African Journal of Biotechnology Vol. 11(98), pp. 16405-16411, 6 December, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB12.2569 ISSN 1684–5315 ©2012 Academic Journals Full Length Research Paper Population genetic study on common kilka (Clupeonella cultriventris Nordmann, 1840) in the Southwest Caspian Sea (Gilan Province, Iran) using microsatellite markers Mehrnoush Norouzi1*, Ali Nazemi2 and Mohammad Pourkazemi3 1Department of Marine Biology and Fisheries Sciences, Islamic Azad University- Tonekabon Branch, Tonekabon, 46817, Iran. 2Department of Biology Sciences, Islamic Azad University- Tonekabon Branch, Tonekabon, 46817, Iran. 3International Sturgeon Research Institute, Rasht, 41635-3464, Iran. Accepted 16 October, 2012 This study represents population genetic analysis of the common kilka Clupeonella cultriventris (Nordmann, 1840) in the southwest Caspian Sea (Gilan Province). A total of 60 specimens of adult common kilka were sampled from two seasons (spring and summer), 2010. Fifteen pairs of microsatellites previously developed for American shad (Alosa sapidissima), Pacific herring (Clupea pallasi), Atlantic herring (Clupea harengus) and Sardine (Sardina pilchardus) were tested on genomic DNA of common kilka. Alleles frequencies, the fixation index RST, observed and expected heterozygosity were determined at disomic loci amplified from fin tissue samples. Five pairs of primers (Cpa6, Cpa8, Cpa104, Cpa125 and AcaC051) as polymorphic loci were used to analyze the genetic variation of the common kilka population. Analyses revealed that an average of alleles per locus was 14.4 (range 5 to 21 alleles per locus in regions). All sampled regions contained private alleles. The average observed and expected heterozygosity were 0.153 and 0.888, respectively. All loci significantly deviated from Hardy-Weinberg equilibrium (HWE). Based on AMOVA, RST values was found to be 0.113 (Nm=1.96, P<0.01). The genetic distance between populations was 0.344, which indicates that the genetic difference among the studied populations is pronounced. These results support the existence of different genetic populations along the Caspian Sea coast (Guilan Province). Key words: Population genetic, Southwest Caspian Sea, microsatellite, Clupeonella cultriventris. INTRODUCTION Common kilka, Clupeonella cultriventris, belongs to the and 1998, the number of fishing vessels and fishing family Clupeidae, lives in the Caspian Sea, feeds on activities increased progressively and Iran increased its zooplankton, crustaceans such as copepods, quota of kilka catches to 95000 mt up until 1999.In the cladocerans and spawn in spring (Abdoli and Naderi, next few years, however, the catch sharply decreased to 2009; http://www.fishbase). Common kilka is faced with 19500 mt in 2004 due to overexploitation and invasion of the challenges resulting from overfishing and invader jelly fish Mnemiopsis leidyi. Ctenophore Mnemiopsisleidyi (Karimzadeh, 2011; During the past 30 years the environmental status of Velikova et al., 2012). Kilka fishing is an important source the Caspian Sea changed significantly due to the impacts of income and protein for Iranians inhabiting in the of various factors, such as fluctuations in sea level and Caspian Sea’s coastal regions. The collapse of kilka pollution of various toxicants (Ivanov,2000). Recent fisheries has adverse effects on both the economy and introductions of invasive species via ballast water from regional protein consumption. Between the years 1989 ships have also had a negative impact on fish stocks in the Caspian Sea (Ivanov et al. 2000). In particular, an invasive jellyfish (Ctenophora, Mnemiopsis leidyi), which *Corresponding author. E-mail: [email protected]. Tel: +98 had appeared by November 1999 (Ivanov et al. 2000), 911 371 4251. Fax: +98 192 427 4409. affected kilka stocks (Fazli, 2011; Daskalov 16406 Afr. J. Biotechnol. Table 1. Loci, repeat motif, primers sequence, gene bank number, touchdown protocol, PCR product size range (bp)and primer sources used at the present study in common kilka. Touchdown Repeat GenBank Primer Loci Primer sequence protocol motif No. sources Actual size (bp) 40 F: 5'-GTGTGAGTTTGCTCCAAA-3' 52 °C/ Miller et al. Cpa6 (GATA)14 AF309801 R: 5'-GTTTGTACCAATGAATGATTACAA-3' 104 - 216 (2001) F: 5'-GATCCTTCTTTTAAGGAAAA-3' 52°C/40 Cpa8 (GACA)27 AF309804 R: 5'-GTTTGACAGAACTTACTATCTCAGA-3' 104 - 216 F: 5'-ACGTAGGCGCAGACAT-3' 51.5°C/40 Cpa104 (TG)54 AF309791 R: 5'-GTTTGCTCAAGTCAATGTGATTTTTA-3' 312 - 390 40 (GA)32i F: 5'-GCAAGAAAGAGCAGCAGA-3' 59°C/ Cpa125 AF309796 (GT)26 R: 5'-GTTTCGACTCAACAGCTGGAA-3' 216 - 280 40 (GAAT)7 F: 5'-GTAAGTCGCTTTGGACTACCAG-3' 54°C/ Julian and AsaC051 EF014992 (GTAT)13 R: 5'- TCTAAATGCCCAGGTAAAGATG-3' 160 - 180 Barton (2007) and Mamedov, 2007). Because of the recent decline in MATERIALS AND METHODS common kilka populations, several management actions Sample collection and DNA isolation have been implemented, which include the closure of select fisheries (Brown et al. 2000). For the sustainable A total of 60 specimens of adult common kilka were sampled from a management of this unique species, characterization of single sampling location, Anzali port (37° 29′ N, 49° 17′ E; Iran), but genetic variability of wild stocks is essential. Molecular the fish were caught during different seasons (spring and summer) genetic studies on the common kilka in the Caspian Sea and preserved in 95% ethanol. were so far limited to a few studies using RFLP (Lalouei et al., 2006). Microsatellites recently have become an DNA extraction extremely popular marker type in a wide variety of genetic investigations (Sekar et al., 2009). Microsatellites Genomic DNA was extracted from the fin tissue using High Pure PCR Template Preparation Kit (Roche Applied Science, Germany) are abundantly distributed across the genome, according to manufacturer’s instructions. The quality and demonstrate high levels of allele polymorphism and can concentration of DNA were assessed by 1% agarose gel easily be amplified with polymerase chain reaction electrophoresis and spectophotometry (CECIL model CE2040) and (PCR). stored at −20°C until use. These features provide the underlying basis for their successful application in a wide range of fundamental Microsatellite data set and applied fields of fisheries and aquaculture (Sekar et al., 2009). Microsatellite genotypes are particularly helpful Genomic DNA was used as a template to amplify microsatellite loci to detect structure in closely related populations, by touchdown PCR. Totally, 15 primer pairs were designed for regardless of whether they are in an evolutionary Alosa (AsaC051, 059, 249, 334, Julian and Barton, 2007), Clupea (Cpa6, 8, 100, 104, 107, 120, 134, 125, Miller et al., 2001; 1235, equilibrium. Additionally, primers designed for one 1014, McPherson et al., 2001) and Sardina (SAR1.12, Gonzalez species can often be used with other related species and Zardoya, 2007a). (Chistiakov et al., 2005). For all primer sets, amplification was performed in a reaction Recently, many microsatellite loci were used to volume of 25 µl containing 0.2 mM of deoxynucleotide investigate the genetic structure of various Clupeidae triphosphates (dNTPs), 0.2 to 0.4 µM of each primer, 200 ng of template DNA; 0.3 to 0.4 unit of HotStarTaqTM DNA polymerase; 1x species including Atlantic herring (Shaw et al., 1999; TM HotStarTaq PCR buffer and 2.5 to 4.5 mM of MgCl2. McPherson et al., 2001), Sardine (Gonzalez and Microsatellites were amplified (Table 1 for specific annealing Zardoya, 2007a), Pacific sardine (Pereyra et al., 2004), temperatures) using a thermocycler (MyCycler, BioRad). An initial Pacific herring (Miller et al., 2001; Semenova et al., 2012) denaturing step of 10 min at 95°C was followed by amplification for and American shad (Julian and Barton, 2007). 40 cycles at the following conditions: 30 s at 95°C, 40 to 120 s at The objectives of the present study were to investigate 51.5 to 59°C and 45 to 120 s at 70 to 72°C. A final 5-min extension at 72°C completed the protocol (Table 1). on genetic structure of common kilka and also to test the PCR products were separated on 10% polyacrylamide gels (29:1 hypothesis that common kilka has identical population in acrylamide: bis-acrylamide; 1×TBE buffer) followed by silver different seasons in the Southwest Caspian Sea. staining. Gels were run at 40 mA for 14h. Alleles were sized using Norouzi et al. 16407 Figure 1. Microsatellite banding profile of C. cultriventris from Anzali spring using primer pair AcaC051. Uvitec software, and each gel contained an allelic ladder (100 bp) bands displayed a characteristic disomic banding pattern to assist in consistent scoring of alleles. (Figure 1). Data analysis Genetic variation within sampling Allelic frequencies, observed and expected heterozygosities, genetic distance (Nei, 1972), genetic identity (Nei, 1972), FIS, FST The total number of alleles found in each population and RST value, Nm, Hardy-Weinberg (HW) tests of equilibrium, analysis of molecular variance (AMOVA) via codominant data were ranged from 68 in spring to 73 in summer (Table 2). Out computed in the GeanAlex 6 software (Peakall and Smouse, 2006). of 93 observed alleles, in spring and summer, 35 and 33 alleles, respectively occurred at frequencies of >0.05 in all samples. AsaC051 showed the maximum variability RESULTS ranging in frequency from 0.067 to 0.333. All sampled populations contained private alleles at the significant Amplification and banding patterns level (Table 2). For example,