|Johnson, Nathan - VIRGINIA POLYTECH INST. &|
|Hallerman, Eric - VIRGINIA TECH|
Submitted to: World Aquaculture Society Meeting
Publication Type: Abstract Only
Publication Acceptance Date: October 1, 2005
Publication Date: February 26, 2007
Citation: Johnson, N., Palti, Y., Rexroad III, C.E., Silverstein, J., Vallejo, R.L. 2006. Critical evaluation of a new multiplex system used for parental allocation and broodstock management in rainbow trout (oncorhynchus mykiss) selection programs. World Aquaculture Society Meeting. Technical Abstract: Examination of microsatellite variation at several polymorphic loci is the predominant method for determining parentage and examining genetic diversity within rainbow trout (Oncorhynchus mykiss). The genotyping costs associated with these analyses are considerable for large-scale commercial breeding programs; therefore, we utilized a high throughput and high-resolution single-step method of co-amplifying multiple microsatellite markers to reduce costs and improve efficiency. A protocol for co-amplifying twelve DNA microsatellite loci in two hexaplex reactions was developed for rainbow trout. The protocol was applied to collect genotypic data from samples previously analyzed in order to evaluate the accuracy of the multiplexes as tools for determining parentage and for managing hatchery broodstocks. Each locus within the multiplex system was evaluated based on criteria of non-duplication, absence of null alleles, un-linked, and low probability of genotyping errors. Four of the twelve markers were excluded from parental analysis based on these criteria. Parental assignments were calculated using the “no error” model of PAPA software and compared to the results of a previous study that used five independently amplified microsatellite loci to assign parentage in a mixed-family stock. Percentage of correct parental assignment was > 96% for the 8 multiplexed loci and approximately 92% for the 5 markers used in the previous study. Through the utilization of multiplexing, additional markers were used to improve parental allocation while also improving efficiency by reducing the number of required PCR reactions. We evaluated our methods further through the estimation of F- statistics between and genetic distances within even and odd brood-years of rainbow trout (Oncorhynchus mykiss) at the National Center for Cool and Cold Water Aquaculture (NCCCWA) facility in Leetown, WV. These measures of genetic variability were compared to estimates derived from a set of nine independently amplified microsatellite loci used in a previous study. Fst results calculated between brood-years show similar values of genetic differentiation in both marker sets. Estimates of individual pairwise genetic distances, using the transformed proportion of shared alleles (-ln(PSA)) were used to detect strain sub-division through the construction of Neighbor-joining trees. Both marker-sets yielded trees that show similar sub-population structuring. This approach of detecting population sub-structuring is particularly useful for new breeding programs where the founders’ relatedness is unknown.