Development, characterization and application of microsatellite markers of mangrove horseshoe crab, Carcinoscorpius rotundicauda Latreille in peninsular Malaysia

Horseshoe crab populations are said to be declining worldwide. However, there is still no published  report on the status of horseshoe crabs in Malaysia. In this study, microsatellite markers were  developed using 5'anchored ISSR-PCR enrichment procedure to diagnose population genetic structure  of the mangrove horseshoe crab, Carcinoscorpius rotundicauda. A total of 134 microsatellite regions  have been succesfully isolated using this technique and 36 novel microsatellite markers were  developed. Out of these, eleven informative microsatellite loci were identified in an analysis of  127 samples representing five C. rolundicauda populations namely Kuala Juru, Kg. Pennatang, Kg.  Chuah, Kg. Sekokoh and Kg. Sg. Pulai  from Peninsular Malaysia. The mean expected (He) and observed  heterozygosity (Ho) values obtained ranged between 0.299-0.42 l and 0.148-0.197 respectively. Significant departures from Hardy-Weinberg equilibrium were detected m C. rotundicauda populations  at Kg. Permatang, Kg. Chuah and Kg. Sg. Pulai. These populations also show moderate genetic  differentiation with overall FsT values of 0.108. Pairwise genetic distance values analysed between the  five populations studied  were close to zero which  indicate high genetic similarity between populations.  Five polymorphic inter-simple-sequence-repeat (ISSR) markers tested also generated comparable results. Thus, samples  in each population are presumed to be genetically similar although the populations studied were  geographically distant. Due to the unknown migratory ability of C. rotundicauda species, existing habitats must be  conserved for the survival of this species especially in Peninsular Malaysia. Findings from this  study provide information on the genetic diversity of C. rotundicauda in Peninsular Malaysia and  are hoped to assist in the development of conservation program for the mangrove horseshoe crabs in  Southeast Asia.