Rapd profile of horseshoe crabs, tachypleus Gigas

Abstract

Horseshoe crabs are prehistoric marine arthropods belonging to the family Limulidae and are still living today. Tachypleus gigas is one of the species of horseshoe crabs which are still abundant in Peninsular Malaysia. The purposes of conducting this study are to detect the DNA polymorphism among T gigas individuals and to verify the genetic variability of this species. Genomic DNA from 33 samples were extracted using the Wizard® Genomic DNA Purification Kit protocol. Screening of primers were done to determine the suitable Random Amplified Polymorphic DNA (RAPD) markers. These samples were then amplified using the Polymerase Chain Reaction (PCR) technique. The DNA purity obtained ranged from 1.00-3.50 while the DNA quantity ranged from 37.5-350.0 ng/μl. 40 primers were screened to test their amplification abilities and three primers: OPC5, OPD18, and OPD20, were chosen for further analysis. From these three primers, polymorphism was only detected using primer OPC5 and OPD20. A total of 31 bands were obtained with 90.32% polymorphism. The bands obtained ranged from 2-12 bands with 300-2000 bp. The mean of the similarity index among individuals of T gigas calculated was 0.61 ± 0.16. The genetic similarity ranged from 0.32-0.97 while the genetic distance ranged from 0. 03-1. 03. These results show that the genetic variety among individuals of Malaysian T gigas is quite diverse as they do not share the same genetic makeup. From this study, RAPD technique was found to be a suitable technique to generate genomic DNA profiles of horseshoe crabs.