Isolation And Characterization Of Microsatellites In The Glauconome Rugosa

The Glauconome rugosa or Rugose Tagelus belongs to the Phylum Mollusca, Class Bivalvia, Order Veneroida, Family Glauconomidae and Genus Glauconome. It is also known as Wrinkled Sea-green Mussel or Brown or Mud Mussel. According to the record of World Register of Marine Species (WRMS), it was fist discovered and record by Hanley in1843.The Glauconome rugosa is mainly distributed in tropical waters of the Indo-pacific region of Asia, for example:Australia, East Indies, Malaysia, Singapore and others, and it thrives in habitat with shallow water and mud bay. Due to its delicious taste, Glauconome rugosa is harvested as one of the food resource in Southeast Asia. Till date, there is no relevant record of its aquaculture in China, and the research on its biology genetics has not been explored.Genetic markers are used to track any kind of genetic characteristics in the chromosome or on a segment or locus. Genetic markers which are widely used in the present study include morphological marker, cytological marker, biochemical marker, immunology marker and molecular marker.Microsatellites, as one of the second generation molecular markers, also known as short tandem repeats (STRs) or simple sequence repeats (SSRs), As early as1974, Skinner found them in the genome study of the hermit crab (Pagurus policaris).Because the number of simple sequence repeat is highly variable and abundant between individuals, the microsatellites are highly polymorphic. Simple operation and efficiency of microsatellite isolation make it widely used in eukaryotes’genome study. Currently the application in shellfish mainly include:genetic map construction, genetic diversity assessment, population genetic structure analysis, germplasm evaluation and protection and genetic relationship identification etc.In this study,17polymorphic microsatellites were isolated with the protocol of enrichment by magnetic beads and were also characterized,which are depicted with the details as following:Twenty-four Glauconome rugosa individuals were collected in Singapore Island;Hybridization between three types of biotinylated probe and the digested genomic DNA of upon one individual was done. Due to the strong affinity property between biotin and streptavidin, streptavidin-coated magnetic beads were used for enrichment of target repeat fragment through magnetic adsorption.The enrichment product was amplified using the21-mer adaptor as the primer. After checking with agrose gel, the PCR product was ligated to the pGEM-T vector. The ligated product was transformed into blue supercompetent cells (Strategene) and cultured on LB agar plates, containing ampicillin.192positive clones containing inserts were sequenced in both directions using sequencer ABI3730x1(Applied Biosystems) after PCR amplification using M13and M13R as primers and checking by agarose gel electrophoresis.Fifty unique microsatellites were identified after the sequencing results’ analysis and assembling by software Sequencher (GeneCodes). Primers were designed using software Primer Select(DNA Star) for25microsatellites containing enough flanking sequences for primer design, and one primer of each pair was labeled with either FAM or HEX (Sigma) fluorescent dyes. These25pairs of primers were used for PCR amplification and genotyping analysis with the24Glauconome rugosa individuals’DNA. After assembly of the sequences using software GeneMapper Version3.5(Applied Biosystems) and GDA, seventeen pairs of primers amplified specific and polymorphic products. The average allele number was8/locus with a range of5to14. The locus Glu01is the most polymorphic with14alleles, whereas Glu04is the least polymorphic with only five alleles. Extreme size difference (over50bp) of alleles was seen at five loci. The average observed heterozygosity was0.798with a range of0.62to0.96, and the expected heterozygosity was0.801with a range of0.65to0.91, which was slightly higher than the observed one. The exact tests for HWE revealed that11of the17microsatellites conformed to the Hardy-Weinberg equilibrium (P>0.05).The Glauconome rugosa tastes delicious with rich nutrition, but it is still a wild variety without aquaculture breeding and improvement, and its genetic basis is also wild type. The17polymorphic microsatellites isolated in this study facilitate a started understanding of its genetic diversity. To further improve the efficiency of genotyping of microsatellites, multiplex-PCR for the developed microsatellites should be developed. These microsatellite markers developed here will also facilitate the future study of the genetic diversity、population structure and germplasm resource conservation of Glauconome rugosa, and will support the superior germplasm selection to carry out large scale artificial breeding and aquaculture.