Patrick M. Gaffney

The oyster genome reveals stress adaptation and complexity of shell formation

The Pacific oyster Crassostrea gigas belongs to one of the most species-rich but genomically poorly explored phyla, the Mollusca. Here we report the sequencing and assembly of the oyster genome using short reads and a fosmid-pooling strategy, along with transcriptomes of development and stress response and the proteome of the shell. The oyster genome is highly polymorphic and rich in repetitive sequences, with some transposable elements still actively shaping variation. Transcriptome studies reveal an extensive set of genes responding to environmental stress. The expansion of genes coding for heat shock protein 70 and inhibitors of apoptosis is probably central to the oyster’s adaptation to sessile life in the highly stressful intertidal zone. Our analyses also show that shell formation in molluscs is more complex than currently understood and involves extensive participation of cells and their exosomes. The oyster genome sequence fills a void in our understanding of the Lophotrochozoa.

Genetic heterozygosity and growth rate in Mytilus edulis

Individuals of Mytilus edulis of the same age (ca 2 months) were collected as spat from natural populations. Relative growth rates were determined among individuals differing in heterozygosity at five enzyme loci. Growth rate was positively correlated with individual heterozygosity and each of the five loci contributed about equally to the relatinship. More heterozygous individuals also achieved more uniform average growth rates. Although there was a deficiency of heterozygotes at each locus, relative to Hardy-Weinberg expectations, the magnitude of the deficiency, measured as FIS, was less among faster growing mussels. Our results conform closely with those of Zouros et al. (1980) on the American oyster. We conclude that the relationship between multiple locus heterozygosity and growth rate is one that is general to a diversity of outbreeding plant and animal populations. Other studies indicate that this relationship is due to a greater average metabolic efficiency of more heterozygous individuals. This relationship does not emerge from experimental designs in which there has been limited genetic sampling of the natural genetic variation.

Genetic heterozygosity and production traits in natural and hatchery populations of bivalves

Abstract New data are presented and published data reviewed for several bivalve species regarding the correlations of electrophoretic phenotypes with performance characteristics such as growth and viability in natural and hatchery-reared populations. The relationship between allozyme phenotype and physiological traits depends strongly on the genetic structure of the population.

THE CASE FOR SEQUENCING THE PACIFIC OYSTER GENOME

Abstract An international community of biologists presents the Pacific oyster Crassostrea gigas as a candidate for genome sequencing. This oyster has global distribution and for the past several years the highest annual production of any freshwater or marine organism (4.2 million metric tons, worth

Hybridization among Crassostrea species: a review

3.5 billion US). Economic and cultural importance of oysters motivates a great deal of biologic research, which provides a compelling rationale for sequencing an oyster genome. Strong rationales for sequencing the oyster genome also come from contrasts to other genomes: membership in the Lophotrochozoa, an understudied branch of the Eukaryotes and high fecundity, with concomitantly high DNA sequence polymorphism and a population biology that is more like plants than any of the model animals whose genomes have been sequenced to date. Finally, oysters play an important, sentinel role in the estuarine and coastal marine habitats, where most humans live, environmental degradation is substantial, and oysters suffer intense fishing pressures and natural mortalities from disease and stress. Consumption of contaminated oysters can pose risks to human health from infectious diseases. The genome of the Pacific oyster, at 1C = 0.89 pg or ~824 Mb, ranks in the bottom 12% of genome sizes for the Phylum Mollusca. The biologic and genomic resources available for the Pacific oyster are unparalleled by resources for any other bivalve mollusc or marine invertebrate. Inbred lines have been developed for experimental crosses and genetics research. Use of DNA from inbred lines is proposed as a strategy for reducing the high nucleotide polymorphism, which can interfere with shotgun sequencing approaches. We have moderately dense linkage maps and various genomic and expressed DNA libraries. The value of these existing resources for a broad range of evolutionary and environmental sciences will be greatly leveraged by having a draft genome sequence.

Differences in mitochondrial 16S ribosomal gene sequences allow discrimination among American [Crassostrea virginica (Gmelin)] and Asian [C. gigas (Thunberg) C. ariakensis Wakiya] oyster species

Abstract Numerous attempts at hybridization in the genus Crassostrea have been reported. Most suffer from one or more of the following: (1) ambiguities in the classification of oysters; (2) contamination of experimental cultures by extraneous gametes or larvae; (3) the absence of control experiments for assessing the quality of gametes as well as larval viabilities; (4) the absence of genetic confirmation of hybrid status. We conclude that to date there is no unequivocal evidence for the existence of viable interspecific hybrids among Crassostrea species. Examples of taxonomic ambiguities that cloud interpretation of hybridization experiments include the frequent misclassification of the Kumamoto oyster ( C. sikamea ) as the Pacific oyster ( C. gigas ), the probably unjustified distinction between C. gigas and the Portuguese oyster C. angulata , and the lumping of C. iredalei with C. rivularis . Low-level contamination of cultures is a common occurrence that may account for many reports of successful hybridization. Experimental controls are essential to properly assess gamete quality and zygote growth and survival, yet are often not included. Finally, few reports provide genetic confirmation that the zygotes produced are actually hybrids. To date, most genetic analyses have demonstrated that putative interspecific hybrid oysters were not hybrids. Use of adequate controls and genetic analysis of parents and offspring are essential to hybridization experiments, and are demonstrated elsewhere.

Genetic effects of artificial propagation: signals from wild and hatchery populations of red abalone in California

Abstract Limited field trials for potentially disease resistant Asian cupped oysters were recently initiated in the central Atlantic coastal region of North America. We have developed a simple and sensitive genetic assay that unambiguously distinguishes the native Crassostrea virginica (Gmelin) from two of its exotic Asian congeners, C. gigas (Thunberg) and C. ariakensis (Wakiya). Homologous fragments (443 nt) of the mitochondrial rRNA large subunit were amplified from, and sequenced for, these three commercially important species of oysters. C. virginica exhibits 85.5 and 84% sequence similarity to C. gigas and C. ariakensis (respectively) and contains seven additional nucleotides in the amplified 16S gene fragment. The two Asian species do not differ in gene length and display 95.1% sequence similarity. Numerous endonuclease restriction site differences are apparent among the three oyster species and we present a polymerase chain reaction/restriction fragment length polymorphism strategy that can distinguish these species. This methodology will prove valuable in monitoring the ecological consequences, at all life history stages, of trial introductions of Asian Crassostrea species into this region.

Assessment of drift and selection in hatchery populations of oysters (Crassostrea virginica)

Abstract Numerous attempts have been made to restore the declining California abalone fisheries by outplanting of hatchery-produced seed. Poor survival of planted seed has been generally attributed to predation and emigration, lessening enthusiasm for reseeding efforts. We present genetic evidence that one large-scale outplanting of red abalone (Haliotis rufescens) in southern California was effective, and document changes in the genetic composition of red abalone populations accompanying hatchery propagation. Allozymes appeared to behave as neutral and therefore effective markers for tracking population bottlenecks in hatchery production and reseeded populations. Careful monitoring of hatchery breeding practices is essential for both commercial production and artificial reseeding programs.

Pearl, a novel family of putative transposable elements in bivalve mollusks

Abstract Allozyme frequencies in three hatchery sublines of oysters ( Crassostrea virginica ) after one generation of selection for rapid juvenile growth were compared to their respective unselected control sublines. Using genotypic data for six enzyme-coding loci, we analyzed (a) differences in allelic frequencies between sublines, (b) departures from Hardy-Weinberg proportions within sublines, and (c) distributions of multilocus genotypes. Because gene frequencies in the parents and parental source populations were unknown, Monte Carlo simulations based on the census number of spawning adults were used to obtain expected genotypic distributions. Comparison of expected and observed distributions indicated that estimated effective breeding numbers were significantly lower than the census number of spawning adults in mass-spawned populations. The discrepancy between nominal and estimated N e was reduced in sublines formed by pooling the progeny of multiple small group spawns. Control over N e in hatchery populations of cultured aquatic species may be improved by following this simple expedient.

Genetic Variability Assessed by Microsatellites in a Breeding Program of Pacific White Shrimp (Litopenaeus vannamei)

While genome sequencing projects have discovered numerous types of transposable elements in diverse eukaryotes, there are many taxa of ecological and evolutionary significance that have received little attention, such as the molluscan class Bivalvia. Examination of a 0.7-MB genomic sequence database from the cupped oyster Crassostrea virginica revealed the presence of a common interspersed element, CvA. CvA possesses subterminal inverted repeats, a tandemly repeated core element, a tetranucleotide microsatellite region, and the ability to form stable secondary structures. Three other less abundant repetitive elements with a similar structure but little sequence similarity were also found in C. virginica. Ana-1, a repetitive element with similar features, was discovered in the blood ark Anadara trapezia by probing a genomic library with a dimeric repeat element contained in intron 2 of a minor globin gene in that species. All of these elements are flanked by the dinucleotide AA, a putative target-site duplication. They exhibit structural similarity to the sea urchin Tsp family and DrosophilaSGM insertion sequences; in addition, they possess regions of sequence similarity to satellite DNA from several bivalve species. We suggest that the Crassostrea repetitive elements and Ana-1 are members of a new MITE-like family of nonautonomous transposable elements, named pearl. Pearl is the first putative nonautonomous DNA transposon to be identified in the phylum Mollusca.