Genetic inviability is a major driver of type III survivorship in experimental families of a highly fecund marine bivalve.

The offspring of most highly fecund marine fish and shellfish suffer substantial mortality early in the life cycle, complicating prediction of recruitment and fisheries management. Early mortality has long been attributed to environmental factors and almost never to genetic sources. Previous work on a variety of marine bivalve species uncovered substantial genetic inviability among the offspring of inbred crosses, suggesting a large load of early-acting deleterious recessive mutations. However, genetic inviability of randomly bred offspring has not been addressed. Here, genome-wide surveys reveal widespread, genotype-dependent mortality in randomly bred, full-sib progenies of wild-caught Pacific oysters (Crassostrea gigas). Using gene-mapping methods, we infer that 11-19 detrimental alleles per family render 97.9-99.8% of progeny inviable. The variable genomic positions of viability loci among families imply a surprisingly large load of partially dominant or additive detrimental mutations in wild adult oysters. Although caution is required in interpreting the relevance of experimental results for natural field environments, we argue that the observed genetic inviability corresponds with type III survivorship, which is characteristic of both hatchery and field environments and that our results, therefore, suggest the need for additional experiments under the near-natural conditions of mesocosms. We explore the population genetic implications of our results, calculating a detrimental mutation rate that is comparable to that estimated for conifers and other highly fecund perennial plants. Genetic inviability ought to be considered as a potential major source of low and variable recruitment in highly fecund marine animals.

Detection of mitochondrial DNA from domestic cattle in bison on Santa Catalina Island.

In 1924, 14 American bison (Bison bison) were introduced to Santa Catalina Island, California and sporadically supplemented thereafter with additional animals. To reduce the herd and its impact on native vegetation, over 2000 animals have been exported during the past four decades. Today, the herd is estimated to contain around 250 individuals. Genetic analysis was performed on 98 animals removed from the island in 2004. Forty-four samples (45%) had domestic cattle mitochondrial DNA (mtDNA), 12 (12%) had previously reported bison haplotypes and 42 (43%) had a new haplotype differing by one base pair from a previously reported bison haplotype. A complement of five restriction enzymes was found to be useful in identifying bison with domestic cattle mtDNA.

High genetic load in the Pacific oyster Crassostrea gigas.

The causes of inbreeding depression and the converse phenomenon of heterosis or hybrid vigor remain poorly understood despite their scientific and agricultural importance. In bivalve molluscs, related phenomena, marker-associated heterosis and distortion of marker segregation ratios, have been widely reported over the past 25 years. A large load of deleterious recessive mutations could explain both phenomena, according to the dominance hypothesis of heterosis. Using inbred lines derived from a natural population of Pacific oysters and classical crossbreeding experiments, we compare the segregation ratios of microsatellite DNA markers at 6 hr and 2-3 months postfertilization in F(2) or F(3) hybrid families. We find evidence for strong and widespread selection against identical-by-descent marker homozygotes. The marker segregation data, when fit to models of selection against linked deleterious recessive mutations and extrapolated to the whole genome, suggest that the wild founders of inbred lines carried a minimum of 8-14 highly deleterious recessive mutations. This evidence for a high genetic load strongly supports the dominance theory of heterosis and inbreeding depression and establishes the oyster as an animal model for understanding the genetic and physiological causes of these economically important phenomena.

The impact of supplementation in winter-run chinook salmon on effective population size.

Supplementation of young raised at a protected site, such as a hatchery, may influence the effective population size of an endangered species. A supplementation program for the endangered winter-run chinook salmon from the Sacramento River, California, has been releasing fish since 1991. A breeding protocol, instituted in 1992, seeks to maximize the effective population size from the captive spawners by equaling their contributions to the released progeny. As a result, the releases in 1994 and 1995 appear not to have decreased the overall effective population size and may have increased it somewhat. However, mistaken use of non-winter-run chinook spawners resulted in artificial crosses between runs with a potential reduction in effective population size, and imprinting of the released fish on Battle Creek, the site of the hatchery, resulted in limiting the contribution of the released fish to the target mainstem population. Rapid genetic analysis of captured spawners and a new rearing facility on the Sacramento River should alleviate these problems and their negative effect on the effective population size in future years.

Fixation, segregation and linkage of allozyme loci in inbred families of the Pacific oyster Crassostrea gigas (Thunberg): implications for the causes of inbreeding depression.

The effect that inbreeding has on the fixation and segregation of genes has rarely been confirmed by direct observation. Here, fixation, segregation, and linkage of allozymes is investigated in the progeny of self-fertilized hermaphrodites of the normally outcrossing Pacific oyster Crassostrea gigas. The estimate of fixation pooled over loci, individuals, and families, F = 0.462, is significantly lower than the expected value of 0.5. Log-likelihood ratios reveal significant heterogeneity in fixation among individuals, among families, and among loci. In addition, the grand pooled segregation ratio, 127:243:54, deviates significantly from 1:2:1, with a bias against homozygotes for alleles of lesser frequency in the natural population. Segregation ratios for 11 of 14 loci are significantly heterogeneous among families, and exact tests for segregation within families reveal 16 significant results out of 51 tests. Thus, fixation and segregation of allozyme markers in inbred oyster families deviates from the expectations of neutral inbreeding theory. Di-genic disequilibria are significant for four of 74 di-locus pairs revealing two linkage groups. Strong viability selection is apparently conditional on the genotype of the hermaphrodite-founders and is largely focused on these two linkage groups. These genetic effects are explained by interaction between cis-linked factors and polymorphic regulatory backgrounds.

On the potential for estimating the effective number of breeders from heterozygote-excess in progeny.

The important parameter of effective population size is rarely estimable directly from demographic data. Indirect estimates of effective population size may be made from genetic data such as temporal variation of allelic frequencies or linkage disequilibrium in cohorts. We suggest here that an indirect estimate of the effective number of breeders might be based on the excess of heterozygosity expected in a cohort of progeny produced by a limited number of males and females. In computer simulations, heterozygote excesses for 30 unlinked loci having various numbers of alleles and allele-frequency profiles were obtained for cohorts produced by samples of breeders drawn form an age-structured population and having known variance in reproductive success and effective number. The 95% confidence limits around the estimate contained the true effective population size in 70 of 72 trials and the Spearman rank correlation of estimated and actual values was 0.991. An estimate based on the heterozygote excess might have certain advantages over the previous estimates, requiring only single-locus and single-cohort data, but the sampling error among individuals and the effect of departures from random union of gametes still need to be explored.

Morphogenesis of Maternal and Paternal Genomes in Fertilized Oyster Eggs (Crassostrea gigas): Effects of Cytochalasin B at Different Periods During Meiotic Maturation.

Fertilized oyster (Crassostrea gigas) eggs, treated with and without cytochalasin B (CB) at varying periods during meiotic maturation, were examined following 7-aminoactinomycin D (7-AAD) and anti-{beta} tubulin staining for DNA and microtubular patterns, respectively. Electron microscopic observations of untreated fertilized eggs revealed the development of an extensive network of endoplasmic reticulum during germinal vesicle breakdown. Germinal vesicle breakdown was not accompanied by the disappearance of the nucleolus; the latter persisted morphologically unchanged throughout fertilization. The first meiotic spindle [12.4 +/- 1.0 {mu}m (1) x 5.4 +/- 0.55 {mu}m (diameter (d); metaphase plate)] was oriented with its long axis perpendicular to the egg's surface. In contrast, the second meiotic spindle was approximately one half the size of the first [7.4 +/- 0.65 {mu}m (1) x 2.91 +/- 0.29 {mu}m (d)] and initially oriented with its long axis parallel to the egg's surface. Just prior to anaphase II, the spindle rotated so that its long axis became perpendicular to the egg's surface. Following its incorporation into the egg cytoplasm, the sperm nucleus dispersed but did not form a nuclear envelope until the completion of polar body formation. Just prior to pronuclear migration, an array of microtubules assembled around the female pronucleus, and then regressed; concomitantly, a sperm aster formed in conjunction with the centrosome associated with the developing male pronucleus. Following their migration and apposition with one another, both pronuclei underwent prophase as independent structures. Chromosomes from the male and female pronuclei became organized on a mitotic spindle in preparation for first cleavage. In zygotes treated with CB continuously or for just meiosis I, anaphase I occurred; however, a polar body failed to form. Consequently, all the diads were retained within the zygote and became situated on a tripolar spindle. In such cases, anaphase II resulted in the production of a variable number of chromosomal aggregations that developed into pronuclei (3 to 6). In contrast, zygotes treated with CB during meiosis II developed spindles characteristic of untreated specimens; however, polar body formation was blocked, resulting in the development of usually two maternal pronuclei. Morphogenesis of C. gigas fertilized eggs is discussed in reference to similar processes in other species and with respect to strategies of triploid embryo production using CB.

Discrimination between closely related Pacific oyster species (Crassostrea) via mitochondrial DNA sequences coding for large subunit rRNA.

Mitochondrial DNA sequence variation was characterized for the large subunit rRNA-coding gene (16SrDNA) in two closely related Pacific oyster species (Crassostrea gigas and C. sikamea) and an out group, the Olympia oyster (Ostrea lurida). Although each species was shown to have a single, fixed haplotype for the DNA sequence under study, 7 nucleotide differences were found between C. gigas and C. sikamea, and these two species differed from the O. lurida haplotype at 62 and 60 nucleotide sites, respectively. Nucleotide differences for the two Crassostrea species showed a notable transition bias (85.7%) in contrast to the marginal transversion bias (54.5%) in nucleotide differences between Crassostrea haplotypes and the more distantly related O. lurida. Conservation of primary sequence in all three oyster species as well as other published 16SrDNA sequences was noted for regions with apparent functional significance. We developed DNA sequence-specific discrimination techniques and employed sequence-specific PCR primers, dot-blot hybridization, and restriction digests as alternate techniques for rapid diagnosis of Crassostrea oyster larvae.

Population variation of human mtDNA control region sequences detected by enzymatic amplification and sequence-specific oligonucleotide probes.

A method for detecting sequence variation of hypervariable segments of the mtDNA control region was developed. The technique uses hybridization of sequence-specific oligonucleotide (SSO) probes to DNA sequences that have been amplified by PCR. The nucleotide sequences of the two hypervariable segments of the mtDNA control region from 52 individuals were determined; these sequences were then used to define nine regions suitable for SSO typing. A total of 23 SSO probes were used to detect sequence variants at these nine regions in 525 individuals from five ethnic groups (African, Asian, Caucasian, Japanese, and Mexican). The SSO typing revealed an enormous amount of variability, with 274 mtDNA types observed among these 525 individuals and with diversity values, for each population, exceeding .95. For each of the nine mtDNA regions significant differences in the frequencies of sequence variants were observed between these five populations. The mtDNA SSO-typing system was successfully applied to a case involving individual identification of skeletal remains; the probability of a random match was approximately 0.7%. The potential useful applications of this mtDNA SSO-typing system thus include the analysis of individual identity as well as population genetic studies.

Genetic variation in two widespread species of salamanders, Taricha granulosa and Taricha torosa.

Two species of the genus Taricha are widely distributed. T. granulosa ranges from southern Alaska to central California. T. torosa is comprised of two described subspecies, T. t. torosa, which occupies much of the coast ranges of California, and T. t. sierrae, which inhabits the western slopes of the Sierra Nevada Mountains. A starch gel electrophoretic survey for genetic variation at 34 loci in four population samples of T. granulosa and at 40 loci in five population samples of T. torosa reveals differences among these taxa both in amounts of intrapopulational variability and in patterns of geographic variation. Average observed heterozygosity is 9.6% +/- 0.3% in T. granulosa, 3.3% +/- 0.5% in T. t. torosa, and 7.2% +/- 1.2% in T. t. sierrae. Average numbers of alleles per lon T. t. sierrae, and lowest in T. t. torosa. Oregon and California granulosa are genetically nearly as different as the subspecies of torosa, but geographic variation is continuous in the former. T. torosa on the other hand is comprised of three distinct gene pools--T. t. sierrae and northern and southern races of T. t. torosa. Strikingly different amounts of intrapopulational genetic variation and patterns of geographic variation may be explained by steady-state species differences, but historical causes may also exist.