Development of microsatellite markers in tropical abalone Haliotis asinina

Abstract

Three genomic libraries were constructed for microsatellite isolation in the tropical abalone Haliotis asinina. The first genomic library was constructed from DNA fragments obtained from AluI digestion. The second library was from vortexed and sonicated genomic DNA. The third library was from mixed-enzyme (Alu I, Rsa I and Hinc II) digested genomic DNA. After screening with (GT)15-, it was found that the third library gave the highest percentage of positive clones (1.46%) whereas the first and the second libraries gave 0.2% and 0.43% of positive clones, respectively. Dot blot hybridization suggested that (GT)n -repeats were more abundant than (CT)n repeats. Nucleotide sequencing of positive clones revealed that perfect, imperfect and compound microsatellites were found in the same proportion. From 33 microsatellite loci, 10 pairs of specific primers were designed for microsatellie amplification. Preliminary analysis of polymophism of 10 loci (Has1-10) showed that numbers of alleles ranged from 3-26 alleles and observed heterozygosity were 0.27-0.85. Genotypes from these microsatellite were associated randomly (p > 0.0031). All pairs of primers could not be used for cross-amplification in other tropical abalone species, H. ovina and H. varia. Sensitivity of PCR using 32P labeled primer indicated that genotyping of 2 week-old (spat) samples can be detected and only 50 pg of DNA is enough for detection.Three microsatellite loci, Has2, Has3 and Has8 were used for examination of genetic variation in natural and hatchery stocks of H. asinina. Three natural samples were collected from the Andaman Sea (Talibong Island) and the Gulf of Thailand (Samet Island and Cambodia). Hatchery samples were brought from 3 hatcheries where broodstocks originated from Samet Island, Cambodia and Philippines respectively. The effective number of alleles (ne) and observed heterozygosity (hobs) indicated that natural samples from the Gulf of Thailand (Semet: ne = 5.73, hobs = 0.78; Cambodia: ne = 7.65, hobs = 0.62) have higher genetic variation than that of the Andaman sea (Talibong: ne = 5.10, hobs = 0.58) and hatchery populations from Samet (ne = 6.16, hobs = 0.82) and Cambodia (ne = 6.63, hobs = 0.79) still have high genetic variation. Gene frequencies of each population conform to Hardy-Weinberg equilibrium (p > 0.0027) except natural population from Cambodia (at Has2 loci (p < 0.0001) and Has3 loci (P < 0.0001)) and the Philippines samples (at Has8 locus (p < 0.0001)). Analysis of geographic heterogeneity suggested that samples within the Gulf of Thailand did not showed significant heterogeneity to one another (p > 0.0027) but significant heterogeneity was observed between the Gulf of Thailand, the Andaman Sea and hatchery stock from Philippines (p < 0.0027). Phylogenetic reconstruction using the Neighbor-joining approach divided the 6 geographic samples to three different gene pools constituting of the Gulf of Thailand (A), the Andaman Sea (B) and the Philippines (C).