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.

Transcriptome profiling of selectively bred Pacific oyster Crassostrea gigas families that differ in tolerance of heat shock.

Sessile inhabitants of marine intertidal environments commonly face heat stress, an important component of summer mortality syndrome in the Pacific oyster Crassostrea gigas. Marker-aided selection programs would be useful for developing oyster strains that resist summer mortality; however, there is currently a need to identify candidate genes associated with stress tolerance and to develop molecular markers associated with those genes. To identify candidate genes for further study, we used cDNA microarrays to test the hypothesis that oyster families that had high (>64%) or low (<29%) survival of heat shock (43 degrees C, 1 h) differ in their transcriptional responses to stress. Based upon data generated by the microarray and by real-time quantitative PCR, we found that transcription after heat shock increased for genes putatively encoding heat shock proteins and genes for proteins that synthesize lipids, protect against bacterial infection, and regulate spawning, whereas transcription decreased for genes for proteins that mobilize lipids and detoxify reactive oxygen species. RNAs putatively identified as heat shock protein 27, collagen, peroxinectin, S-crystallin, and two genes with no match in Genbank had higher transcript concentrations in low-surviving families than in high-surviving families, whereas concentration of putative cystatin B mRNA was greater in high-surviving families. These ESTs should be studied further for use in marker-aided selection programs. Low survival of heat shock could result from a complex interaction of cell damage, opportunistic infection, and metabolic exhaustion.

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.

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

BACKGROUND: Since 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. RESULTS: As 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. CONCLUSION: This 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.

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.

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: F(ST)=0.042, P=0.02; Ots515NWFSC: F(ST)=0.058, P=0.003). In contrast, the fall and threatened spring runs are genetically homogenous based on neutral microsatellite data (F(ST)=-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: F(ST)=0.083, P-value=0.001; Ots515NWFSC: F(ST)=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 F(ST )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.

MUTATIONAL CONTRIBUTIONS TO GENETIC VARIANCE-COVARIANCE MATRICES: AN EXPERIMENTAL APPROACH USING INDUCED MUTATIONS IN ARABIDOPSIS THALIANA.

Genetic potential for evolutionary change and covariational constraints are typically summarized as the genetic variance-covariance matrix G, and there is currently debate over the extent to which G remains effectively constant during the course of adaptive evolution. However, G provides only a temporally restricted view of constraints that ignores possible biases in how new mutations affect multivariate phenotypes. We used chemical mutagenesis to study the effect of mutations as summarized by the mutational covariance matrix, M, in Arabidopsis thaliana. By introducing mutations into three isogenic strains of A. thaliana, we were able to quantify M directly as the genetic variance-covariance matrix of mutagenized lines. Induced mutations generally did not alter the means of the six morphology and life-history traits we measured, but they did affect the levels of available genetic variation and the covariances among traits. However, these effects were not consistent among the three isogenic lines; that is, there were significant differences among the lines in both the number of mutations produced by ethyl-methane-sulfonate treatment and the M matrices they induced. The evolutionary implications of the dependence of M on the number of mutations, the particular genetic background, and the mutagenic sampling of loci in the genome are discussed in light of commonly applied models of multivariate evolution and the potential for the genetic architecture itself to change in ways that facilitate the coordinated evolution of complex phenotypes.

A RECENT HOST RANGE EXPANSION IN JUNONIA COENIA HÜBNER (NYMPHALIDAE): OVIPOSITION PREFERENCE, SURVIVAL, GROWTH, AND CHEMICAL DEFENSE.

This paper reports on an investigation of two populations of Junonia coenia, the buckeye butterfly, one that feeds on the species' typical host plant (Plantago lanceolata) and one that utilizes a novel host plant (Kickxia elatine). I examined these populations for local adaptive responses in terms of oviposition behavior, growth, and chemical defense, on both P. lanceolata and K. elatine. In addition, I examined the genetic architecture underlying these traits using a full-sib quantitative genetic analysis. I found that a significant majority of females prefer the host plant species found at their collection sites in oviposition tests, but that there is no evidence that they are locally adapted in growth performance, as measured by fifth-instar and pupal weights and development times. Neither are there correlations between oviposition preferences of females and the growth performance or levels of chemical defense of their offspring. The two populations studied do, however, show specialization in terms of the levels of chemical defense they sequester from their host plants. I argue that these results indicate that natural enemies are the normal barriers to host range expansion in this oligophagous herbivore because a breakdown in those barriers results in genetic changes that enhance resistance to predation. This is despite the fact that adaptive responses in physiology are unlikely to be limited by a lack of genetic variability; the genetic architecture among traits would be conducive to specialization in growth performance; and there are costs to chemical defense in this species. All these conditions would tend to argue that J. coenia harbors considerable potential for coevolutionary interactions with its chemically defended hosts, but this potential is not realized, probably because natural selection on diet breadth by natural enemies is much stronger than selection from host plants in this system.