Genetic analysis
The nonlethal genetic tagging method known as parentage-based tagging
(PBT) was used to identify parent-pair offspring relationships among
sets of adult and offspring samples based on patterns in Mendelian
inheritance (Steele et al. 2013; 2019). Results from PBT were then used
to characterize mating systems and reproductive success among
Yellowstone Cutthroat Trout.
Genomic DNA was extracted from fin clips using the nexttec Genomic DNA
Isolation Kit (XpressBio, Thurmont, Maryland) following manufacturer’s
protocol. Samples were screened with a panel of 134 single nucleotide
polymorphic (SNP) loci which included a sex-linked marker
(2017SDYCUT ) used to identify genetic sex of each fish (see Roth
et al. 2018 for sex-linked primer details). Genetic sex data was used to
verify that parentage assignments were attributed to a male and female
combination. Primer sequences for all SNPs are available from the
authors upon request. Genotyping of SNPs was performed following the
genotyping-in-thousands protocol described by Campbell et al. (2015). We
screened all genotypes for duplicates and completeness; only unique
genotypes that were ≥50% complete (minimum of 66 loci) were retained
for analyses. Two forms of parentage error, Type I and II error, were
quantified by Roth et al. (2018) to assess the reliability of parentage
assignments for this data set. Type I error measures the rate that
untrue parent pairs were assigned as a true parent, and Type II error
refers to the number of offspring for which a true parent pair was
present but not assigned. Type I error was estimated using two separate
methods and was determined to be 0.000 and 0.001. Type II error was
estimated to be 0.001.
Parentage-based tagging was applied by Roth et al. (2018) to identify
parent pair-offspring relationships. Specifically, adults were
exhaustively sampled during their migration to spawning grounds and SNP
genotype profiles were created for each adult. This approach effectively
genetically tagged all offspring that would be the byproduct of matings
among sampled adults. Parentage assignments in trios (both parents and
one offspring) were estimated using the likelihood approach implemented
within SNPPIT (Anderson and Garza 2005; Anderson 2010) assuming a
per-allele genotyping error rate of 0.5%. The confidence of assignments
was assessed using the logarithm of odds (LOD) score generated by
SNPPIT. The LOD score represents the natural logarithm of the likelihood
of the parental trio hypothesis divided by the likelihood of the
nonparental hypothesis for a trio. Only parentage assignments with a LOD
score ≥ 18 were retained as previous analysis has identified this
threshold minimized false positive and false negative assignments (Roth
et al. 2018).