Isolation by the 5′ anchored PCR technique and characterization of eighteen microsatellite loci in horseshoe crab (Tachypleus gigas)

Abstract

The horseshoe crab, a well-known ‘living fossil’, is represented by two species (Tachypleus gigas and Carcinoscorpius rotundicauda) in peninsular Malaysia. One of these, T. gigas inhabits shallow marine waters and migrates to intertidal sandy beaches for breeding during high tides at every full and new moon throughout the year (Hajeb et al. 2009). This species is being used as bait by local fishermen to catch crabs or shrimps by netting or cage fishing methods. Due to its low commercial value in comparison to other marine economic species such as fishes, crabs and shrimps, most of the animals caught in fishing nets or cages are released immediately back to sea by fishermen or left to die in the sun. In certain states of Malaysia, such as Kelantan, Penang and Johore, adult female horseshoe crabs are sold to local seafood restaurants and in fish markets due to consumption of their eggs by the local people. Horseshoe crab populations are rapidly declining around the world due to various reasons such as water pollution, loss of their living and spawning habitats and human exploitation of adult horseshoe crabs (Li et al. 2009; Shin et al. 2009). Assessing the genetic variability of horseshoe crab is important as it has direct benefit for the conservation and management of wild populations of the species. However, there is no information available on the population genetic structure and diversity of T. gigas in Malaysia. To address this research need, we have isolated and developed the first set of polymorphic microsatellite markers for T. gigas by using the 5′ anchored PCR technique. A total of 130 individuals of horseshoe crabs were collected from five locations along the coastal areas of peninsular Malaysia, namely Port Dickson (Negeri Sembilan), Pantai Bersih (Penang), Sungai Muar (Johor), Pantai Balok