Development of two new microsatellite multiplex PCRs for three sparid species: Gilthead seabream ( Sparus auratus L.), red porgy ( Pagrus pagrus L.) and redbanded seabream ( P. auriga, Valenciennes, 1843) and their application to paternity studies

Abstract

This study reports two new and highly informative multiplex PCRs of microsatellite markers, with redesigned interspecific primer sets for three sparid species: gilthead seabream ( Sparus auratus L.), red porgy ( Pagrus pagrus L.) and redbanded seabream ( P. auriga, Valenciennes, 1843). The evaluation and validation of the two multiplex PCRs, named RimA and RimB (Redesigned Interspecific Multiplex), were carried out upon: 148 individual gilthead seabream (66 breeders of unknown gender, and 82 of their descendents obtained by mass-spawning), 37 red porgy and 125 redbanded seabream. From 15 and 12 redesigned microsatellite markers for RimA and RimB, respectively, the number of markers included in the final multiplex PCRs were 10 in RimA and 7 in RimB for gilthead seabream, 6 in each multiplex PCR for red porgy and 8 in RimA and 5 in RimB for redbanded seabream. The a priori combined parental exclusion probability for each multiplex in the three species was 0.999. For gilthead seabream, it was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with at least seven microsatellite markers for each multiplex PCR. Null alleles were found only for marker PbMS2, through familial segregation, with a frequency similar to the expected one (0.09 vs 0.14). Results revealed that the multiplex reaction no more than one-sixth the cost of single reactions even when these reactions were performed in a unique run, and that genotyping errors were minimized due to automation. These robust multiplex PCRs will be a fundamental tool for the industry to introduce selection programs and to manage their broodstocks under industrial conditions.