Development and evaluation of a new microsatellite multiplex system for parental allocation and management of rainbow trout (Oncorhynchus mykiss) broodstocks

Examination of microsatellite variation at several polymorphic loci is a widely accepted method for determining parentage and examining genetic diversity within rainbow trout (Oncorhynchus mykiss) breeding programs. Genotyping costs are considerable; therefore, we developed a single-step method of co-amplifying twelve microsatellites in two hexaplex reactions to reduce costs and improve efficiency. The protocol is explicitly described to ensure reproducible results. We applied the protocol to samples analyzed at the National Center for Cool and Coldwater Aquaculture (NCCCWA) with previously reported marker sets for a comparison of results. Each marker within the multiplex system was evaluated for duplication, null alleles, physical linkage, and probability of genotyping errors. Data from four of the 12 markers were excluded from parental analysis based on these criteria. Parental assignments were compared to those of a previous study that used five independently amplified microsatellites. Percentages of progeny assigned to parents were higher using the subset of eight markers from the multiplex system than with five markers used in the previous study (98% vs. 92%). Through multiplexing, additional markers were used to improve parental allocation while also improving efficiency by reducing the number of required PCR reactions and genotyping runs. We evaluated our methods further through estimation of F-statistics, pairwise genetic distances, and cluster analysis among brood-years at the NCCCWA facility. These measures were compared to estimates from nine independently amplified microsatellites used in a previous study. The F-st results calculated between brood-years show similar values of genetic differentiation using both marker sets. Estimates of individual pairwise genetic distances were used for constructing neighbor-joining trees. Both marker sets yielded trees that show similar subpopulation structuring and agreed with results from a model-based cluster analysis and available pedigree information. These approaches for detecting population substructure and admixture portions within individuals are particularly useful for new breeding programs where the founders' relatedness is unknown. Published by Elsevier B.V.