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

Abstract

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 PCR-RFLP of 16S TDNA560 whereas P. semisulcatus and M. japonicus shared a BABB mitotype. These shrimps were successfully discriminated by SSCP analysis of 16S TDNA560. Nevertheless, the amplification success for L. vannamei and F. merguiensis was not consistent when tested against larger sample sizes. As a result, 16S rDNA560 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 rDNA312 was as effective as that of 16S TDNA 560. Differentiation of all shrimp species were successfully carried out by SSCP analysis.