Time is of the essence: using archived samples in the development of a GT-seq panel to preserve continuity of ongoing genetic monitoring

Genotyping-in-Thousands by sequencing (GT-seq) is a promising tool for genetic monitoring. For the past 25 years, genetic monitoring of Rio Grande silvery minnow (<i>Hybognathus amarus</i>) has been conducted annually by surveying variation at microsatellite loci. This is the first study describing the development of a GT-seq panel using archived samples that maintains the analytical and inferential continuity of long-term genetic monitoring. A total of 2,983 microhaplotypes in 373 individuals were identified using nextRAD-seq from samples spanning 20 years and a conspecific reference genome. Using this data, estimates of genetic diversity and temporal genetic structure across the time-series were used as a baseline to test subsets of loci that effectively tracked those changes. A panel including 250 loci with higher F_ST across temporal samples and 250 loci selected randomly offered the highest power and was used for GT-seq optimization. A sex-linked marker validated previously 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%. 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 is explored. The strategy used here, effectively preserved the long-term genetic monitoring of the endangered Rio Grande silvery minnow while transitioning to a more efficient and cost-effective marker system.