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United States Department of Agriculture

Agricultural Research Service

Research Project: IDENTIFICATION AND CHARACTERIZATION OF GENES AFFECTING COOL AND COLD WATER AQUACULTURE PRODUCTION Title: Development and Critical Evaluation of a New Multiplex System for Parental Allocation and Broodstock Management in Rainbow Trout

Authors
item Johnson, Nathan
item Rexroad, Caird
item Eric, Hallerman - VIRGINIA POLYTECH INST
item Vallejo, Roger
item Palti, Yniv

Submitted to: Aquaculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 26, 2007
Publication Date: April 27, 2007
Citation: Johnson, N., Rexroad III, C.E., Eric, H., Vallejo, R.L., Palti, Y. 2007. Development and critical evaluation of a new multiplex system for parental allocation and broodstock management in rainbow trout. Aquaculture 266:53-62.

Interpretive Summary: Many commercial aquaculture breeding programs start rearing individuals from different families before they are large enough to be tagged and are in search of affordable and accurate genetic tagging method that will enable family base analysis of production efficiency performance. Examination of microsatellite DNA markers variation is the predominant 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. Parental assignments were compared to those of a previous study that used five independently amplified microsatellites. Percentages of progeny assigned to parents were higher with the new marker system. 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.

Technical Abstract: Examination of microsatellite variation is the predominant 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 previously analyzed to evaluate the accuracy of the multiplexes. Each locus within the system was evaluated for duplication, null alleles, physical linkage, and probability of genotyping errors. Parental assignments were compared to those of a previous study that used five independently amplified microsatellites. Percentages of progeny assigned to parents were higher with eight multiplexed loci than with five markers used in the previous study. 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. These measures were compared to estimates from nine independently amplified microsatellites used in a previous study. The Fst 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 STRUCTURE analysis. These approaches of detecting population substructuring are particularly useful for new breeding programs where the founders’ relatedness is unknown.

Last Modified: 10/19/2014
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