Iranian Journal of Fisheries Sciences 10(4) 596-606 2011 Application of microsatellite markers to determine populations of the Persian ( persicus) in the South of

Chakmehdouz Ghasemi F.*; Pourkazemi M. ; Tavakolli M. ; Yarmohammadi M. ; Hassanzadeh Saber M. ; Baradaran Noveiri S.

Received: November 2010 Accepted: April 2011

Abstract The objective of this study was to analyse the population genetic structure of the Persian sturgeon (Acipenser persicus) in Sefidrud and Gorganrud rivers watershed based on the characterization of microsatellite markers during 2006 – 2008. 100 samples of Persian sturgeon were collected from two regions. Four microsatellite loci (Ls68, Spl168, Spl173 and Afu68) were analyzed for the molecular characterization of this species which resulted in polymorphic patterns. DNA bands were analysed using Biocapt and GenAlex software package. A total of 109 alleles were observed of which the maximum number of alleles (17) were found in Spl168 locus which belonged to from Sefidrud river,s watershed and the minimum number of alleles (10) in Ls68 locus belonging to the sturgeons from Gorganrud river,s watershed. Results of microsatellite analysis revealed that the differences between samples of two regions were not statistically significant (p>0.05), neither for the average number of alleles per locus nor for observed heterozygosities. The calculated Fst and

Rst between two regions was 0.07 and 0.17 showing that the genetic difference was significant (p< 0.01). Samples from Sefidrood river,s watershed in Spl173, Afu68 and Spl168 loci and samples of other regions in Afu68 and Spl168 loci were at Hardy-Weinberg equation. The genetic distance was calculated as 0.4 which represents a significant genetic difference between samples of two studied areas. In conclusion, this study suggests that the Persian sturgeons in two regions of the southern part of the Caspian Sea are genetically differentiated, thereforeArchive fisheries management of thisof unique SID stocks for restocking and conservation of gene pools is highly recommended.

Keywords: Persian sturgeon, Acipenser persicus, Caspian Sea, Microsatellite, Genetic structure, Population genetic

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Dr. Dadman International Sturgeon Research institute, Genetic Dept. P.O.Box: 41635-3464 Rasht, Iran. *Corresponding author,s email: chakmehdouz13@ yahoo.com

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Introduction Sturgeons are one of the oldest fishes disease resistance (Beardmore et al., existing from 200 million-years ago and 1997). Sturgeon population genetic studies being referred as live fossils by several provide important information which can authors (Grande and Bemis, 1996; Bemis be used for management, sustainable use et al., 1997). Recently, sturgeon and conservation of the species concerned populations in the Caspian Sea are (Rosenthal et al., 2006). Among molecular endangered due to various factors, such as markers, microsatellite methods are new over-exploitation, habitat alternation, techniques which are commonly applied pollutions, barriers to migration and loss of for many commercially important aquatic spawning habitat. (Pourkazemi, 2006). species that can be examined by using Therefore all sturgeon species have been samples harvested without endangering the listed in the 1996 IUCN Red List life of the individual. Microsatellite assessment as an endangered species markers were used to study sturgeon (Dugo et al., 2004; Pourkazemi, 2006). At population structure and can also identify present, 27 sturgeon species are found different brood stocks for management in living throughout the world (Ustaoglu and hatcheries to conserve diversity and Okumus, 2004), of which six species minimize inbreeding in artificial belonging to two genera ( and propagation (Norouzi et al., 2008). Acipenser), are found in the Caspian Sea Pourkazemi (2001) and Shabani et al. and its drainage basin which provide the (2006) studied mtDNA variation of A. bulk of the world’s yield today stellatus populations in south Caspian Sea

(Pourkazemi, 2006). In the recent years, by RFLPs method using ND 5/6 gene Acipenser persicus has comprised the regions. They found low genetic variation largest proportion of the total Iranian among studied areas and mentioned that commercial catch (Moghim et al., 2006). the RFLPs method is not a powerful This species is also artificially propagated technique to determine genetic diversity in the Iranian hatcheries and the sufficiently to fisheries management of fingerlings are released into the rivers of this species. Safari et al. (2008) using the south Caspian Sea for restocking. microsatellite markers showed high level An understandingArchive of the genetic diversity of of variety of SIDA.nudiventris between Ural in aquatic organisms can be useful in stock River and samples from the southern part conservation. Genetic diversity is of the Caspian Sea. Following the results, important in both natural and cultural they expressed there are two independent populations because it provides the populations of this species, so necessary spectrum of genotypes for conservation policy and restocking adaptive response to changing conditions programs are necessary. Sturgeon family and heterozygous individuals usually are structure and relationships can easily be superior to less heterozygous individuals identified using microsatellite markers. in many economically important Using these methods allows appropriate characteristics like growth, fertility and broodstock management both in

www.SID.ir Iranian Journal of Fisheries Sciences, 10(4), 2011 598 aquaculture and in sturgeon restocking microsatellite DNA markers. It can also be programs (Rosenthal et al., 2006). In applied for future genetic improvement recent years, genetic researches have been and assessment of this species in limited to a few studies on the Caspian Sea hatcheries and to design suitable sturgeon species using a variety of management guidelines for artificial molecular markers (Rezvani, 1997; breeding activities. Pourkazemi et al., 1999; Shabani, 2005; Qasemi et al., 2006; Khoshkholgh, 2007; Materials and methods Norouzi et al., 2008; Safari et al., 2008) Sample collection but despite the commercial importance of A total of 100 fin clips of adult Acipenser the Persian sturgeon, study on its genetic persicus were collected from 2 regions and population structure in south Caspian including 50 samples from Sefidrud Sea is scarce and more studies are watershed and 50 samples from Gorganrud necessary. The goal of this study was to watershed (Fig. 1). For each sample, 2-3 determine the genetic diversity population cm caudal fin tissue was cut and kept in and provide a genetic background of this absolute ethanol and then transferred to the species for management in the southern Genetic department of International part of the Caspian Sea using Sturgeon Research Institute, Rasht, Iran.

Archive of SID Figure1: Sampling regions map: Gorganrud watershed () and Sefidrud watershed ()

Genomic DNA extraction For each sample, approximately 50 mg of et al. (1996). The quantity of DNA was fin tissues was cut into small pieces and measured in 260 and 280 nm using UV- genomic DNA was extracted by standard spectrophetometry method by Nanodrop SDS proteinase-K digestion, phenol: (ND 1000 model) and the quality of the chloroform: isoamylalcohol extraction and extracted DNA sample was checked by ethanol precipitation as described by Hillis

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1% agarose gel electrophoresis. Purified temperatures were optimized for each o DNA was stored at -20 C until use. microsatellite loci to produce scorable amplification products (Table 1). The PCR PCR profiles and primer sequences was performed in a 20 µl reaction volume In this study, we used 4 microsatellite loci: containing 100 ng of template DNA, 30 Ls68, Afu68 (May et al., 1997), Spl168 and pmol of forward and reverse primer, 200

Spl173 (McQuown et al., 2000) all of µm of dNTPs, 5 u / µl of taq DNA which produced polymorphic patterns. The polymerase, 1 µl of 1.5 mM Mgcl2 and 2 PCR conditions, especially the annealing µl of 10X reaction buffer (Table 2).

Table 1: Characteristics of 4 polymorphic microsatellite loci of Acipenser persicus used in this study Loci Primer (5/-3/) Size (bp) Annealing Reference (o ) temp. C Ls68 F- TTATTGCATGGTGTAGCTAAAC 198 – 264 60 May et al., R- AGCCCAACACAGACAATATC 1997 Spl168 F- CACTGATTCGCTACAACCGT 232 - 310 59 McQuown et R- AGAAGGACTTGCAGTCCGAA al., 2000 Spl173 F- GGCTTTTGTCTGAAACGTCC 232 - 292 61 McQuown et R- TGGTGTGTCATTTTGAAGGC al., 2000 Afu68 F- AACAATATGCAACTCAGCATAA 106 - 168 55 May et al., R- AGCCCAACACAGACAATATC 1997

Table 2: Concentration and kind of materials used in this study Material Concentration Amount for 20 µl reaction DNA 100 ng 1 µl PCR buffer 10 X 2 µl

dNTPs 10 mM 0.5 µl

Mgcl2 50 mM 1 µl Primer (F) 30 pm 1 µl Primer (R) 30 pm 1 µl Archivetaq DNA polymerase 5 u / µl of0.2 µl SID dH2o - 13.3 µl

o PCR reactions were performed with an 30 cycles of 30 s denaturation at 94 C, 60 eppendorf thermal cycler (Mastercycler ep s at the respective annealing temperature, o gradient, 96 plus, eppendorf, Germany) and 60 s extension at 72 C, ending with 10 o under the following conditions: initial min at 72 C as the elongation period. o denaturation of 5 min at 95 C followed by

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Table 3: Variability of 4 microsatellite loci in two areas of the Persian sturgeon (A, number of alleles; Ho, observed heterozygosity; He, expected heterozygosity; P, P-values of X2 tests for Hardy-Weinberg equilibrium)

Sefidrud Gorganrud Locus Parameters watershed watershed A 14 10 H 0.670 0.520 Ls68 o He 0.840 0.850 P 0.00 0.000 A 17 15 H 1 0.950 Spl168 o He 0.840 0.840 P 0.095 0.363 A 16 14 H 0.708 0.710 Spl173 o He 0.880 0.860 P 0.1 0.008 A 11 12 H 0.250 0.500 Afu68 o He 0.780 0.640 P 0.113 0.285 Average number 14.5 ± of alleles per 12.75 ± 2.22 2.64 locus rage H 0.66 ± o 0.67 ± 0.21 0.31 Average H 0.83 ± e 0.80 ± 0.10 0.04

Gel electrophoresis and staining Following polyacrylamid gel The PCR products with a standard 100 bp electrophoresis and silver nitrate staining, DNA markers ladder (Fermentas) were one or two clear bands were observed at separated by electrophoresis on 6% (w/v) each locus for each specimen. The bands denatured polyacrylamideArchive gel (29:1 representing of allelesSID were manually scored acrylamid: bis acrylamid; 1X TBE buffer) based on their sizes. The recorded using a Hoefer gel electrophoresis system microsatellite genotypes were used as the (Pharmacia-Biotech, USA). Gel run input data for the GENALEX software carried out at 120 V until the loading (Peakall and Smouse, 2006). Allele and buffer reached the bottom of the plate. genotype frequencies, observed After electrophoresis, the gel was stained heterozygosity (Ho), expected with silver nitrate protocol (Pourkazemi, heterozygosity (He) and test of deviations 1996). from Hardy-Weinberg equilibrium were Microsatellite data analysis calculated. Genetic distance between two areas was estimated using Nei standard

www.SID.ir 601 Chakmehdouz Ghasemi et al., Application of microsatellite markers to determine populations of…. genetic distance index (Nei, 1972). in two regions, respectively. The Genetic differentiation between two areas differences between two regions were not was also evaluated by the calculation of statistically significant (p> 0.05), neither pairwise estimates of Fst and Rst values. All calculations were conducted using the for the average number of alleles per locus GENALEX version 6. nor for observed heterozigosities. Results Significant deviations from Hardy- Allele frequencies at all loci in both areas Weinberg equilibrium at the locus level in are shown in Table 3. Overall, 109 alleles two regions are shown in Table 4. Except resulted in 4 microsatellite loci in which Ls68 locus in Sefidrud region and Ls68 the locus Spl168 presented the highest and Spl173 loci in Gorganrud region, the number of alleles (17) in Sefidrud other loci in both regions were at Hardy- watershed, while the locus Ls68 in Weinberg equilibrium. The Fst (0.07) and Gorganrud watershed was the lowest (10). Rst (0.17) values from pairwise The average number of alleles per locus comparisons were significant (p<0.01), was 14.5 for samples of Sefidrud indicating that the populations of two watershed and 12.75 for Gorganrud regions were divergent from each other. watershed. The average observed The genetic distance computed by Nei heterozygosities (H ) were 0.66 and 0.67 o (1972) was 0.4.

Table 4: Hardy-Weinberg equilibrium Test of 4 microsatellite loci in two areas of Persian sturgeon in this study Regions Locus DF X2 (Chi Sq) Level of Probability of significant significant Ls68 21 76.11 0.00  Sefidrud ArchiveSpl168 45 57.83 of0.095 SIDns watershed Afu68 10 15.55 0.113 ns Spl173 22 57.51 0.1 ns Ls68 15 52.78 0.000  Gorganrud Spl168 36 38.37 0.363 ns watershed Afu68 6 7.41 0.285 ns Spl173 55 83.65 0.008  p≤ 0.05 , p≤ 0.001 , ns: no significant

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Discussion

Anthropogenic influence and other factors levels of Fst between samples of A. caused the dramatical decrease of sturgeon stellatus in four fishery regions are stocks such as Acipenser persicus in the significant (Norouzi et al., 2008). In this

Caspian Sea. This required the study, the Fst was low (0.07) and Rst value development of conservation programs was 0.17 but Fst and Rst were significant and genetic studies to distinguish different between samples of two regions (p<0.01). populations. Among molecular methods, Although the lowest value of Fst can be microsatellite markers allow the evaluation considered as an important genetic of intraspecific genetic diversity and offer difference between populations (Wright, the possibility to distinguish differences 1978; Hartl and Clark, 1997). According between populations due to their high level to decisions of CITES (Convention on of allelic variation (Dudu, et al., 2008). For International Trade in Endangered this purpose, we evaluated the genetic Species), setting annual quotas of caviar in diversity in Persian sturgeon from two the Caspian Sea is based on sturgeon stock regions in the southern part of the Caspian assessment. In the future, it is possible to Sea, using microsatellite markers. determine quotas of caviar based on Polymorphic patterns produced by primers molecular genetic methods for the in the present study confirmed that the identification of sturgeon populations. The microsatellite markers used are suitable for data presented here suggests that the genetic diversity studies. According to the populations of these two studied areas are results there are no significant differences genetically differentiated and do not between samples of the two regions represent a single panmictic population. (p>0.05) neither for the average number of The genetic distance value based on Nei, alleles per locus nor for observed (1972) for conspecific populations heterozygosities. The high level of averaged 0.05 (range: 0.002-0.07) and for heterozygosity in this species can be congeneric species averaged 0.30 (range: attributed to use of most breeders in 0.03-0.61). The distance value obtained in hatcheries and fingerlings released into the this study (0.4) falls within the average south Caspian Sea Rivers (Sefidrud and value of congenerics and indicated that Gorganrud rivers)Archive for restocking. However genetic of differ SIDences among these two regular monitoring of genetic variability studied populations is pronounced among the offspring of each region is (p<0.01). essential to avoid the loss of current Samples of the two studied areas polymorphism due to inbreeding and were mostly of loci at Hardy-Weinberg outbreeding problems. equilibrium test. The significant

The value of Fst and Rst based on differences from Hardy-Weinberg ANOVA test is a useful measure of equilibrium could be explained either by genetic differentiation among populations sample bias, migration, artificial breeding (Ballox and Lugan-Moulin, 2002). or the presence of null alleles. The Previous research has showed that low inheritance of microsatellite null alleles

www.SID.ir 603 Chakmehdouz Ghasemi et al., Application of microsatellite markers to determine populations of…. have been reported by most of researchers markers. Molecular Ecology, 11, 155- (Pyatskowit et al., 2001; Welsh et al., 165. 2003; Rodzen et al., 2004; Zhao et al., Beardmore, A.L., Mair, C.G. and Lewis, 2005; Norouzi et al., 2008). It may also be C.G., 1997. Biodiversity in aquatic related to not using species specific systems in relation to aquaculture. primers and the most important reason, Aquaculture Research, 28, 829-839. sampling from mixtures of migrating Bemis, W.E., Findeis, E.K. and Grande, population and sampling methodology L., 1997. An overview of (McQuown et al., 2003). To detect such a . Environmental population structure, samples must be Biology of Fishes, 48, 25-71. collected from spawning sites. Samples Dewoody, J. A. and Avise, J. C., 2000. from non-spawning adults may reflect Microsatellite variation in marine, mixtures of migrating population freshwater and anadramous fishes (McQuown et al., 2003). compared with other . Journal The data generated in this study of Fish Biology, 56, 461-473. showed that populations of A.persicus in Dudu, A., Georgescu, S. E., Luca, C., two studied areas in the southern part of Suciu, R. and Costache, M., 2008. the Caspian Sea are genetically Microsatellite DNA variation in the differentiated. Accurate species Black Sea Stellate sturgeon (Acipenser identification is the first step in each stellatus). Lucrari stiintifice Zootehnie successful conservation program. This si Biotehnologii, 41(1), 78-82. information can be applied for future Dugo, M. A., Kreiser, B. R., Ross, S. T., genetic improvement and the assessment Slack, W. T., Heise, R. J. and of this species in hatcheries. We strongly Bowen, B. R., 2004. Conservation and suggest special consideration on management implications of fine-scale conservation policy and restocking genetic structure of Gulf sturgeon in program of this species in Sefidrud and the Pascagoula River, Mississippi. Gorganrud. Journal of Applied Ichthyology, 20, Acknowledgments 243-251. We thank M.R. Noruzfashkhami, head of Grand, L. and Bemis, W. E., 1996. the Genetic departmentArchive of Dr. Dadman ofInterrelationships SID of Acipenseriformes International Sturgeon Research Institute. with comments on “”. IN: Special thanks to: Leila Azizzadeh for her Interrelationships of Fishes. M.L.J. laboratory help, Mr. Sohrab Dejandian and Stiassny, L.R. Parenti and G.D. Mr. Mohammad Khoshghalb and Mr., Ali Johnson (eds). Academic Press, San Hallajian for their help in collecting the Diego. 496 pages. pp. 85-115. samples. Hartl, D. L. and Clark, A. G., 1997. References Principles of Population Genetics, 3th Ballox, F. and Lugon-Moulin, N., 2002. edition. Sinauer Assoc Inc. Pub., The estimate of population Massachusetts, USA. 542 pp. differentiation with microsatellite

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