A Reliable and Cost-Efficient PCR-RFLP Tool for the Rapid Identification of Cetaceans in the Mediterranean Sea

Abstract

Twenty-five species of cetaceans have been reported throughout the Mediterranean Sea, eight of them are commonly distributed in the whole basin and are regularly found beached or adrift in the sea. Stranded animals are frequently found in poor conservation status, preventing reliable identification; identification is thus often based solely on morphological features. Therewith, molecular tools are especially useful to provide taxonomic identification. In this work, a four-enzymes PCR-RFLP in silico protocol, based on a fragment of the mitochondrial gene cytb, has been designed for cetacean species occurring in the Mediterranean Sea. Moreover, beached or floating specimen samples belonging to the eight common species have been tested in the laboratory, providing evidence that this approach represents a reliable, cost- and time-effective tool for their specific identification.

Long Eared Owls (Asio otus Linnaeus, 1758) as Field-Assistants in an Integrative Taxonomy Survey of a Peculiar Microtus savii (Rodentia, Cricetidae) Population

“…Samples were incubated at 55 • C for 24 to 72 h with occasional vortex mixing, adding 20 µL of Proteinase K every 12 h until complete lysis of the bone fragments was achieved. The obtained solution was then processed using the Wizard Genomic DNA Purification Kit (Promega) following a modified protocol [27,28]. For the final step, 50 µL of DNA Rehydration Solution were aliquoted in each sample, which were then incubated at 37 • C for 20 to resuspend DNA.…”

“…Samples were incubated at 55 • C for 24 to 72 h with occasional vortex mixing, adding 20 µL of Proteinase K every 12 h until complete lysis of the bone fragments was achieved. The obtained solution was then processed using the Wizard Genomic DNA Purification Kit (Promega) following a modified protocol[27,28]. For the final step, 50 µL of DNA Rehydration Solution were aliquoted in each sample, which were then incubated at 37 • C for 20 to resuspend DNA.PCR amplification targeting the mitochondrial gene cytochrome b (cytb), widely used for similar molecular analyses[3,6,29,30], was performed using four sets of primers that enabled the amplification of the whole gene and of three partially overlapping fragments of the gene, the latter were used to amplify samples that failed to…”