Genetic analysis of radio-tagged westslope cutthroat trout from St. Mary s River and Elk River April 9, 2002 Report prepared for: Angela Prince, M.Sc., R.P. Bio Westslope Fisheries 517 13 th Avenue South Cranbrook, B.C. V1C 2W5 Prepared by: Emily Rubidge Native Fish Research Group Department of Zoology University of British Columbia 6270 University Blvd Vancouver, B.C. V6T 1Z4 (604) 822-1301
Summary Tissue samples taken from 40 radio-tagged westslope cutthroat trout caught in two rivers in the Upper Kootenay drainage were analysed using molecular techniques. The objective of the analysis was to identify any hybrid individuals and to determine if the tagged trout were in fact pure westslope cutthroat trout. Four species-specific nuclear loci were used to differentiate between westslope cutthroat trout, rainbow trout and their hybrids. One hybrid was found in the St. Mary s River (Sample ID St. Mary s #11). This fish was classified as a backcrossed individual and mitochondrial DNA analysis indicated that the mother of this fish was a westslope cutthroat trout. All other individuals were classified as pure westslope cutthroat trout. DNA Analysis Genomic DNA was extracted from 40 fin clips (20 from St. Mary s River and 20 from Elk River) following standard Proteinase K/salt extraction techniques. Trout species and hybrids were identified using four diagnostic nuclear markers: two restriction fragment length polymorphisms (RFLPs) and two species-specific dual primer PCR (dppcr) markers developed by Ostberg and Rodriguez (2002). The RFLP markers are based on sequence variation in the Ikaros (IK) intron and the Heat Shock protein (HSC). These intron regions were amplified using the polymerase chain reaction (PCR) and incubated with restriction enzymes (Hinf I for IK and Taq I for HSC) following the enzyme supplier protocol (New England Biolabs) with 6 ul of PCR product in a 15 ul reaction volume (see Rubidge et al. 2001 for more detail). The two dppcr markers (OCC16 and OM13) were amplified following conditions detailed in Ostberg and Rodriguez (2002). Annealing temperatures and allele sizes are shown for all markers in Table 1. A portion of the mitochondrial DNA genome including the cytochrome b gene, the control region and a portion of the 12rRNA gene, was amplified in individuals of hybrid descent. Because mtdna is maternally inherited, RFLP haplotypes diagnostic for each species can be used to identify the female parent in each hybrid mating. Fragments were incubated with the restriction enzyme Ava II, following the enzyme supplier protocol (New England Biolabs). PCR products and restriction fragments were visualised on 1.5%-2.5% agarose gels stained with ethidium bromide. Table 1. Primers, PCR conditions (annealing temperatures/cycle number), species-specific restriction fragments and dppcr allele sizes for molecular markers used in DNA analysis of westslope cutthroat trout (WCT), rainbow trout (RBT) and their hybrids. Primer Source? Annealing temp and Cycle no. Enzyme for RFLPs & Allele Size IK 48/45 Hinf I, 500 (WCT), 750 (RBT) HSC 60,56/8,32 Taq I, 550 (WCT), 590 (RBT) OCC16 50/35 380 (WCT), 280 (RBT) OM13 56/35 190 (WCT), 175 (RBT)
Identification of Hybrid Class The number of molecular markers required to accurately identify hybrid genotypes from their parental genotypes is unclear. Boecklen and Howard (1997) showed that only about four to five markers are needed to provide a coarse classification of individuals in hybrid zones. Individuals were classified as pure O. clarki lewisi (WCT) or O. mykiss (RBT) only if they contained the respective diagnostic alleles at all loci. Individuals were considered to be hybrids if they contained any combination of alleles from the two parental species. The hybrids were then subdivided into F 1, F n or backcross genotype classes: F 1 genotype individuals are heterozygous at all loci; F n genotype individuals (or reverse backcrosses) are homozygous for alternate species alleles at two or more loci; and backcross genotypes (BC) are heterozygous at one to three loci, with the remaining loci homozygous for alleles from one species. These three hybrid classes are intended to describe the individuals' genotypes, but do not necessarily reflect their parentage. For example, an individual classified as a backcross genotype could be anything from a first generation backcross to an n th generation backcross (the latter would essentially be complete introgression of alleles).
Results St. Mary s River Fish Of the 20 individuals assayed, 17 were classified as pure WCT (homozygous for WCT alleles at all four loci), two were classified as pure WCT at two loci but failed to amplify at the other two loci, and one individual (SM-11) was classified as a backcross hybrid (heterozygous at one locus and homozygous for WCT alleles at the other three loci). The mitochondrial DNA haplotype of this backcrossed individual, SM-11, matched that of a westslope cutthroat trout. Because we cannot distinguish between a first generation backcross or an n th generation backcross this haplotype could have resulted in different ways. Two mating scenarios come to mind 1) the mother was a WCT that mated with a hybrid male; or 2) the mother was a hybrid with WCT mtdna that mated with a WCT male. The two scenarios cannot be distinguished without the genotype of SM-11's mother, but either way it is certain that mtdna was inherited from a WCT female. This indicates that when the mating between RBT and WCT took place to produce the hybrid relative of SM-11, the male was RBT and the female was WCT. Table 2. Genotypes of radio-tagged trout form the St. Mary s River using four species-specific nuclear loci. Sample # IK HSC OCC16 OM13 Classification SM-1 WCT WCT WCT WCT Westslope SM-2 WCT WCT WCT WCT Westslope SM-3 WCT WCT WCT WCT Westslope SM-4 WCT WCT WCT WCT Westslope SM-5 WCT WCT WCT WCT Westslope SM-6 WCT WCT WCT WCT Westslope SM-7* Failed Failed WCT WCT Westslope SM-8 WCT WCT WCT WCT Westslope SM-9 WCT WCT WCT WCT Westslope SM-10 WCT WCT WCT WCT Westslope SM-11 WCT HYB WCT WCT Backcross SM-12 WCT WCT WCT WCT Westslope SM-13 WCT WCT WCT WCT Westslope SM-14 WCT WCT WCT WCT Westslope SM-15 WCT WCT WCT WCT Westslope SM-16 WCT WCT WCT WCT Westslope SM-17 WCT WCT WCT WCT Westslope SM-18 WCT WCT WCT WCT Westslope SM-19 WCT WCT WCT WCT Westslope SM-20 WCT WCT WCT WCT Westslope * tissue from individual was degraded; low DNA quality most likely reason for failed RFLP
Elk River Fish All 20 individuals assayed were classified as pure WCT, however three individuals did not amplify at one of the four loci (most likely due to poor quality DNA). No hybrid individuals were detected. Table 2. Genotypes of radio-tagged trout form the St. Mary s River using four species-specific nuclear loci. Sample # IK HSC OCC16 OM13 Classification ER-1 WCT WCT WCT WCT Westslope ER-2 WCT WCT WCT WCT Westslope ER-3 WCT WCT WCT WCT Westslope ER-4 WCT WCT WCT WCT Westslope ER-5 WCT WCT WCT WCT Westslope ER-6 WCT WCT WCT WCT Westslope ER-7 WCT WCT WCT WCT Westslope ER-8 WCT WCT WCT WCT Westslope ER-9 WCT WCT WCT WCT Westslope ER-10 WCT Failed WCT WCT Westslope ER-11 WCT WCT WCT WCT Westslope ER-12 WCT WCT WCT WCT Westslope ER-13 WCT WCT WCT WCT Westslope ER14 WCT WCT WCT WCT Westslope ER-15 WCT WCT WCT WCT Westslope ER-16 WCT WCT WCT WCT Westslope ER-17 WCT WCT WCT WCT Westslope ER-18 WCT WCT WCT WCT Westslope ER-19 Failed WCT WCT WCT Westslope ER-20 WCT Failed WCT WCT Westslope References Boecklen, W. J., and D. J. Howard. 1997. Genetic Analysis of Hybrid Zones: Numbers of markers and power of resolution. Ecology 78:2611-26116. Ostberg, C. O., and J. Rodriguez. 2002. Novel molecular markers differentiate Oncorhynchus mykiss (rainbow trout and steelhead) and the O.clarki (cutthroat trout) subspecies. Molecular Ecology in press Rubidge, E., P. Corbett, and E. B. Taylor. 2001. A molecular analysis of hybridization between native westslope cutthroat trout and introduced rainbow trout in southeastern British Columbia, Canada. Journal of Fish Biology 59(Supplement A):42-54.