Inland walleye genotypes

Targeted amplicon sequencing methods, such as genotyping-in-thousands by
sequencing (GT-seq), facilitate rapid, accurate, and cost-effective
analysis of hundreds of genetic loci in thousands of individuals.
Development of GT-seq panels is non-trivial, but studies describing
trade-offs associated with different steps of GT-seq panel development are
rare. Here, we construct a dual-purpose GT-seq panel for walleye (Sander
vitreus), discuss trade-offs associated with different development and
genotyping approaches, and provide suggestions for researchers
constructing their own GT-seq panels. Our GT-seq panel was developed using
an ascertainment set consisting of restriction site-associated DNA data
from 954 individuals sampled from 23 populations in Minnesota and
Wisconsin. We then conducted simulations to test the utility of all loci
for parentage analysis and genetic stock identification and designed 600
primer pairs to maximize joint accuracy for these analyses. We conducted
three rounds of primer optimization to remove loci that overamplified and
our final panel consisted of 436 loci. We also explored different
approaches for DNA extraction, multiplexed polymerase chain reaction (PCR)
amplification, and cleanup steps during the GT-seq process and discovered
the following: (1) inexpensive Chelex extractions performed well for
genotyping, (2) the exonuclease I and shrimp alkaline phosphatase (ExoSAP)
procedure included in some current protocols did not improve results
substantially and was likely unnecessary, and (3) it was possible to PCR
amplify panels separately and combine them prior to adapter ligation.
Well-optimized GT-seq panels are valuable resources for conservation
genetics and our findings and suggestions should aid in their construction
in myriad taxa.