Abstract

not-yet-known not-yet-known not-yet-known unknown Genotyping-in-Thousands by sequencing emerged as a promising tool for genetic monitoring. For the past 25 years, genetic monitoring of Rio Grande silvery minnow (Hybognathus amarus) has been conducted annually by surveying variation at microsatellite loci. Here we developed a GT-seq panel to maintain the analytical and inferential continuity of the long-term genetic monitoring program for this species. We identified 2,983 microhaplotypes in 373 individuals using nextRAD-seq from archived samples spanning 20 years using a conspecific reference genome. This dataset provided estimates of genetic diversity and temporal genetic structure across the time-series. These results were used as a baseline to test subsets of loci that effectively tracked those changes. A panel including 250 loci with higher FST and 250 loci selected randomly offered the highest power and was used for GT-seq optimization. A sex-linked marker from another study was also included for sex assignment. The optimized GT-seq panel included 284 loci. Comparisons of genotypes from those loci obtained for the same samples with nextRAD-seq and GT-seq revealed high genotype accuracy (98.3%). Estimates of genetic diversity and patterns of temporal genetic structure were similar between datasets and accuracy of sex assignment was 100%. We discuss the utility of using a conspecific genome for both loci identification and primer design in the face of reduced genetic diversity, and the importance of temporal metrics representative of ongoing genetic monitoring. The strategy used here, effectively preserved the long-term genetic monitoring while transitioning to a more efficient and cost-effective marker system.