Single locus microsatellite marker development for the green-lipped mussel, Perna viridis.

The green lipped-mussel, Perna viridis belongs to the order Mytilidae. It is abundant in Southeast Asia, has two green to dark green chitinous outer shells and is commercially harvested and eaten. P. viridis is commercially important because of its rapid growth rate and high population densities. In the present study, a novel method, Random Amplified Microsatellites (RAMS) was used to develop single locus microsatellite markers for the green lipped-mussels. RAMs or Random Amplified Microsatellites, originally described by Zietkiewicz et al. (1994), is a dominant multiloci genetic marker system which can also be used as part of the process to isolate microsatellite regions and develop single locus microsatellite markers. The development of a 5' anchoring procedure allows the consistent anchoring of PCR primers at the 5' ends of microsatellites, amplifying two close and inverted simple sequence repeats and the region between them. The technique of RAMs is sensitive enough to even differentiate between closely related individuals. Single locus microsatellite is a highly informative genetic marker system for analyzing intraspecific phenomena related to both natural genetic substructuring and anthropogenic changes. It is a class of nuclear DNA loci, consisting of tandemly repeated sequence motifs of two to six base pairs in length. It is based on the polymerase chain reaction (PCR) and easy to score. The procedures included DNA extraction, PCR amplification, cloning, plasmid extraction and finally automated DNA sequencing. DNA from the green mussel was isolated by the protocol of Winnepenninckx et al. (1993) with some modifications. Then the PCR products were cloned into the TOPO Cloning vector. Positive clones of transformed E. coli cells were extracted using the method of Sambrook et al. (1989). Finally the plasmids were sequenced using a DNA sLquencer. A total of 14 RAMS primers will be screened but at this stage of the study only two different degenerate primers containing (CAG)₅ and (TACA)₅ microsatellite repeat motifs had been used to detect the presence of microsatellite sequences in the genomic DNA of p. viriclis from different individuals. 13 clones from the first primer had been sent for sequencing and a total of 16 microsatellite regions were detected while for the second primer, 23 clones had been sent for sequencing and a total of 50 microsatellite regions were detected. Single locus microsatellites are highly polymorphic repeat DNA sequences that have become the markers of choice in many research fields in biology. The findings of our study showed the success of using this method as a starting point for detecting microsatellite sequences in p. viridis.The work will be continued with the designing of the forward and reverie primers for each microsatellite region and they will then be tested for their abilities to amplify single locus microsatellite markers in this species.