Int. J. Aquat. Biol. (2016) 4(1): 57-68 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2016 Iranian Society of Ichthyology Original Article Genetic characterization of Garra rufa (Heckel, 1843) populations in Tigris Basin, Iran using microsatellite markers Hamed Kolangi Miandare1, Ghasem Askari*1, Ali Shabany1, Hamid Reza Rezaei2 1Department of Fisheries, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. 2Department of Environmental Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. Article history: Received 7 November 2015 Accepted 29 January 2016 Available online 2 8 February 2016 Abstract: The isolation-by-distance theory states that the genetic differentiation between individuals raised by increasing geographic distance. Therefore, this study tested this hypothesis for Garra rufa, a freshwater fish species of Iranian inland waters, from six rivers located at the different distances in Tirgis basin. For this purpose, eight variable microsatellite loci were applied to identify geographic-based population structure of G. rufa. From 240 fish of six populations, 102 alleles were found with a mean number of 11.625 to 13.250 alleles. Heterozygosity was ranged 0.567-0.638 in six studied populations. Moreover, a significant deviations from Hardy-Weinberg were found in the studied populations. Unweight pair group analysis indicated that the six studied populations could be divided into four major clusters. The results revealed a fairly high level of genetic variation in the microsatellite loci within six studied populations. Wright’s fixation index (Fst) ranged between 0.013-0.044 indicating little genetic differentiation between populations. Within this range, however, we found a strong positive relation between Fst and geographical distance lending support to the isolation-by-distance theory. Keywords: Genetic diversity Microsatellite Doctor fish Tigris Basin Iran Introduction Genetic diversity is one of the important indicators of ecological condition in aquatic ecosystems and has been considered as a useful and powerful tool for evaluation and management of biological communities (Avise, 2000). It is one of three levels of biodiversity, proposed by the global conservation organization for the stocks conservation program (Lucentini et al., 2009). Since higher genetic diversity may lead to an increase in the survival rate of natural populations (Zoller et al., 1999), maintaining genetic diversity in these populations is crucial for conservation biologists. Of the common DNA markers used to study genetic diversity at the molecular levels, microsatellite markers are especially informative (Chen et al., 2008). Because of unique features such as high polymorphism, high scope in genome, and high mutation rates, the microsatellite markers have * Corresponding author: Ghasem Askari DOI: http://dx.doi.org/10.7508/ijab.2016.01.008 E-mail address: Askarighasem82@gmail.com been widely applied in population genetic studies (Li et al., 2009). Microsatellite markers have been identified in the genomes of many species, and widely used in relation to species with an economic value (Wang et al., 2012). The application of these markers includes aspects of evolutionary biology, population genetics, ecology and pedigree identification in populations (Cui et al., 2005; Cruz et al., 2005; Maremi et al., 2005). There are 257 fish species in the inland waters of Iran (Joulade-Roudbar et al., 2015), which mostly belong to the family Cyprinidae. Garra rufa is the member of Cyprinidae and occurs in the river basins of the Northern and Central Middle East (Keivany et al., 2015; Mousavi-Sabet and Eagderi, 2016). This species has been utilized in psoriasis treatment (Ündar et al., 1990) and are preyed by piscivorous fishes such as Anguilla anguilla and Clarias gariepinus in their habitats (Yalçin-Özdilek, 2007). 58 Kolangi Miandare et al./ Genetic characterization of G. rufa populations in Tigris Basin, Iran Despite the ecological importance of G. rufa, the studies on its genetic structure and demography remain rudimentary. Isolation by distance (IBD) model has been extensively used to study spatial patterns of genetic variation in natural populations (e.g., Crispo and Hendry, 2005; Storfer et al., 2010). According to the IBD theory, the genetic distance between individuals or populations will increase with decreasing in gene flow as a result of increasing geographic distance between them (Wright, 1943; Rousset, 1997). This restriction of gene flow can finally result in speciation events (Coyne and Orr, 2004). Although IBD has been detected in a range of species groups, including fishes, plants, oysters, beetles, and mammals across both small and large geographical scales (Angers and Bernatchez, 1998; Launey et al., 2002; Bockelmann et al., 2003; Peakall et al., 2003; Buonaccorsi et al., 2004; Oleksa et al., 2012), some studies have not supported this theory (e.g., Peterson, 1995; Peter and Slatkin, 2013). Here, we tested IBD hypothesis by comparing genetic structure of six populations of G. rufa from six rivers located in different distances from each other in the Tirgis basin of Iran. Materials and Methods During October and November 2011, a total of 240 samples (40 specimens per population) were collected using cast net from six rivers belonging to three river drainages, including Kabkiyan River (30°51'N, 51°19'E; Population 1), Berim River (30°19'N, 51°15'E; Population 2), Fahlyan River (30°11'N, 51°31'E; Population 3), Beshar River (30°26'N, 51°46'E; Population 4); Sarab Bahram River (30°05'N, 51°26'E; Population 5), and Shahpour River (29°45'N, 51°33'E; Population 6) (Fig. 1). The average distance (± SD) between sampling sites was 98±44 Km (range 30-180) (Table 1). A small piece of the pelvic and pectoral fins of the specimens were removed and fixed in 95% ethanol and the fish were released. Total genomic DNA was extracted and stored at -20°C from tissues using the traditional proteinase-K digestion and standard phenol/chloroform techniques based on Hillis et al. (1996). The extracted DNA was analyzed by electrophoreses via a 0.6% agarose gel containing 5 μg ml-1 ethidium bromide. According to Crooijmans et al. (1997) and Matura et al. (2012), eight microsatellite loci, comprising MFW17, GGM002, GGM007, GGM023, GGM024, GGM027, GGM034 and GGM045 were used (Table 2). PCR amplification was performed in total reaction volume of 25 ml containing 2.0 mM MgCl2, 0.2 mM dNTP mix, 0.2 mM each primer, 1 U Taq DNA polymerase, 1 X PCR buffer, approximately 100 ng template DNA and deionized water. Initial denaturation was achieved at 94°C for 3 min followed by 30 cycles of denaturation in 30 seconds at 94°C, 30 seconds at the respective annealing temperatures, and extension to 72°C for 1 min. The final step was extended for 3 min at 72°C. PCR products were separated using 8% polyacrylamide gels stained with silver nitrate (Bassam et al., 1991). The observed number of alleles (Na), the effective number of alleles (Ne), observed heterozygosity Figure 1. Sampling sites of Garr rufa from Tirgis Basin in Iran (1- Kabkiyan River, 2-Berim River, 3-Fahlyan River, 4-Beshar River, 5-Sarab Bahram River, and 6-Shahpour River). Pop2 Pop3 Pop4 Pop5 Pop6 Pop1 130 125 30 150 180 Pop2 - 80 100 90 100 Pop3 - - 90 30 90 Pop4 - - - 90 150 Pop5 - - - - 40 Pop6 - - - - - Table 1. Sampling locations and distance between sampled rivers (Km). 59 Int. J. Aquat. Biol. (2016) 4(1): 57-68 (Ho), expected heterozygosity (He), number of migrant (Nm), Nei’s genetic distance, Wright’s fixation index (Fst), genetic identity, inbreeding coefficient (FIS) and Hardy–Weinberg equilibrium (HWE) were calculated by Genealex (ver.6.5) Software (Peakall and Smouse, 2012). The Unweighted pair group method with arithmetic mean (UPGMA) diagram based on the matrix of genetic distances between populations was produced by NTSYS (ver.2.2) (Smýkal et al., 2008). The null allele frequency was estimated using MICRO- CHECKER (ver.2.2.3) software (Van-Oosterhout et al., 2004). The Hardy-Weinberg equilibrium tests were adjusted using the sequential Bonferroni correction (Rice, 1989). Analysis of molecular variance (AMOVA) was calculated using ARLEQUIN (ver. 3.1) to evaluate genetic diversity (Excoffier et al., 2005). AMOVA is a suitable approach to determine the population structure and genetic differentiation between populations (Grassi et al., 2004). Effective population size reductions were evaluated using BOTTLENECK (ver. 1.2.02) (Cornuet and Luikart, 1996). The distance matrix was then used to construct a UPGMA dendrogram using the software PopGene (ver.1.31) (Yeh et al., 1999). This program examines discrepancy between the observed heterozygosity and expected heterozygosity based on the observed number of alleles using two- tails model (S.M.M.). Results In 240 individuals of the six studied populations, 102 alleles were observed. The average number of alleles per locus was 12.750. The highest (0.638) and lowest (0.567) average heterozygosity was found in the populations 1 and 2, respectively, whereas the highest (0.876) and lowest (0.840) expected heterozygosity (He) were found in the populations 2 and 6, respectively (Table 3). Thirty-seven out of 48 loci showed consistent significant deviations from Hardy-Weinberg Equilibrium expectations in populations after the probability level (P<0.05) (Table 3). Locus GGM002 and GGM024 in the populations 1, 3, 5 and 6; GGM002 and GGM045 in the population 2, and GGM024 in the population 4 were within HWE. Fixation index (FIS), a measure of heterozygote deficiency or over-plus (inbreeding co-efficient), was often larger than zero, showing a deficiency of heterozygotes in most of loci in all populations (Table 3). According to FIS values, there were no significant differences between regions. There were Microsatellite Loci Gene Bank Accession no. Primer sequence N Size (bps) Anneal (°C) MFW17 MFW17 F: CTCAACTACAGAGAAATTTCATC R: GAAATGGTACATGACCTCAAG 9 112 - 232 46 GGM002 HQ288485 F:CACTTTGTCCTTGCCATTGA R:CTCAACACCGTGGACTCTCA 25 200 - 344 55 GGM007 HQ288490 F:GCTGTGCTGACTGGCACTT R:CAAACCAACATTTCATCAAAAA 11 232 - 300 52 GGM023 HQ288506 F:TCACCATCCACTGAAGACCA R:GAAATATGTAACGTCATTAATTGTGTG 9 96 - 136 53 GGM024 HQ288507 F:TCCCTCTTTTTGCTCTCAGG R:TAGGTGAACAAATGGCATGG 14 128 - 208 52 GGM027 HQ288510 F:TCGGTGCACCCCTAGTAAAC R:CCAAGTGTGTGTTTGGATGG 12 188 - 252 54 GGM034 HQ288517 F:CGCGCAAGTTTCTTTCAGTT R:GCTGTGAGACAAGCCTAAACC 11 128 - 184 56 GGM045 JF268662 F:TCTCATGGGTCTCTGGGTTC R:TGTGCAGAAAGGCTGTTGAG 11 152 - 200 53 N: number of alleles Table 2. Characteristics of the used microsatellite loci in this study. 60 Kolangi Miandare et al./ Genetic characterization of G. rufa populations in Tigris Basin, Iran no significant indications of a recent reduction in the effective population size according to the S.M.M test in any of the populations and there was no evidence of any genetic bottlenecks (Table 4). Examination of genotyping errors revealed no evidence for large allelic dropout or stutter-band scoring at any of the eight loci. At the loci GGM007, GGM023, GGM027 and MFW17, null alleles might have appeared. For removing possible bias in results we repeated our analysis, excluding loci that showed null alleles in all populations. Since the results remained the same, therefore we retained all loci for the analysis. Population Number MFW17 GGM002 GGM007 GGM023 GGM024 GGM027 GGM034 GGM045 Na 10 23 12 8 13 13 10 9 Pop 1 Ne 5.939 17.231 9.011 6.644 9.446 8.711 4.795 5.407 Ho 0.393 1.000 0.357 0.750 0.929 0.321 0.643 0.714 He 0.832 0.942 0.889 0.849 0.894 0.885 0.791 0.815 FIS 0.528 - 0.062 0.598 0.117 - 0.039 0.637 0.188 0.124 PHw *** ns *** *** ns *** *** ** Na 7 24 9 8 15 8 11 11 Pop 2 Ne 4.159 18.892 5.851 5.091 12.346 4.709 7.502 5.244 Ho 0.250 0.964 0.107 0.500 0.857 0.429 0.714 0.714 He 0.760 0.947 0.829 0.804 0.919 0.788 0.867 0.809 FIS 0.671 - 0.018 0.871 0.378 0.067 0.456 0.176 0.117 PHw *** ns *** *** ** *** *** ns Na 11 22 13 8 15 15 10 9 Pop 3 Ne 9.064 16.860 7.127 3.655 10.116 9.924 7.362 5.580 Ho 0.714 1.000 0.250 0.357 0.929 0.357 0.857 0.464 He 0.890 0.941 0.860 0.726 0.901 0.899 0.864 0.821 FIS 0.197 - 0.063 0.709 0.508 - 0.030 0.603 0.008 0.434 PHw *** ns *** *** ns *** *** ** Na 7 25 10 9 11 11 13 11 Pop 4 Ne 4.780 18.667 6.701 4.284 8.760 5.521 9.503 8.859 Ho 0.000 1.000 0.143 0.714 0.857 0.393 0.643 0.786 He 0.791 0.946 0.851 0.767 0.886 0.819 0.895 0.887 FIS 1.000 - 0.057 0.832 0.068 0.032 0.520 0.282 0.114 PHw *** ** *** *** ns *** *** *** Na 10 30 12 8 14 10 10 12 Pop 5 Ne 5.297 24.889 7.362 5.209 11.281 6.426 6.348 5.620 Ho 0.321 1.000 0.393 0.571 0.964 0.197 0.750 0.464 He 0.811 0.960 0.864 0.808 0.911 0.844 0.842 0.822 FIS 0.604 - 0.042 0.545 0.293 - 0.058 0.789 0.110 0.435 PHw *** ns *** *** ns *** *** *** Na 9 23 11 10 17 15 10 11 Pop 6 Ne 6.426 16.505 7.575 6.759 11.701 9.800 5.502 7.840 Ho 0.393 1.000 0.250 0.714 0.857 0.357 0.429 0.929 He 0.844 0.939 0.868 0.852 0.915 0.898 0.818 0.872 FIS 0.535 - 0.064 0.712 0.162 0.063 0.602 0.476 - 0.064 PHw *** ns *** *** ns *** *** *** Na, number of observed alleles; Ne, Effective number of alleles; Ho, observed heterozygosity; He, Expected heterozygosity; Fis, fixation indices; PHW, Hardy-Weinberg probability test (*P< 0.05, **P<0.01,***P<0.001, n.s., non-significant). Table 3. Genetic variability of eight microsatellite loci in six studied populations for Garra rufa. Wilcoxon test S.M.M Mode-shift Pop 1 0.8432 No Pop 2 0.7421 No Pop 3 0.5468 No Pop 4 0.7421 No Pop 5 0.1953 No Pop 6 0.7426 No Table 4. Analysis of the possibility of a recent bottleneck under two tails for H excess or deficiency. 61 Int. J. Aquat. Biol. (2016) 4(1): 57-68 The average level of genetic differentiation between populations, as indicated by FST, was 0.039. There was a significant relationship between genetic divergence (Pair-wise Fst) and geographical distance (r2 = 0.93, P<0.001, Fig. 2). Pair-wise FST estimates between population pairs differed significantly (P<0.01) from zero for all the pairs in populations (Table 5). Analysis of molecular variance (AMOVA) showed that 97% of the observed variation was found within populations and only 3% of the variation was observed between populations. The results revealed high levels of gene flow (Nm) between populations (Table 6). On the basis of Nei's (1978) genetic distance values (Table 7), an UPGMA dendrogram was created displaying four major clusters (Fig. 3). Most of the similarity was observed between populations 1 and 4 in cluster D and between populations 3 and Pop 1 Pop 2 Pop 3 Pop 4 Pop 5 Pop 6 Pop 1 - 0.035** 0.033** 0.013** 0.039** 0.044** Pop 2 - 0.023** 0.031** 0.025** 0.027** Pop 3 - 0.027** 0.013** 0.030** Pop 4 - 0.025** 0.040** Pop 5 - 0.023** Pop 6 - **P<0.01 Table 5. Pairwise Fst between six studied populations of Garra rufa based on eight microsatellite loci. Pop 1 Pop 2 Pop 3 Pop 4 Pop 5 Pop 6 Pop 1 0.000 Pop 2 12.386 0.000 Pop 3 9.831 13.888 0.000 Pop 4 6.192 7.422 5.949 0.000 Pop 5 9.157 9.748 10.460 6.802 0.000 Pop 6 19.106 7.691 10.788 5.385 9.043 0.000 Table 6. Pairwise Population Nm Values Based on Fst Values between six studied populations. Pop 1 Pop 2 Pop 3 Pop 4 Pop 5 Pop 6 Pop 1 - 0.272 0.316 0.404 0.354 0.208 Pop 2 0.762 - 0.260 0.355 0.348 0.374 Pop 3 0.729 0.771 - 0.426 0.330 0.293 Pop 4 0.668 0.701 0.653 - 0.402 0.452 Pop 5 0.702 0.706 0.719 0.669 - 0.351 Pop 6 0.812 0.688 0.746 0.636 0.704 - Table 7. Nei genetic distance (above diagonal) and genetic identity (below diagonal) on the studied Garra rufa populations Figure 6. The relationship between geographical distance and genetic differentiation between six studied populations of Garra rufa Figure 2. UPGMA dendrogram based on Nei’s genetic distance, summarizing the data on differentiation between six studied populations of Garra rufa, according to microsatellite DNA marker analysis. 62 Kolangi Miandare et al./ Genetic characterization of G. rufa populations in Tigris Basin, Iran 5 in cluster C. The UPGMA revealed a distinct population structure for sixth population of G. rufa in Tigris Basin (Fig. 3). Discussion Structure and genetic diversity of G. rufa has been studied using eight microsatellite loci in six populations that showed an average FST of 0.039 (range 0.013-0.044) indicating little genetic differences between studied populations (Wright, 1987). In general, the fixation index can range from 0 to 1, where values close to 0 indicate that the populations are sharing their genetic material through high level of breeding and values close to 1 indicate that population do not share any alleles with one another (DeWoody and Avise, 2000). The population structure of freshwater organisms depends on the distribution of the river systems (Nagarajan et al., 2006). The previous studies showed that genetic structuring can happen even across short geographical distances (e.g., Angers and Bernatchez, 1998; Koskinen et al., 2001, 2002; Primmer et al., 2006) as similar to the results of the present study. The positive significant relationship between genetic variation and geographical distance revealed that the genetic differentiation between studied populations is raised by increasing geographic distance. In addition, UPGMA dendrogram revealed that the six studied populations can be divided into four clusters. The populations 1 and 4 were in one group and were not significantly different (cluster D). Similarly, populations 3 and 5 were categorized in cluster C and the population 6 (cluster A) was a distinct population from others. These findings indicated that the majority of migration occurred between populations being located at about 30 Km from each other. Thus, our study lends support to IBD theory because Garra populations that lived in close spatial proximity were genetically similar. Similarly, using 17 microsatellites, Primmer et al. (2006) identified the relatively high level of genetic structuring and significant isolation-by-distance signal between Atlantic salmon, Salmo salar sampled from the tributaries and main stream of the Varzuga river system. In this study, the average waterway distance between sampling site was 60 Km (range 5-165 Km) and the level of genetic differentiation between sampling locations (FST values) ranged 0.006-0.07, with the global FST being 0.014. Our results revealed a fairly high level of genetic variation in the microsatellite loci within six studied populations. Although no information is available on genetic diversity of G. rufa using microsatellite markers, Durna et al. (2010) applied RFLP markers in this fish species. Here, the mean observed and expected heterozygosity, and number of alleles per locus were 0.46, 0.56 and 9.1, respectively. The values obtained in this study are higher than those reported by Durna et al. (2010) and accordingly represent high genetic diversity of this species. The average observed heterozygosity among six regions was 0.567-0.638, higher than those generally reported in freshwater fish (DeWoody and Avise, 2000). The average observed heterozygosity across all populations was less than the expected average heterozygosity. The microsatellite data showed that allele diversity and heterozygosity levels are high in studied populations. In genetic diversity investigations, allelic richness is higher than heterozygosity values and high allelic richness represents effective population size (Diz and Persa, 2009). According to AMOVA analysis, the mean observed number of alleles in the populations 1 to 6 was 12.250, 11.625, 12.875, 12.125, 13.250 and 13.250, respectively. The primer of GGM002 revealed the highest number of allele (25) compared to other primers. Mean number of alleles per locus was 12.75 across the 8 microsatellite loci which is generally higher than rates reported for freshwater fish (DeWoody and Avise, 2000). However, the finding is similar to those of Kanapen et al. (2006) in Gobio gobio as the average number of alleles per locus ranged from 2 to 13. Furthermore, Kim et al. (2007) reported a positive linear relationship between microsatellite length and number of alleles as well as the average number of 63 Int. J. Aquat. Biol. (2016) 4(1): 57-68 alleles 11.7 per locus in Hemibarbus mylodon. Many of the variation in polymorphism at microsatellite loci that exist between species can be ascribed to differences in their population biology and life history traits (Neff and Gross, 2001). This may be the reason for the observed differences in the number of alleles in the studied populations. In wild populations, fish are often seen deviating from the Hardy-Weinberg equilibrium (HWE) (Lucentini et al., 2006; Yue et al., 2004). Based on the results, the studied populations deviated significantly from HWE at most of the microsatellite loci (37 out of 48 tests). Zhuo et al. (2012) and Quan et al. (2007) reported similar results for Channa argus and Silurus soldatovi, respectively. Factors such as inbreeding, intra-population structure (Wahlund effect), non-random sampling, fishing pressure, and fish migration have been reasons for deviation from HWE (Bergh and Getz, 1989; Garcia DeLeon et al., 1995; Castric et al., 2002; Shao et al., 2002; Ruzafa et al., 2006; Gopalakrishnan et al., 2009; Abbas et al., 2010). Non-significant deviation from HWE was reported by Israel et al. (2004) as a result of the presence of several stocks of green sturgeon that, in turn, showed the existence of one or more of the ovipositor population. Heterozygote deficits could have resulted in null alleles and real biological phenomena including mixing of differentiated wild populations (Mandal et al., 2012). The results indicated a null allele in all studied Garra populations. Examination of the genotyping errors revealed no evidence for large allelic dropout or stutter-band scoring at any of the eight loci. Deviation from Hardy-Weinberg expectations observed in the present study might be a result of non-specific primers usage, mistakes in reading alleles (Borrell et al., 2008), presence of migration, genetic drift (Bhassu et al., 2004), and the occurrence of null alleles in the populations. As conclusion, this study revealed that the populations have a high genetic diversity and thus its ecological value requires adequate protection. The protection of G. rufa populations in Iran, as an important economic and ecological species, requires genetic monitoring to track changes in their genetic diversity. Therefore, the results of the present study could be useful as a reference to monitor any future genetic change created by environmental or anthropogenic factors. 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(2016) 4(1): 57-68 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2016 Iranian Society of Ichthyology چکیده فارسی ریزماهواره نشانگرهای از استفاده با دجله حوضه در( Garra rufa) چراغ گل ماهی هایجمعیت ژنتیکی خصوصیات 2، حمیدرضا رضایی1، علی شعبانی*1قاسم عسکری، 1میاندره کلنگی حامد .ایران ،گرگان گرگان، طبیعی منابع و کشاورزی علوم دانشگاه ،آبزیان پرورش و تکثیر گروه1 .ایران ،گرگان گرگان، طبیعی منابع و کشاورزی علوم دانشگاه ،محیط زیست گروه2 چکیده: ماهی عنوانهب چراغگل ماهی برای فرضیه این. یابدمی افزایش جغرافیای فاصله افزایش با افراد بین ژنتیکی تمایز ،فاصله با جدایی تئوری اساس بر ساختار مطالعه جهت ماهواره ریز جایگاه هشت تعداد منظور این برای. گرفت قرار ارزیابی مورد حوضه از مختلف رودخانه شش در ،ایران داخلی آبهای تا 635/11 بین للیآ میانگین گردید، مشاهده نمونه 240 در للآ 102 تعداد. گردید استفاده جغرافیای پراکنش پایه بر چراغگل ماهی ژنتیکی هایجمعیت که گردید مشخص عالوههب. گشت محاسبه 638/0-567/0 حدود در مطالعه مورد جمعیت شش در هتروزیگوسیتی میزان. بود 250/13 جمعیت شش که داد نشان( UPGMA) وزن بدون جفت گروه تحلیل و تجزیه. دارند واینبرگ-هاردی تعادل از داریمعنی انحراف مطالعه مورد نشان یقتحق این در استفاده مورد هایجایگاه در را ژنتیکی تنوع از باالیی سطح نتایج. گیرند قرار جداگانه کالستر چهار در توانندمی شده مطالعه از لحاص نتایج اساس بر. باشدمی مطالعه مورد هایجمعیت بین پایین ژنتیکی تماییز بیانگر که بود 044/0-013/0 دامنه در Fst میزان. دهدمی .نمایدمی تقویت را فاصله با جدایی نظریه که دارد وجود جغرافیای فاصله و Fst بین قوی بسیار ای رابطه که گردید مشخص تحقیق این .ایران دجله، حوضه ،ماهی دکتر ماهواره، ریز ژنتیکی، تنوع :کلمات کلیدی