Species identification of five penaeid shrimps using PCR-RFLP and SSCP analyses of 16S ribosomal DNA

DNA-based molecular markers for differentiation of five penaeid shrimps (Penaeus monodon, P. semisulcatus, Feneropenaeus merguiensis, Litopenaeus vannamei and Marsupenaeus japonicus) were developed based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single-stranded conformation polymorphism (SSCP) of 16S ribosomal (r) DNA. Differentiation of P. monodon, P. semisulcatus and L. vannamei can be unambiguously carried out by PCRRFLP of 16S rDNA(560) whereas P. semisulcatus and M. japonicus shared a BABB mitotype. These shrimps were successfully discriminated by SSCP analysis of 16S rDNA(560). Nevertheless, the amplification success for L. vannamei and F. merguiensis was not consistent when tested against larger sample sizes. As a result, 16S rDNA(560) of an individual representing the most common mitotype of each species was cloned and sequenced. The new primer pair was designed and tested against the large sample sizes (312 bp product, N = 185). The amplification success was consistent across all species. PCR-RFLP of 16S rDNA(312) was as effective as that of 16S rDNA(560). Differentiation of all shrimp species were successfully carried out by SSCP analysis.     

Identification of reproduction-related proteins and characterization of the protein disulfide isomerase A6 cDNA in ovaries of the giant tiger shrimp Penaeus monodon

Proteomic analysis was carried out for identification of proteins functionally involved in ovarian development of the giant tiger shrimp (Penaeus monodon). A total of 335 protein spots including 183 spots from vitellogenic (stage II) and 152 spots from mature (stage IV) ovaries of intact P. monodon broodstock were examined. Of these, 75 (40.98%) and 59 (38.82%) spots significantly matched known proteins in the databases, respectively. In addition, 270 protein spots including 167 and 103 spots from respective ovarian stages of eyestalk-ablated broodstock were also characterized. A total of 95 (56.89%) and 62 (60.19%) spots matched known proteins, respectively. Among differentially expressed reproduction-related proteins, the full-length cDNA of protein disulfide isomerase A6 (PmPDIA6) was further characterized by RACE-PCR. PmPDIA6 was 1946 bp in length containing an open reading frame (ORF) of 1293 bp corresponding to a polypeptide of 430 amino acids. PmPDIA6 was up-regulated at stage III ovaries in intact shrimp (P < 0.05). Interestingly, eyestalk ablation resulted in a lower expression level of PmPDIA6 in each stage of ovarian development compared to that of intact broodstock (P < 0.05). Results in this study clearly indicated the potential of cellular proteomic studies and gene expression analysis for identification of proteins/genes differentially expressed during ovarian development of P. monodon.     

Genetic Diversity and Molecular Markers of the Tropical Abalone (<Emphasis Type="Italic">Haliotis asinina</Emphasis>) in Thailand

Genetic diversity of abalone in Thailand, Haliotis asinina, H. ovina, and H. varia, was analyzed by polymerase chain reaction (PCR) of 18S and 16S rDNAs, with randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). Species-specific RAPD markers were found in each abalone species. Restriction analysis of 18S (nuclear) ribosomal DNA with Alu I, Taq I, and Hae III and 16S (mitochondrial) rDNA with Bam HI, Eco RI, Hae III, and Alu I gave 12 and 13 digestion patterns, respectively. A total of 49 composite haplotypes were found. A dendogram obtained by the unweighted pair-group method with arithmetic mean, constructed from divergence between pairs of composite haplotypes, revealed reproductively isolated gene pools of these abalone and indicated that H. asinina and H. ovina are genetically closer than H. varia. When H. varia was discovered owing to small sample sizes, geographic heterogeneity analysis and F_ST estimate indicated clear genetic differentiation between H. ovina originating from the Andaman Sea (west) and the Gulf of Thailand (east, P < 0.0001), whereas partial differentiation was observed between the Philippines and the remaining H. asinina samples (P < 0.0021). The amplified 16S rDNAs of individuals representing composite haplotypes found in this study were cloned and sequenced. A neighbor-joining tree constructed from sequence divergence of 16S rDNA accurately allocated those sequences according to species origins of abalone. Species-specific PCR based on 16S rDNA polymorphism was successfully developed in H. asinina and H. varia but not in H. ovina.     

The development of species-specific AFLP-derived SCAR and SSCP markers to identify mantis shrimp species

Molecular Biology Reports - Mantis shrimp has become commercially valuable in many countries, while the commercially aquaculture still unsuccessful. The stable supply of the species-specific...     

Development of polymorphic microsatellites for genetic studies of white scar oyster (Crassostrea belcheri) using paired-end shotgun sequencing

Molecular Biology Reports - White scar oyster Crassostrea belcheri is a commercially important bivalve species in Thailand. Appropriate genetic markers are needed for effective management to...