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Hybridization has been frequently observed among 3 species of greenlings (genus Hexagrammos) common in waters off Japan. In order to estimate the frequency of hybridization events from egg masses collected from male territories, efficient maternal identification of numerous egg masses is required. A novel streamlined approach for maternal identification of 3 Hexagrammos spp. was developed using multiplex amplified product length polymorphism (APLP) analysis of the mitochondrial cytochrome b (Cytb) and the 12S and 16S ribosomal RNA (12-16S rRNA) regions. Concurrent use of species-specific primer sets permits the amplification of different-sized PCR products, diagnosing each species through one procedure of PCR in a single reaction tube. The APLP method produced more rapid, reliable, and cost-efficient species identifications compared to those from an established restriction fragment length polymorphism (RFLP) protocol.

Maternal identification of hybrid eggs in Hexagrammos spp. by means of multiplex amplified product length polymorphism of mitochondrial DNA