Development of molecular markers for studies of genetic polymorphism and expression of genes related with metabolisms in Thai abalone Haliotis asinina

Abstract

A total of 2876 ESTs were in silico analyzed and 178 EST sequences consisting of microsatellite regions were found. Four loci (DW455, DW503, PHe177, and PT102) of type I and 2 loci (Haµ9 and Haµ10) of type II microsatellites were used for determination of genetic diversity in wild and domesticated H. asinina and generated 10, 7, 6, 6, 10, and 12 alleles across all samples, respectively. Allele distribution frequencies revealed that the domesticated stocks are majorly contributed by the Gulf of Thailand founders. Observed and expected heterogeneity in wild populations ranged from 0.0625 - 1.0000 and 0.0625 - 0.8448 whereas those in CTRGH and CSMaRTH were 0.3441 - 0.7769 and 0.3333 - 0.8750, respectively. Lower allele and genotype discrimination capacity in domesticated than wild stocks suggested slightly reduced genetic diversity even though high observed heterozygosity was found in the domesticated stocks of H. asinina. FST-statistics and the exact test between pairs of samples at each locus clearly revealed significant differences between wild abalone from the east (SAME, and CAME) and west (TRGW) coasts of Thai waters and the Philippines. Both domesticated stocks (CTRGH and CSMaRTH) were genetically different from TRGW (west) and the Philippines (PHI) at all loci. For AFLP analysis, 64 selective amplification primers were screened. Nine candidates AFLP were converted to sequence-characterized amplified region (SCAR) markers. HaSCAR320, HaSCAR295, and HaSCAR327 exhibited 3, 2, and 3 SSCP genotypes respectively. SSCP genotypes of H. asinina from the Gulf of Thailand and Andaman Sea did not overlap at the HaSCAR327 locus. Genotypes AAA, AAC, and CAA were restrictively observed in the Gulf of Thailand whereas ABB, BAB, and BBB were only distributed in the TRGW sample. Almost all of the CSMaRTH and CTRGH stocks exhibited the east coast genotypes (131/135; 97.03%). Distribution frequencies of composite SSCP genotypes and genetic heterogeneity analysis suggested that offspring of abalone from the east coast population exhibited better adaptability to the cultivated conditions than those from the west coast population. Correlation between genotypes and the body weight of H. asinina (group B) was tested at each microsatellite. A significant genetic heterogeneity between BL - BS subgroups was observed at DW455 and Haµ10 loci (P < 0.05). Significant differences between the body weights of the B sample having different genotypes were only observed at Haµ10 (P < 0.05). The full length cDNA of α-methylacyl-CoA racemase (AMACR), carnitine O-palmitoyltransferase 1A (CPT-1A), hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/ enoyl-CoA hydratase (trifunctional protein) α-subunit and vacuolar H+ ATPase (V-ATP) 14 kDa subunit were successfully characterized by RACE-PCR. AMACR, CPT-1A, hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/ enoyl-CoA hydratase and V-ATP contained the open reading frame (ORF) of 1140, 2304, 2292 and 369 bp corresponding to a polypeptide of 380, 768, 764 and 123 amino acids, respectively. Quantitative real-time PCR analysis indicated that the relative expression level of AMACR and V-ATP 14 kDa subunit of fast- and slow-growing subgroups of domesticated abalone were not significantly different. In contrast, the expression level of CPT-1A in hepatopancreas of the former was significantly greater than that of the latter subgroup (P < 0.05).