Thus, the PINE technique coupled with PCR is used to search for evidence of genetic variation based on the 

 presence or absence of particular DNA fragments. The fragments are labeled by the primers used to produce " 

 them and their length in terms of the number of nucleotides in the fragment. 



The fragments are made using dye labeled nucleotides and after PCR are separated from each other via 

 electrophoresis in polyacrylamide gels. Smaller fragments move through the gels at a faster rate than larger 

 fragments. The use of dye labeled nucleotides allows one to visualize the position of the fragments in the gels 

 after electrophoresis using a spectrophotometer and the size of the fragments is determined by comparison to the 



position of synthetic fragments of known size that were also migrated into the gel. 



■ ' ' , . ■' 



When DNA from westslope cutthroat trout, Oncorhynchus clarki lewisi, and rainbow trout, O. mykiss. is 

 compared with PINE analysis and three different pairs of primers seven fragments are characteristic of westslope 

 cutthroat trout and six fragments are usually characteristic of rainbow trout (Table 1). Likewise, when DNA from 

 westslope and Yellowstone cutthroat trout, O. c. bouvieri, is compared using the same procedure one fragment is 

 characteristic of westslope cutthroat trout and four fragments are characteristic of Yellowstone cutthroat trout 

 (Table 1). - - f - 



Fragments produced from the DNA of one taxon and not another are commonly termed diagnostic or marker loci 

 because they can be used to help determine whether a sample came from a non-hybridized population of one of 

 the taxa or a population in which hydridization between them has or is occurring. Individuals from a non- 

 hybridized population will possess fragments characteristic of only that taxon. In contrast, since half the DNA of 

 first generation hybrids comes from each of the parental taxa the DNA from such individuals will yield all the 

 fragments characteristic of the two parental taxa. In later generation hybrids, the amount and particular regions of 

 DNA acquired from the parental taxa will vary among individuals. Thus, DNA from later generation hybrid 

 individuals will yield only a subset of the parental fragments and the particular subset will vary among 

 individuals. In a sample from a random mating hybrid swarm, that is a population in which the genetic material 

 (i.e. fragments) of the parental taxa is randomly distributed among individuals such that essentially all of them are 

 of hybrid origin, the frequency of the fragment producing allele from the non-native taxon is expected to be 

 nearly equal among the diagnostic loci since their presence can all be traced to a common origin or origins. Thus, 

 if a sample contains variation at only a single marker locus where the presence of the fragment is usually 

 characteristic of a non-native taxon and lacks such fragments at all other markers this is probably not indicative of 

 hybridization. Rather, it much more likely represents the existence of genetic variation for the presence or 

 absence of the fragment within this particular population of the native taxon. 



An important aspect of PINE marker loci is that individuals homozygous for the presence allele (pp) or 

 heterozygous (pa) will both yield the fragment. That is, p is dominant to a. Thus, in order to estimate the genetic 

 contribution of the native taxon to a hybrid swarm we concentrate on the marker loci at which thep allele is 

 characteristic of the non-native taxon. Furthermore, we must assume that genotypic distributions in the 

 population reasonably conform to expected random mating proportions. Under this assumption the frequency of 

 the native a allele is approximately the square root of the frequency of individuals in the population lacking the 

 fragment (aa). The frequency of the non-native allele then is one minus this value. We focus on thep alleles 

 characteristic of the non-native taxon because with low levels of hybridization it is the presence of these alleles 

 that are likely to provide evidence of hybridization. With low levels of hybridization, it is likely all individuals in 

 the sample will genotypically hepp or pa where the/? allele is characteristic of the native taxon. Thus, like in 

 non-hybridized populations all individuals in the sample will yield the fragment providing no evidence of 

 hybridization. 



Failure to detect evidence of hybridization in a sample does not necessarily mean the population is non- 

 hybridized because there is always the possibility that we would not detect evidence of hybridization because of 



