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Development of Disomic Single-Locus DNA Microsatellite Markers for Persian Sturgeon (Acipenser Persicus) of the Caspian Sea

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Development of Disomic Single-Locus DNA Microsatellite Markers for Persian Sturgeon (Acipenser Persicus) of the Caspian Sea Iranian Journal of Fisheries Sciences 12(2) 389-397 2013 __________________________________________________________________________________________ Development of disomic single-locus DNA microsatellite markers for Persian sturgeon (Acipenser persicus) of the Caspian Sea Moghim M.1,3∗; Pourkazemi M.2; Tan S. G.3; Siraj S. S.4; Panandam J. M.5; Kor D.1; Taghavi M. J.1 Received: December 2011 Accepted: February 2012 Abstract Understanding the scale at which wild stock of Persian sturgeon (Acipenser persicus) are genetically discrete is necessary for effective management of this commercially important species. Disomic DNA microsatellite markers are among the best tools for determining stock structure in fishes. As all sturgeon species have a polyploid ancestry of all sturgeons, most gene loci exhibit more than two alleles per individual, limiting the use of powerful analytical methods that commonly assume disomic inheritance. We scored products from 38 sets of microsatellite primers developed in lake (Acipenser fulvescens) and Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) to determine whether they would amplify disomic loci in Persian sturgeon. Samples of 45 individuals were detected.Thirty six loci (95%) were amplified successfully in Persian sturgeon. We identified; a single monomorphic locus, 12 disomic, 19 tetrasomic, three octosomic, and one locus that was ambiguous. This is the first report on development of disomic single-locus DNA microsatellite markers in Persian sturgeon. These loci could be used to characterize variation in geographically discrete populations of the Persian sturgeon in their native ecosystem including in the Caspian Sea. Downloaded from jifro.ir at 8:38 +0330 on Friday October 1st 2021 Keywords: Acipenser persicus, Caspian Sea, Single-locus DNA microsatellite markers ______________________________________ 1- Genetic Department of the Caspian Sea Ecology Research Center, P.O.Box: 961 Sari, Iran 2- International Sturgeon Research Institute, P.O. Box: 41635-3464 Rasht, Iran 3- Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4- Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 5- Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia * Corresponding author’s email: [email protected] 390 Moghim et al., Development of disomic single-locus DNA microsatellite markers… _________________________________________________________________________________________ Introduction seine by-catch in Iranian waters of the Persian sturgeon (Acipenser persicus Caspian Sea after 1990 ( Moghim et al., Borodin 1897) was first described from the 2005, 2006). Many experts believe the Southern Caspian Sea by Borodin (1897) increasing Persian sturgeon stock in the with its range later extended to the south Caspian Sea in 1990's result from northern Caspian Sea (Holcik, 1989). The extensive hatchery production and release Persian sturgeon is an anadromous fish programmes by the Iranian Fisheries which enters rivers for spawning, mainly Organization (Abdolhay and Baradaran the Sefid-Rud, Tajan and Gorgan-Rud Tahori., 2006; Moghim et al., 2006). rivers in Iran and Kura River in Azerbaijan Restorative propagation programs and to a lesser extent the Volga, Ural, can be harmful to wild stocks if they lead Samur, Terek, Lenkoranka and Astara to inbreeding via use of low numbers of rivers (Berg, 1948). It is one of the most parents relative to the number of fish economically important species in Iran stocked or if sites are stocked with fish (Moghim et al., 2006). Persian sturgeon is from a genetically-depauperate stock now listed as a critically endangered (Drauch and Rhodes, 2007). DNA species by the International Union for the microsatellite loci can provide powerful Conservation of Nature (IUCN, 2011). tools for monitoring genetic variation Like most sturgeons, Persian sturgeon levels and for detecting genetic variation density in the wild have declined due to among discrete sturgeon stocks (e.g. over-fishing, spawning habitat destruction Schrey and Heist, 2007). The major , and pollution (Birstein 1997; Pikitch et problem with applying microsatellite al., 2005; Moghim et al., 2006; markers to sturgeon stock management are Pourkazemi, 2006). Between years 1927 the polysomic nature of inheritance (e.g., and 1956, the total catch (expressed as tetrasomy or octosomy) and the presence Downloaded from jifro.ir at 8:38 +0330 on Friday October 1st 2021 flesh weight) declined from about 930 to of null alleles at some loci (Pyatskowit et only 217 tons. Catch rates for this species al., 2001). that currently comprises the majority of An earlier attempt to develop sturgeon landings in the Caspian Sea in disomic microsatellite markers for Persian Iran, have remained relatively stable over sturgeon trialed cross-species the past few decades but have not returned amplification using microsatellite primers to pre-1950s levels (Moghim et al., 2006). developed for Scaphirhynchus spp., that Recruitment in the wild is possess a lower ploidy level than Persian extremely low, in spite of stocking sturgeon (Ludwig et al., 2001). No millions of artificially propagated amplified loci however, exhibited disomic juveniles released from Iranian sturgeon inheritance (Moghim et al., 2009). hatcheries to adjacent rivers in the Caspian Disomic microsatellite loci have been Sea since 1972. This practice has resulted developed successfully in some other in a significant increase in catch of both sturgeon species with high ploidy levels adult and juvenile Persian sturgeon in the (e.g. white, green, and lake sturgeon) commercial catch, trawl surveys and beach although the majority of loci identified Iranian Journal of Fisheries Sciences 12 (2) 2013 391 __________________________________________________________________________________________ were polysomic. For example Welsh and (2003), Welsh and May (2006) and May et May (2006) found that only 9 of 254 al. (1997) (Table1). We amplified genomic primer pairs tested in Lake sturgeon DNA from 12 individuals in an Quanta exhibited disomic inheritance. When Biotec master cycler gradient thermocycler combined with loci from other studies, (Quanta Biotech Ltd, Surrey, United Welsh and May (2006) reported a total of Kingdom) trialing annealing temperatures 13 polymorphic disomic loci in lake ranging that ranged from 48° to 66° to sturgeon, a species with the same ploidy determine the optimal annealing level as Persian sturgeon (Ludwig et al., temperature for each primer pair. The 20- 2001). Recently we developed and tested μl PCR reactions contained approximately 68 microsatellite primer pairs from a 1-10 ng genomic DNA, 0.15 units Taq Persian sturgeon enriched microsatellite DNA polymerase, 1 µM of each primer, library (Moghim et al., 2012 ) and while 200 mM of each dNTP, 1.75 mM MgCl2, none of the markers exhibited disomic and 1× PCR buffer. inheritance in Persian or Russian (A. The amplification protocol for most gueldenstaedtii) sturgeon, several loci loci consisted of a 5 min denaturing step at showed promise in stellate sturgeon (A. 95 °C, followed by 35 cycles of 95 °C for stellatus), ship sturgeon (A.nudiventris) 30 s, 48 - 66 °C for 30 s, and 72 °C for 45 and beluga (Huso huso). s, and a final elongation at 72 °C for 5 In the present study, we tested min. Amplication of AfuGs 9 and 56 cross-species amplifications of followed Welsh and May (2006). PCR microsatellite primer pairs developed in products were suspended 1:1 in 98% lake and Atlantic sturgeon to identify formamide/loading dye, denatured at 95°C disomic microsatellite loci for Persian for 5 min, and separated in a 6% sturgeon. denaturing polyacrylamide gels on a Bio- Downloaded from jifro.ir at 8:38 +0330 on Friday October 1st 2021 Rad SequiGen Sequencing Cell-system Materials and methods with gel size 38 × 30 cm and run at 70 W Experimental materials and Molecular for 45 - 60 min. DNA bands were analysis visualized using a silver staining method Persian sturgeon fin clips (n=45) were (An et al., 2009) and amplified fragments collected from adult broodstocks from were sized by comparing their migration Iranian coastline of the south Caspian Sea against a 50 bp DNA Ladder (Promega, and preserved in 95% ethanol. Genomic Madison, WI, USA). Fragment sizes were DNA for amplification of 38 microsatellite estimated using UVIDoc version 99.04 loci was extracted using the Qiagen software (UVItech limited. UK). Loci that DNeasy Tissue Kit (Qiagen, Valencia, appeared to be disomic were amplified and CA) and stored at –20°C. scored in a minimum of 30 individuals. We interpreted a locus as being PCR reactions and program polymorphic if multiple bands of the Microsatellite primer sequences tested appropriate size range and appearance here were as reported in Welsh et al. were present in most individuals. We 392 Moghim et al., Development of disomic single-locus DNA microsatellite markers… _________________________________________________________________________________________ determined if bands were of the amplification in Persian sturgeon including appropriate sizes based on allele sizes annealing temperature, observed allele size reported by Welsh et al. (2003) and their range in base pairs, number of Persian migrations relative to the dye in the stop sturgeons screened (N), number of alleles solution. Most loci scored
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