Cloning and characterization of pejerrey mitochondrial DNA and its application for RFLP analysis

ABSTRACT

Probes were cloned, characterized, and developed for all regions of the mitochondrial DNA (mtDNA) of pejerrey Odontesthes bonariensis to provide the basis for the study of genetic diversity of South American atherinopsinii and to enable species identification from small amounts of tissue. The mtDNA was extracted from liver and cleaved with Eco RI, producing four fragments (7·4, 3·4, 3·1 and 2·9 kb) which were cloned using pUC118 plasmid vectors. Sequence analysis from both ends of the fragments showed that they encode tRNA (Asp, Phe, and Ser-TGA), 12 S rRNA, cytochrome oxidase (CO) II, NADH 4, 5, and 6, and the d-loop, and that the relative positions of these genes are identical to those in the mtDNA of other teleosts. A comparison of homology with carp mtDNA nucleotide sequences revealed that tRNA (Phe and Ser-TGA) and CO II were relatively conserved, whereas the d-loop region was highly divergent. The cloned mtDNA probes detected mtDNA fragments from about 800 ng of total DNA extracted from liver, muscle, and single embryos of O. bonariensis, and were effective for restriction length fragment polymorphism (RFLP) analysis of Patagonina hatcheri, the most distant atherinopsine relative of pejerrey. The cloned mtDNA probes may be useful for the analysis of genetic diversity and non-destructive species identification, including the examin-ation of eggs, larvae and juveniles. The mtDNA sequences reported here provide the basis for the design of primers for PCR-based RFLP analysis. 1997 The Fisheries Society of the British Isles