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.
Khamnamtong
Bavornlak
B
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Paholyothin Rd., Klong 1, Klong Laung, Pathumthani 12120. sirawut@biotec.or.th
Klinbunga
Sirawut
S
Menasveta
Piamsak
P
D000818:Animals; D001483:Base Sequence; D004275:DNA, Ribosomal / Q000032:analysis*; D003937:Diagnosis, Differential; D005819:Genetic Markers / Q000235:genetics*; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000737:chemistry* / Q000145:classification*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational; D012689:Sequence Homology, Nucleic Acid
D004275:DNA, Ribosomal; D005819:Genetic Markers