI 370 automated sequencer (Seq/Teqh/California, USA). Chromatograms of each of the forward and reverse sequences were checked using ChromasPro and Chromas LITE softwares (Technelysium Pty Ltd, Australia). The sequences confirmation and homologies were searched using Blast (NCBI). The sequences were aligned using the multiple– alignment program ClustalW2 (Larkin et al., 2007). Base composition was calculated using MEGA6 software (Tamura et al., 2013). The sequences distance matrix was calculated using Kimura 2- Parameter (K2P) (Kimura, 1980) and subjected to 276 International Journal of Aquatic Biology (2015) 3(4): 274-281 the construction of neighbor-joining tree with 1000 bootstrap replicates (Tamura et al., 2013). Other conditions, for calculation of the genetic distance and tree construction include: transition and transversion substitutions, uniform rate among sites, homogeneous (same) pattern among lineages and complete deletion. For phylogenetic evaluation, the 5.8S rRNA of other available species of the decapoda were retrieved from GenBank database following Blast search. At least two species of each genus were selected (Table 1). Then, their 114 bp of 5.8S rRNA 3’ end were selected for phylogenetic reconstruction. All molecular analyses include sequence alignment, nucleotide composition, the pattern of nucleotide substitution, pairwise sequence distance and phylogenetic tree were conducted in MEGA6 (Tamura et al., 2013). Results and Discussion The nuclear DNA marker has been widely recruited for studies of phylogenetic relationship of crustacean (Ahyong et al., 2007; Liu and Cordes, 2004; Porter et al. 2005). The DNA-based nuclear molecular markers can be classified into two types, the nuclear ribosomal RNA (rRNA or rDNA) genes and protein –coding genes (Ma et al., 2009; Tsang et al., 2008; Blanck et al., 2013). The nuclear ribosomal DNA has three rRNA genes (5.8S, 18S and 28S rRNA) and two internal transcribed spacers (ITS-I and ITS-II). The ITS-I and ITS-II are located between 18S and 5.8S rRNA and 28S rRNA, respectively (Gillespie et al., 2006). Analysis of 5.8S rRNA between P. semisulcatus and P. semisulcatus persicus: The 5.8S rRNA section of PCR products were well-sequenced using ITS2F primer pairs, and reverse sequencing was failed. Therefore, only 114 bp section of the 3’ end of 5.8S rRNA, corresponds to more than 70% of the 5.8S rRNA, was obtained from 6 studied samples. The base composition of the 5.8S rRNA fragment of P. semisulcatus and P. s. persicus samples was as Taxonomic designation Abbreviation Accession number Region used Penaeus vannamei P.vann AF124597 853-967 Macrbrchium rosenbergii M.ros HM804252 1180-1294 Macrbrchium nipponense M.nipp GQ369796 1519-1633 Exopalaemon carinicauda E.car GQ369794 469-583 Exopalaemon cf. modestus E.cfmod GQ369793 685-799 Pandalus goniurus Pa.gon EF035129 450-564 Pandalus hypsinotus Pa.hip AB193480 970-1021 Pandalus eous Pa.eou AB193477 790-904 Eriocheir japonica E.jap AF316381 382-496 Eriocheir leptognathus E.lept AF316385 385-499 Eriocheir formosa E.for AF316375 389-503 Epilobocera sinuatifrons Ep.sin FN395447 616-779 Sesarma meridies S.mer FN396099 457-571 Sesarma dolphinum S.dol FN396039 468-582 Chionoecetes japonicus Ch.jap HQ909101 866-980 Chionoecetes opili Ch.opi HQ909100 942-1056 Table 1. Accession numbers of the materials of decapod species retrieved from GenBank database. Table 2. Maximum composite likelihood estimation of the pattern of nucleotide substitution. Rates of different transitional substitutions are shown in bold and those of transversionsal substitutions are shown in italics. 277 Noroozi and Hosseini/ Evaluation of 5.8S rRNA to identify Decapod species follows: A:17.5, T:24.6, G:26.3 and C:31.6. The multiple sequence alignment (Fig. 1) indicated that the 5.8S rRNA gene of both studied taxa were exactly identical, and no variation was observed. Analysis of 5.8S rRNA variation among Decapoda: The pattern of nucleotide substitution between analyzed decapoda is shown in Table 2. The rate of substitution of Thymine by Cytosine was 18.41%. The nucleotide frequencies were 23.16% (A), 21.08% (T/U), 29.00% (C), and 26.75% (G). The transition/transversion rate ratios were k1 = 1.273 (purines) and k2 = 2.453 (pyrimidines). The overall transition/transversion bias is R = 0.915, where R = [A*G*k1 + T*C*k2]/[(A+G)*(T+C)]. The alignment of 5.8S rRNA resulted 129 sites. The aligned sequences showed that the GC content was more than AT content (50.4% to 57.9% in Pandalus and Penaeus, respectively) and GC content average was calculated as 55.7%. The 129 sites of the 5.8S rRNA gene were containing 74 conserved and 55 variable and parsimony informative sites. The average distance between all taxa was 0.339 and ranged from 0.00 between the species of one genus to 0.707 between Penaeus and Exopalaemon (Table 3). The phylogenetic tree was inferred using the neighbor-joining method (Saitou and Nei, 1987) in Table 3. The 5.8S rRNA gene distance among some decapoda species analyzed by pairwise distance calculation using Kimura two-parameter model. Standard error estimate(s) are shown above the diagonal and were obtained by a bootstrap procedure (1000 replicates). Figure 1. Alignment of Penaeus senisulcatus, and its subspecies based on a 114 pb fragment of 5.8S rRNA (Sem3, 4 and 6= P. senisulcatus and Per3, 4 and 6= P. s. persicus). 278 International Journal of Aquatic Biology (2015) 3(4): 274-281 MEGA6 software (Tamura et al., 2013) based on K2P (Fig. 2). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Fleckenstein, 1985). The same tree topology was obtained by minimum evolution, UPGMA and maximum likelihood (Tamura et al., 2013). The results of molecular analyses indicated that except for Chionoecetes, no genetic variation observed between species of same genus among the studied members of Decapoda. The constructed neighbor-joining tree separated taxa into three major clades, including clade A consisting Sesarma (Sesramidea), Epilobocera (Pseudothelphusidae), Eriocheir (Varunidae) and Chionocetes (Oregoniidae), clade B consists of Pandalus (Pandalidae), Macrobrachium and Exopalaemon (Palaemonidae) and clade C consists of Penaeus (Penaeidae). The results indicated that clades A and B are closer to each other than to clade C. This result predictable, because, the members of the clades A and B taxa belong to suborder Pleocyemata and clades C to suborder Dendrobranchiata. The divergences within clades A, B, and C were 0.0- 0.039, 0.0-0.136 and 0.0, respectively. The highest genetic diversity was estimated between the genus Penaeus and Exopalaemon (Table 3), and the average distance between all taxa was 0.339. The maximum genetic distance (0.515) between the genus Penaeus and exopalaemon, and minimum genetic distance (0.00) between the genus Eriocheir and Epilobacera were calculated, and the average genetic distance was calculated to be 0.185. When the full length of 5.8S rRNA was used, compared with 114 bp section of the 3’ end of 5.8S rRNA gene, genetic diversity is decreased. This suggests that 5.8S rRNA 5’ end, compared with 3’ end, is the most conserved. Similar to other nuclear genes, the 5.8S, 18S and 28S rRNA genes evolve relatively slowly and are useful in addressing broad phylogenetic hypotheses involving a broad range of organisms i.e. a high level taxonomy (Gulling and Voglar, 1998). Molecular studies using nuclear protein-coding genes indicated that they are highly informative for phylogeny estimation across all taxonomic levels of Decapoda (Chu et al., 2009). In addition, this study suggests that evolutionary rate of protein-coding genes are more than rRNA genes. This phenomenon could be due to the fundamental role played by rRNA in translation. The 5.8S rRNA plays an important role in mRNA translation (Elela and Nazar, 1997; Graifer et al., 2005). Studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides have suggested that 5.8S rRNA plays an important role in eukaryotic ribosome function (Elela and Nazar, 1997). References Askari G., Shabani A., Kolangi Miandare H. (2013). Application of molecular markers in fisheries and aquaculture. Scientific Journal of Animal Science, 49(2): 82-88. Ahyong S.T., Lai J.C., Sharkey D., Colgan D.J., Ng P.K. Figure 2. Topologies resulting from the neighbor-joining analysis of the nucleotide sequences of the 114 bp 5.8S RRNA genes in all species of the Decapoda (Numbers above and below branches indicate bootstrap values from NJ analysis. A, B and C refer to three main clades in the tree belongings to Brachyura, Caridea and Penaeadea infraorder, respectively). 279 Noroozi and Hosseini/ Evaluation of 5.8S rRNA to identify Decapod species (2007). Phylogenetics of the brachyuran crabs (Crustacea: Decapoda): the status of Podotremata based on small subunit nuclear ribosomal RNA. Molecular Phylogenetic Evolution, 45(2): 576-86. Blanck D.V., Valenti W.C., Freitas P.D., Junior P.M.G. (2013). Isolation and characterization of SNPs within HSC70 gene in the freshwater prawn Macrobrachium amazonicum. Conservation Genetic Resource, 5(3): 631-633. Bowman T.E., Abele L.G. (1982). Classification of recent crustacea, In: L.G. Abele (Ed.). The Biology of the Crustacea. Systematics, the Fossil Record and Biogeography, Academic Press, New York, pp: 1-27. Brandfass C., Karlovsky P. (2008). Upscaled CTAB- based DNA extraction and Real -TIme PCR assays for Fusarium culmorum and F. graminearum DNA in plant material with reduced sampling error. International Journal of Molecular Sciences, 9: 2306- 2321. Calomata P., Pascoal A., Fernandez I., Bohme K., Gallardo J. (2009). Evaluation of a novel 16S rRNA/tRNAVal mitochondrial marker for the identification and phylogenetic analysis of shrimp species belonging to the superfamily Penaeoidea. Analytical Biochemistry, 91(2): 127-134. Chan T.M., Tong J., Tam Y.K., Chu K.H. (2008). Phylogenetic Relationships Among the Genera of the Penaeidae (Crustacea: Decapoda) Revealed by Mitochondrial 16S rRNA Gene Sequences. Zootaxa, 1694: 38-50. Chauhan T., Rajiv K. (2010). Molecular markers and their applications in fisheries and aquaculture. Advanced in Bioscience and Biotechnology, 1: 281-291. Chu K.H., Tsang L.M., Ma K.Y., Chan T.Y., Ng P.K.L. (2009). Decapoda phylogeny: What can protein- coding gene tell us? In: J.W. Martin, K.A. Kerandall, D.L. Felder (Eds.). Decapoda Crustacean phylogenetic. CRC Press, pp: 89-110. Dall W., Hill B.J., Rothisberg P.C., Sharples D.J. (1990). The biology of the Penaeidae. In: J. H. S. Blaxter, A. J. Southward (Eds.). Advances in Marine Biology. Academic Press, New York, 487 pp. Elela S.A., Nazar R. N. (1997). Role of the 5.8S rRNA in ribosome translocation. Nucleic Acids Research, 25(9): 1788-1794. Felsenstein J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39:783- 791. Ferguson M.M., Danzmann R.G. (1998). Role of genetic markers in fisheries and aquaculture: useful tools or stamp collecting?. Canadian Journal of Fisheries and Aquatic Sciences, 55(7): 1553-1563. Gillespie J.J., Johnston J.S., Cannone J.J., Gutell R.R. (2006). Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements. Insect Molecular Biology, 15: 657-686. Graifer D., Molotkov M., Eremina A., Ven’yaminova A., Repkova M., Karpova G. (2005). The central part of the 5.8 S rRNA is differently arranged in programmed and free human ribosomes. Biochemical Journal, 387: 139-145. Gulling K.W., Voglar D.R. (1998). A 5.8S nuclear ribosomal RNA gene sequence database: application to ecology and evolution. Molecular Ecology, 7: 919- 923. Holthuis L.B. (1980). FAO Species catolog. Shrimp and prawns of the word. FAO Fishery Synopis No. 125: 1- 271. Hosseini S.J., Elahi E., Raei M. (2004). The chromosome number of the Persian Gulf shrimp Penaeus semisulcatus. Iranian International Journal of Science, 5: 13-23. Kimura M. (1980). A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16: 111-120. Larkin M.A., Blackshields G., Brown N.P., Chenna R., McGettigan P.A., McWilliam H., Valentin F., Wallace I.M., Wilm A., Lopez R., Thompson J.D., Gibson T.J., Higgins D.G. (2007). Clustal W and Clustal X version 2.0. Bioinformatic, 23(21): 2947-48. Lavery S., Chan T.Y., Tam Y.K., Chu K.H. (2004). Phylogenetic relationships and evolutionary history of the shrimp genus Penaeus s.l. derived from mitochondrial DNA. Molecular Phylogenetic Evolution, 31(1): 39-49. Liu Z.J., Cordes J.F. (2004). DNA marker technoloigies and their applications in aquaculture genetics. Aquaculture, 238: 1-37. Ma K.Y., Chan T. Y., Chu K.H. (2009). Phylogeny of penaeoid shrimp (Decapoda: Penaeoidea) inferred from nuclear protein – coding genes. Molecular phylogenetic Evolution, 53:45-55 Matinfar A. (1999). Study and specification of species 280 International Journal of Aquatic Biology (2015) 3(4): 274-281 and population of green tiger shrimp in northern Persian Gulf, PhD dissertation, Azad University, Tehran Science and Research Unit Nucleic Acids Research, 25(9): 1788-1794. Nayak S., Umadevi K. (2012). In silica comparative molecular phylogeny of mitochondrial 16S rRNA and COI genes of the spiny lobster genus Panurlirus (Decapoda: Palinuridae). Advanced Bioinformatics Applications and Research, 3: 364-373. Porter M. L., Perez–Losada M., Cranall K. A. (2005). Model-based multi-locus estimation of decapod phylogeny and divergence times. Molecular Phylogenetics and Evolution, 37: 355-369. Porter M.L., Perez-Losada M., Crandall K.A. (2005). Model-based multi-locus estimation of decapod phylogeny and divergence times. Molecular Phylogenetics and Evolution, 37: 355-369. Rahnama R., Hosseini S.J., Qasemi S.A., Yavari V., Zolgharnein H., Matinfar A. (2010). Preiminary molecular comparison of P.(penaeus) semisulcatus of Persion Gulf and its subspecies Penaeus semisulcatus persicus using 16S rRNA. Modern Genetics, 25: 23- 31. Saitou N., Nei M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4: 406-425. Tamura K., Stecher G., Peterson D., Filipski A., Kumar S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30: 2725-2729. Tsang L.M., Ma K.Y., Ahyong S.T. Chan T.Y., Chu K.H. (2008). Phylogeny of Decapoda using two nuclear protein-coding genes: origin and evolution of the Reptantia. Molecular Phylogenetic Evolution, 48(1):359-68 Voloch C.M., Freir P.R., Russo C.A.M. (2005). Molecular phylogeny of penaeid shrimps inferred from two mitochondrial markers. Genetics and Molecular Research, 4: 668-674. International Journal of Aquatic Biology (2015) 3(4): 274-281 ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.NPAJournals.com © 2015 NPAJournals. All rights reserved چکیده فارسی ،و زیرگونه آن Penaeus semisulcatus میگوی سبز ببری در تشخیص 5.8S rRNAارزیابی ژن Penaeus semisulcatus persicus (Penaeidae) ای هو برخی گونهDecapoda 2، 1*، سید جواد حسینی1زهرا نوروزی شناسی سلولی و مولکولی، دانشکده علوم، دانشگاه خلیج فارس، بوشهر، ایران.زیست گروه1 ، دانشگاه خلیج فارس، بوشهر، ایران.خلیج فارس انستیتوبیوتکنولوژی گروه2 چکیده: این مطالعه برای بررسی در خلیج فارس است. Penaeidaeخانواده مهم بسیار یکی از اعضای Penaeus semisulcatusمیگوی سبز ببری، دیگر این هدف .به اجرا درآمد 5.8S rRNAوسیله آنالیز توالی بخشی از ژن هب P. s. persicus و P. semisulcatusفاصله ژنتیکی بین .Pهر دو گروه 5.8S rRNA ژن کهپایان است. نتایج نشان داد های دهبرای تشخیص گونه 5.8S rRNAتحقیق ارزیابی قابلیت ژن semisulcatus و P. s. persicus .ژنهای توالی که کهنشان داد همچنین نتایج کامالً مشابه هستند و تنوعی در توالی آنها مشاهده نشد 5.8S rRNA اهده ای مشو هیچ فاصله ژنتیکی در سطح گونه بودهحفاظت شده پایان های آنالیز شده دههای مشابه گونههای جنسبین گونه .نسبت داده شود ترجمهتواند به نقش آن در فرایند می 5.8S rRNAو حفاظت موثر ژن نرخ پایین تکاملی شد.مین .پایانده، پوستان سخت، ایتهسه نشانگر، تنوع ژنتیکی: کلمات کلیدی