Mark D. Camara

Candidate loci reveal genetic differentiation between temporally divergent migratory runs of Chinook salmon (Oncorhynchus tshawytscha).

Local adaptation is a dynamic process driven by selection that can vary both in space and time. One important temporal adaptation for migratory animals is the time at which individuals return to breeding sites. Chinook salmon (Oncorhynchus tshawytscha) are excellent subjects for studying the genetic basis of temporal adaptation because their high seasonal homing fidelity promotes reproductive isolation leading to the formation of local populations across diverse environments. We tested for adaptive genetic differentiation between seasonal runs of Chinook salmon using two candidate loci; the circadian rhythm gene, OtsClock1b, and Ots515NWFSC, a microsatellite locus showing sequence identity to three salmonid genes central to reproductive development. We found significant evidence for two genetically distinct migratory runs in the Feather River, California (OtsClock1b: FST = 0.042, P = 0.02; Ots515NWFSC: FST = 0.058, P = 0.003). In contrast, the fall and threatened spring runs are genetically homogenous based on neutral microsatellite data (FST = –0.0002). Similarly, two temporally divergent migratory runs of Chinook salmon from New Zealand are genetically differentiated based on polymorphisms in the candidate loci (OtsClock1b: FST = 0.083, P‐value = 0.001; Ots515NWFSC: FST = 0.095, P‐value = 0.000). We used an individual‐based assignment method to confirm that these recently diverged populations originated from a single source in California. Tests for selective neutrality indicate that OtsClock1b and Ots515NWFSC exhibit substantial departures from neutral expectations in both systems. The large FST estimates could therefore be the result of directional selection. Evidence presented here suggests that OtsClock1b and Ots515NWFSC may influence migration and spawning timing of Chinook salmon in these river systems.

Sequence polymorphism can produce serious artefacts in real-time PCR assays: hard lessons from Pacific oysters

BackgroundSince it was first described in the mid-1990s, quantitative real time PCR (Q-PCR) has been widely used in many fields of biomedical research and molecular diagnostics. This method is routinely used to validate whole transcriptome analyses such as DNA microarrays, suppressive subtractive hybridization (SSH) or differential display techniques such as cDNA-AFLP (Amplification Fragment Length Polymorphism). Despite efforts to optimize the methodology, misleading results are still possible, even when standard optimization approaches are followed.ResultsAs part of a larger project aimed at elucidating transcriptome-level responses of Pacific oysters (Crassostrea gigas) to various environmental stressors, we used microarrays and cDNA-AFLP to identify Expressed Sequence Tag (EST) fragments that are differentially expressed in response to bacterial challenge in two heat shock tolerant and two heat shock sensitive full-sib oyster families. We then designed primers for these differentially expressed ESTs in order to validate the results using Q-PCR. For two of these ESTs we tested fourteen primer pairs each and using standard optimization methods (i.e. melt-curve analysis to ensure amplification of a single product), determined that of the fourteen primer pairs tested, six and nine pairs respectively amplified a single product and were thus acceptable for further testing. However, when we used these primers, we obtained different statistical outcomes among primer pairs, raising unexpected but serious questions about their reliability. We hypothesize that as a consequence of high levels of sequence polymorphism in Pacific oysters, Q-PCR amplification is sub-optimal in some individuals because sequence variants in priming sites results in poor primer binding and amplification in some individuals. This issue is similar to the high frequency of null alleles observed for microsatellite markers in Pacific oysters.ConclusionThis study highlights potential difficulties for using Q-PCR as a validation tool for transcriptome analysis in the presence of sequence polymorphism and emphasizes the need for extreme caution and thorough primer testing when assaying genetically diverse biological materials such as Pacific oysters. Our findings suggest that melt-curve analysis alone may not be sufficient as a mean of identifying acceptable Q-PCR primers. Minimally, testing numerous primer pairs seems to be necessary to avoid false conclusions from flawed Q-PCR assays for which sequence variation among individuals produces artifactual and unreliable quantitative results.

p-loci: a computer program for choosing the most efficient set of loci for parentage assignment: COMPUTER PROGRAMS

Determining how many and which codominant marker loci are required for accurate parentage assignment is not straightforward because levels of marker polymorphism, linkage, allelic distributions among potential parents and other factors produce differences in the discriminatory power of individual markers and sets of markers. p‐loci software identifies the most efficient set of codominant markers for assigning parentage at a user‐defined level of success, using either simulated or actual offspring genotypes of known parentage. Simulations can incorporate linkage among markers, mating design and frequencies of null alleles and/or genotyping errors. p‐loci is available for windows systems at http://marineresearch.oregonstate.edu/genetics/ploci.htm.

p-loci: a computer program for choosing the most efficient set of loci for parentage assignment.

Determining how many and which codominant marker loci are required for accurate parentage assignment is not straightforward because levels of marker polymorphism, linkage, allelic distributions among potential parents and other factors produce differences in the discriminatory power of individual markers and sets of markers. p-loci software identifies the most efficient set of codominant markers for assigning parentage at a user-defined level of success, using either simulated or actual offspring genotypes of known parentage. Simulations can incorporate linkage among markers, mating design and frequencies of null alleles and/or genotyping errors. p-loci is available for windows systems at http://marineresearch.oregonstate.edu/genetics/ploci.htm.

Mitochondrial and Nuclear DNA Analysis of Genetic Heterogeneity Among Recruitment Cohorts of the European Flat Oyster Ostrea edulis

Marine species with high fecundity and high early mortality may also have high variance in reproductive success among individuals due to stochastic factors, making successful reproduction a “sweepstakes.” In some cases, the impact is sufficient to reduce the effective number of breeders in wild populations. We tested two predictions of the sweepstakes reproductive success hypothesis in a French Atlantic population of the European flat oyster, Ostrea edulis, by evaluating (1) whether individuals belonging to temporally discrete recruitment cohorts within a single reproductive season displayed reduced genetic variation relative to the entire adult population, and (2) whether these temporal cohorts of recruits were genetically differentiated from each other. We assayed genetic variation at four nuclear microsatellites and a 12S mitochondrial fragment in four recruitment cohorts. Nuclear markers provided no evidence for differentiation between recruitment cohorts and adults or between temporal cohorts. However, mitochondrial data indicate that the first temporal cohort showed significant differentiation with the last (Fst = 0.052, P < 0.05) and with the adult sample (Fst = 0.058, P < 0.05). These differences are most likely due to the smaller effective size of the mitochondrial genome—and hence its increased sensitivity to drift compared to the nuclear genome. This slight mitochondrial signal indicates a certain limitation in the number of contributing female parents in this species. The “sweepstakes” phenomenon was therefore limited in our case. Hypothetically, this phenomenon may occur or not, with a high variance as a result of the interaction between the oyster reproductive biology and different environmental conditions.

Nineteen novel microsatellite markers for the Olympia oyster, Ostrea conchaphila/lurida

Accurate evaluation of remnant Ostrea conchaphila/lurida population structure is critical for developing appropriate restoration efforts. Here we report 19 polymorphic microsatellites suitable for analyses of population differentiation, pedigree reconstruction and linkage map construction. We screened clones from four enriched genomic libraries, identified 73 microsatellite‐containing sequences and designed polymerase chain reaction primers for 44 of these loci. We successfully optimized polymerase chain reaction conditions for 20 loci, including one monomorphic locus. In a Willapa Bay reference sample, mean observed and expected heterozygosities were 0.6729 and 0.8377. Nine loci deviated from Hardy‐Weinberg equilibrium. These markers have proven useful for genetic studies of the Olympia oyster.