Andy R. Beaumont

Combining two-stage testing and interval mapping strategies to detect QTL for resistance to bonamiosis in the european flat oyster Ostrea edulis.

We have identified quantitative trait loci (QTL) in the flat oyster (Ostrea edulis) for resistance to Bonamia ostreae, a parasite responsible for the dramatic reduction in the aquaculture of this species. An F2 family from a cross between a wild oyster and an individual from a family selected for resistance to bonamiosis was cultured with wild oysters injected with the parasite, leading to 20% cumulative mortality. Selective genotyping of 92 out of a total of 550 F2 progeny (i.e., 46 heavily infected oysters that died and 46 parasite-free oysters that survived) was performed using 20 microsatellites and 34 amplification fragment length polymorphism primer pairs. Both a two-stage testing strategy and QTL interval mapping methods were used. The two-stage detection strategy had a high power with a low rate of false positives and identified nine and six probable markers linked to genes of resistance and susceptibility, respectively. Parent-specific genetic linkage maps were built for the family, spanning ten linkage groups (n = 10) with an observed genome coverage of 69–84%. Three QTL were identified by interval mapping in the first parental map and two in the second. Good concordance was observed between the results obtained after the two-stage testing strategy and QTL mapping.

Strategies for the retention of high genetic variability in European flat oyster (Ostrea edulis) restoration programmes

The native European flat oyster Ostrea edulis is listed in the OSPAR Convention for the Protection of the Marine Environment of the North-East Atlantic (species and habitat protection) and in the UK Biodiversity Action Plan. Once extremely abundant in the nineteenth century, European stocks of O. edulis have declined during the twentieth century to rare, small, localised populations due to overexploitation, habitat degradation and, most recently, the parasitic disease bonamiosis. Selective breeding programmes for resistance to bonamiosis have been initiated in France and Ireland. High genetic diversity and bonamiosis-resistance would be important features of any sustainable restoration programmes for O. edulis. Oysters were sampled across Europe from four hatchery sources, four pond-cultured sources and four wild, but managed fisheries and were genotyped at five microsatellite loci. Hatchery-produced populations from small numbers of broodstock showed a significant loss of genetic diversity relative to wild populations and pedigree reconstruction revealed that they were each composed of a single large full-sib family and several small full-sib families. This extremely low effective population size highlights the variance in reproductive success among the potential breeders. Pond-cultured oysters were intermediate in genetic diversity and effective population size between hatchery and wild populations. Controlled hatchery production allows the development of bonamiosis-resistant strains, but at the expense of genetic diversity. Large scale pond culture on the other hand can provide a good level of genetic diversity. A mixture of these two approaches is required to ensure a healthy and sustainable restoration programme for O. edulis in Europe.

Mitochondrial DNA variation in the scallop Pecten maximus (L.) assessed by a PCR-RFLP method.

Two PCR-RFLP mitochondrial DNA (mtDNA) markers were developed through the cloning and sequencing of mtDNA from the scallop Pecten maximus, and were used to study genetic differentiation of UK and Atlantic coast populations of this species. Although no distinct pattern of mtDNA haplotype frequencies was apparent and no diagnostic haplotypes were identified for any population, sequence divergence data provided convincing evidence that a P. maximus sample taken from Mulroy Bay, Eire, a semi-enclosed sea lough, was genetically differentiated from all other samples. However, this could not be unequivocally attributed to a restriction in gene flow, as the sample consisted of an ongrown single spatfall, which may not have been representative of the wild population. Despite the inability to separate populations on the basis of haplotype frequency, it was noteworthy that the frequency pattern of the commonest haplotype varied between sampling sites in a manner similar to that of allozyme allele frequencies in Aequipecten opercularis, a scallop species with a similar distribution and life history, for which there is evidence of population subdivision. Pecten maximus from St Brieuc Bay, reasoned to be a self-recruiting population from reproductive and physiological evidence, could not be separated from other populations using mtDNA markers. Further investigation of this population with alternative markers is warranted.

Production and growth of triploid Mytilus edulis larvae

Abstract Triploidy induction trials were carried out on the eggs of Mytilus edulis using heat shock treatment at three temperatures (20, 25 and 30 °C) and cytochalasin B (CB) treatment at three concentrations (0.1, 0.5 and 1.0 mg CB · l −1 ). Heat shock was administered at meiosis I while CB treatments were applied at both meiosis I and II. Estimates of potential triploidy were obtained by counts of diploid eggs in heat shock experiments and also by chromosome counts in early embryos in CB experiments. Diploid eggs (potential triploid zygotes) were produced at all heat shock temperatures with best results (25%) at 25 °C but all CB treatments regardless of concentration or timing of application produced higher percentages of triploids (26–67%). Both heat shock and CB treatments significantly reduced yield and normality of 72 h larvae. Veliger larvae derived from eggs treated with CB at meiosis I had grown significantly larger after 36 days, than controls or larvae derived from any other treatments. We propose that this is a result of the higher overall genomic heterozygosity expected in meiosis I triploids compared to meiosis II triploids or diploid controls.

Multilocus heterozygosity and size: a test of hypotheses using triploid Mytilus edulis

Cohorts of meiosis I and meiosis II induced triploid Mytilus edulis were produced from mass matings in the laboratory and reared alongside normal diploid cohorts during 1990 and 1991. Diploid cohorts generally exhibited a significant positive correlation between multilocus heterozygosity and size. This correlation was absent or much more weakly expressed in cohorts of triploid mussels and this supports the ‘associative overdominance’ hypothesis rather than the ‘direct involvement’ hypothesis as the major explanation for the correlation. For both diploids and triploids, no correlation between heterozygosity and the coefficient of variation of size was evident and heterozygotes at single loci were not larger than homozygotes. Triallelic triploid heterozygotes were no larger than diallelic triploids. The diploid cohorts were generally in agreement with the Hardy-Weinberg model and there was no trend towards heterozygote deficiency.

Disruption of doubly uniparental inheritance of mitochondrial DNA in hybrid mussels ( Mytilus edulis × M. galloprovincialis )

Blue mussels of the genus Mytilus have an unusual mode of mitochondrial DNA inheritance termed doubly uniparental inheritance (DUI). Females are homoplasmic for the F mitotype which is inherited maternally, whereas males are heteroplasmic for this and the paternally inherited M mitotype. In areas where species distributions overlap a varying degree of hybridization occurs; yet genetic differences between allopatric populations are maintained. Observations from natural populations and previous laboratory experiments suggest that DUI may be disrupted by hybridization, giving rise to heteroplasmic females and homoplasmic males. We carried out controlled laboratory crosses between Mytilus edulis and M. galloprovincialis to produce pure species and hybrid larvae of known parentage. DNA markers were used to follow the fate of the F and M mitotypes through larval development. Disruption of the mechanism which determines whether the M mitotype is retained or eliminated occurred in an estimated 38% of M. edulis × M. galloprovincialis hybrid larvae, a level double that previously observed in adult mussels from a natural M. edulis × M. galloprovincialis hybrid population. Furthermore, reciprocal hybrid crosses exhibited contrasting types of DUI disruption. The results indicate that disruption of DUI in hybrid mussels may be associated with increased mortality and hence could be a factor in the maintenance of genetic integrity for each species.

Are Pecten maximus and Pecten jacobaeus different species

Two members of the scallop genus Pecten (Bivalvia: Pectinidae) occur in European waters. Pecten maximus is largely an Atlantic species whilst Pecten jacobaeus is almost completely confined to Mediterranean waters despite slight overlap of distributions in the western Mediterranean. Genetic distances between these species were estimated using both allozyme electrophoresis and mitochondrial DNA PCR–RFLP and shown to be of a similar magnitude to intraspecific values, considerably lower than expected for congeneric species. These data are discussed in the light of recognised morphological differences, hybrid rearing trials and previous studies examining genetic distance by allozyme methodologies.

Characterization of ten microsatellite loci in the blue mussel Mytilus edulis.

ABSTRACT Mussels of the genus Mytilus are ecologically and commercially important worldwide, and they form hybrid complexes where their distributions overlap. Allozyme and nuclear markers have been used to investigate their genetics over many years, but successful development of reliable highly valuable microsatellite markers has lagged behind other shellfish species. We have developed and characterized ten novel microsatellite loci that amplify reliably for the blue mussel Mytilus edulis. The number of alleles among 30 individuals from a wild population (Menai Strait, North Wales, UK) ranged between 9 and 29 and the observed heterozygosity between 0.300 and 0.954. Significant heterozygote deficiencies against the Hardy-Weinberg model were observed at 6 out of 10 loci. Analyses using MICRO-CHECKER suggested the presence of null alleles at 8 out of 10 microsatellites with estimated null allele frequencies ranging from 0.105–0.305. The 10 newly developed microsatellites will have value to discriminate between Mytilus species, to support studies of introgression and hybridization and to strengthen and improve the available genetic linkage map.

Larval growth, juvenile size and heterozygosity in laboratory reared mussels, Mytilus edulis.

Studies with marine bivalve juveniles have shown a positive correlation between growth and allozyme multi-locus heterozygosity (MLH), and, in some cases, between larval growth and juvenile growth, but there has been little research on the relationship between allozyme heterozygosity and larval growth. Larvae of M. edulis from different mating systems (half-sib families with a single female, or a single male parent, a reciprocal cross of two malesxtwo females and two mass matings of 13x13 and 8x17 females and males, respectively) were reared in the laboratory and selected into fast and slow growing groups when about 10-30% were undergoing metamorphosis. Offspring were reared to the juvenile stage (>3.00 mm) and both groups of each mating were electrophoresed and genotyped at up to 12 allozyme loci. There was generally good agreement with Mendelian inheritance (half-sibs and reciprocal cross) or the Hardy-Weinberg model (mass matings). Null alleles were detected at the Odh and Lap loci but there was no evidence that null allele heterozygotes grew slower than other genotypes. Over all cohorts, juveniles from the fast growing larval group were not significantly larger, or smaller, than juveniles from the slow growing group which suggests that larval growth rate may be independent of juvenile growth rate. This observation agrees with some, but not all, earlier studies and has commercial relevance. Tests of heterozygosity and juvenile shell length indicated no association between average heterozygosity across all allozyme loci and the size of juveniles in any cohort regardless of the mating system used or their larval growth rate. The association between MLH and juvenile growth in bivalves is seldom detected in cohorts from a limited genetic background. The lack of an association between heterozygosity and size might therefore be expected in the half-sib and reciprocal cross cohorts, but not in the mass matings. The results argue against any significant association between heterozygosity and larval size in mussels.

Differences in morphology, survival and size between self- and cross-fertilized larvae of Mytilus galloprovincialis

The serendipitous detection of a rare hermaphrodite individual of Mytilus galloprovincialis during the process of spawning in the laboratory enabled self- and cross-fertilization to be achieved. Self-fertilized larvae exhibited significantly higher percentages of morphological abnormality at 3 d, lower survival at 9 d and decreased size at 31 d of age compared with cross-fertilized larvae. These results are discussed in the light of equivocal evidence for inbreeding depression in larvae derived from self-fertilization, full-sib matings and gynogenesis in other bivalves.