Characterisation of eight dinucleotide microsatellite loci and sequencing of the cytochrome oxidase I (COI) gene in the Asian Horseshoe Crab

Ow, Choong Kit (2007) Characterisation of eight dinucleotide microsatellite loci and sequencing of the cytochrome oxidase I (COI) gene in the Asian Horseshoe Crab. Universiti Malaysia Sabah. (Unpublished)

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Abstract

Population study for the horseshoe crab was performed using microsatellite markers and cytochrome oxidase I (COI) gene. COI gene is used for phylogenetic studies and also in DNA barcoding. A total of 15 samples were collected from Tuaran and Kota Belud for population analysis using dinucleotide microsatellite loci. Samples collected from Tuaran were Carcinoscorpius rotundicauda and samples from Kota Belud were Tachypleus tridentatus. DNA extraction was performed using phenol-chloroform extraction method and characterisation of population was done using six microsatellite loci, TTLR121, TTLR122, TTLR1A, TTLR1B2, TTLR151 and TTLR152. TTLR1B1 and TTLR13 were not used because the bands produced were unreproducible. All tested loci were 100% polymorphic and loci TTLR121, TTLR122 and TTLRIA exhibited heterozygote phenotype in the samples. The mean observed heterozygosity (Ho) for the population is 0.063 and mean of Nei's (1973) expected heterozygosity (HE) for the population is 0.468. All tested loci were found to deviate from Hardy- Weinberg equilibrium with the highest x2 value in locus TTLRIB2 (38.857) and the lowest in locus TTLR152 (18.087). The dendrogram constructed based on Nei's (1972) genetic distance using UPGMA method showed that samples from Tuaran and Kota Belud were clustered separately. A single DNA sample from Tuaran and Kota Belud were used for COI gene sequencing. PCR amplification of COI gene was done using specifically designed primers TTCO 1F and TTCO1R. After PCR, cloning and plasmid minipreparation was done before it was sequenced. COI gene sequence obtained was submitted to GenBank and accession number EF195333 was given for C. rotundicauda and EF460846 for T tridentatus. Sequence alignment of COI gene from the study and from other species of horseshoe crab found in GenBank was done and pairwise nucleotide differences were calculated. The dendrogram and consensus tree using bootstrapping method was constructed and it showed that C. rotundicauda from the study did not form a cluster with the same species in both the phylogenetic trees

Item Type: Academic Exercise
Uncontrolled Keywords: horseshoe crab, cytochrome oxidase I, gene, DNA, micro satellite, GenBank
Subjects: Q Science > QH Natural history > QH426 Genetics
Divisions: SCHOOL > School of Science and Technology
Depositing User: MDM SITI AZIZAH IDRIS
Date Deposited: 02 Aug 2013 05:38
Last Modified: 23 Oct 2017 07:26
URI: http://eprints.ums.edu.my/id/eprint/6693

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