International Council C.M. 2002/U:11 for the Exploration of the Sea Mariculture Committee Trends in genetics of bivalve mollusks: A review S. Stiles and J. Choromanski Abstract Recent advances in genome mapping and the development of linkage maps, as well as the production of polyploids and aneuploids, in bivalve mollusks have led to a resurgence of interest in chromosome organization and manipulation. Knowledge of normal meiosis and baseline data on mitoses in these invertebrates can assist in such undertakings and serve to elucidate genome features and processes. Baseline data will be presented on meiosis and genetic manipulation such as polyploidy, aneuploidy, and tetraploid cloning in the eastern oyster, Crassostrea virginica. For example, approximately 12% of eggs from mass-spawned populations of C. virginica oysters were heteroploid. Haploids were 6%, polyploids 1.5%, hypodiploids 1.5%, hyperdiploids 1.5%, and mosaics 1.5%. Thus, aneuploids averaged 3% in these populations of oysters, which can serve as a reference frequency for current studies on aneuploidy in eastern oysters. Previous chromosome engineering efforts for the induction of cloning or polyploidy in 15 experiments with eastern oysters revealed that the ploidy level of early embryos developed from eggs treated with cytochalasin B, high pressure and/or exposed to irradiated sperm in general ranged from haploidy through pentaploidy. Outcomes depended on the female, experimental conditions, synchronous development and whether or not the sperm were genetically inactivated with irradiation. Triploidy occurred as high as 66%, but generally ranged from 3% to 38%. Some embryos were chromosomal mosaics or aneuploids. Implications for genetic manipulation, including transgenesis, in other bivalves such as bay scallops will be discussed. Results should be considered with regard to efforts for rehabilitating or restocking bivalve populations. Key Words: Chromosomes, polyploidy, aneuploidy, cloning, bivalve mollusks, genome mapping, linkage S. Stiles and J. Choromanski, National Oceanic and Atmospheric Administration (NOAA)/National Marine Fisheries Service (NMFS), Milford Laboratory, Milford, Connecticut, USA (Tel. 203-882-6524; FAX 203-882-6517; email: [email protected], [email protected] 1 INTRODUCTION New developments in genomics are a key component of modern genetics with some understanding of basic concepts such as genome evolution and gene interactions. They also provide a basis for the application of modern genetics to biological, agricultural and aquaculture sciences, including biotechnological advances. For example, relationships are being explored for DNA replication, Mendelian ratios and Hardy-Weinberg equilibrium. Genomic sequence databases are also being explored, providing new insights into how genes function as well as characterization of transposable elements. In addition, an understanding of dimensions between DNA and chromosomes is becoming necessary. However, such relationships between physical and genetic distances of genes and their impact on genetic events, gene expression, and evolution have been little elucidated. Trends in genetics of bivalve mollusks encompass investigations of genome mapping and linkage, the development of inbred lines to subsequently cross for heterosis, the development of hybrids and the induction of polyploidy and aneuploidy. Application of molecular (DNA) and cytological (banding) tools are valuable for population genetics investigations of stock identification and related studies on marker-assisted selection and quantitative trait loci for breeding. Ultimate goals are increased harvests of natural populations and increased production in commercial hatcheries. Aquaculture or controlled culture of bivalve mollusks has provided opportunities for selection and breeding to improve stocks. Approaches have included inbreeding, mass selection, genome manipulation and population analyses for genetic diversity. Overall, some progress has been made through selective breeding for commercially important traits such as growth, survival, and disease resistance. Recent advances in genetics of some commercially valuable bivalve mollusks include induction of triploidy, tetraploidy and aneuploidy, and development of disease-resistant lines in oysters, development of notata clams as genetically-marked stocks, and development of genetically-marked and transgenic lines of scallops. Since gametes of bivalves generally are spawned or released at metaphase I of meiosis, opportunities also exist and are afforded for use of genetic manipulation or biotechnology, not easily done in finfish or other vertebrates. In addition, millions of gametes can be manipulated for polyploid, cloning and transgenic induction. The purpose of this present review, primarily of chromosomal genetics using the eastern oyster (Crassostrea virginica) as a model, was to evaluate status and trends including genome mapping and, particularly, cytological responses of a bivalve to some common means for inducing gynogenesis, androgenesis and polyploidy. The value of genetic manipulations such as gynogenesis in practical breeding and in basic research has been pointed out by Purdom (1983), Kirpichnikov (1981), Stanley (1974) and several researchers as listed in the bibliography. In addition, production of successful gynogenetic progeny offers some special opportunities for studying the quantitative inheritance of economic traits. 2 Chromosome Genetics One of the bases to a better understanding of these approaches involves chromosomal variation and polymorphisms or genomic processes in evolution or selection. Concurrent with advances in molecular genetics has come increased awareness and understanding of the role of chromosomes as a significant part of genomic research in any organism. Additionally, the advent of molecular marker techniques has greatly facilitated the construction of linkage maps. For example, linkage maps have been constructed using morphological traits, isozymes, restriction fragment length polymorphisms, (RFLPs), random amplified polymorphic DNAs (RAPDs), and microsatellite DNA. An important application of linkage maps is that these serve as a starting point for the identification of quantitative trait loci. As more genes are identified, mapped and linked to specific traits, genetic and chromosomal assays will take on new importance. Early development of maps was difficult due to the paucity of marker loci and to genotype environmental interactions which can modify the expression of qualitative traits. In addition, map utility has often been constrained by the lack of linkages to economically important traits. Genome Mapping Genome maps are created from markers - DNA sequences in or near genes whose locations are known - and compared with the occurrence of favorable traits. If the markers and traits appear together more often than would occur by chance, the locations of the genes for the desirable trait are likely to be near the markers. Multiple genes usually govern a single trait of economic importance. The locations of these genes are called quantitative trait loci or QTLs. Once QTLs are identified, DNA tests are conducted on breeding lines to find out whether they have the desired QTLs. If so, marker - assisted selection (MAS) enables the researchers to put these traits into new breeding programs much sooner than if they used trial and error breeding to identify organisms with good genes. Comprehensive genome analyses should entail analyzing genome rearrangements in the context of cytogenetics, molecular genetics, population biology and breeding projects. Advances in the characterization of bivalve chromosomes can contribute to genome mapping. Early studies involved karyotyping and chromosome banding with some degree of success. More recent analyses include computer-assisted karyotyping (Zhang et al, 1999). Early attempts to map the genetic basis of heterosis in bivalves employing quantitative trait loci were met by problems from distortions of Mendelian segregation ratios, as reviewed by Launcey and Hedgecock (2001). Based on microsatellite loci, these investigators reported distorted segregation ratios in Pacific oyster families and estimated the genetic load. It was hypothesized that selection against recessive deleterious mutations at closely linked genes was responsible for non-Mendelian inheritance of markers, beginning in the juvenile stage. They concluded that such distortions resulted from homozygote disadvantage rather than heterozygote advantage. These could be caused by a high mutation rate or by association of a large number of fitness genes with a few markers in a small genome. The haploid number of chromosomes in all Crassostrea and Ostrea species studied thus far is 10. 3 Genomes can evolve by acquiring new sequences and by rearranging existing sequences. Another source of variation is transposable elements or transposons, which are sequences in the genome that are mobile. Recombination also is a key event in the evolution of the genome. Recombinant chromosomes contain different combinations of alleles, providing the raw material for selection. One important mechanism for the genome to change its content of genes rather than a combination of alleles is crossing-over, or when a recombination event occurs between two sites that are not homologous. Crossover events in meiosis can be observed in bivalves as demonstrated in the prometaphase I or diakinesis group and metaphase I bivalents in Figures 1a and 1b. Chromosome