Gynogen induction and diploid restoration in the Pacific oyster Crassostrea gigas

A large proportion of stock improvement in agriculture and more recently aquaculture has come through breeding strategies that work to isolate and fix favourable characters and eliminate undesirable genetic qualities in the target species. The development of homozygous breeding lines, which allow fixing of desirable traits need to be progressed to enhance the effectiveness of breeding strategies. Such lines would also be a valuable research tool for examining genetic versus environmental influence on reproductive biology and stock performance. One potentially rapid and effective method that may produce 100% homozygous individuals is to create what are known as "double-haploids", by suppressing the 1st cell cleavage in haploid zygotes. This project investigated the efficacy of haploid production in the Pacific oyster, Crassostrea gigas, by examining UV irradiation of the paternal DNA, and restoration to the diploid state using the chemicals 6-dimethylaminopurine (6-DMAP) or Cytochalasin B (CB) to suppress either polar body I, polar body II or 1st Cleavage of activated eggs. Additionally, the use of a recessive colour phenotype was examined as a marker to identify parentage of putative gynogen offspring. Sperm Viability via a membrane integrity test ranged between 78-80% viable for controls (no UV exposure) and was significantly greater than all treatment exposures except 60 second low energy exposure (601±22μWcm⁻²s⁻¹), with 300 second high energy exposure (1063±15μWcm⁻²s⁻¹) causing the greatest reduction in sperm viability, ranging between 32-52%. There was no significant difference in egg activation between controls and treatments. In contrast, there was a significant reduction in development of embryos to D larvae with controls averaging approximately 40% development to D stage and the lowest energy treatment (60 seconds @ 601±22μWcm⁻²s⁻¹) averaging a little over 1%. UV radiation of sperm had a significant effect on embryo ploidy levels, with all treatments 180 seconds or longer resulting in less than 20% of cells being diploid or some level of aneuploidy between haploid and diploid. However other published work indicated that, sperm densities and treatment depths are likely to be as important as energy levels when developing irradiation protocols. Survival of putative diploid gynogens was extremely low in all experiments, with the best results yielding 3.9% recovery to D stage larvae and 0.04% recovery to settled spat assessed at 34 days post spawning. Suppression of PB II or 1st Cleavage with CB resulted in the greatest proportion of diploid cells (37.3% and 28.9% respectively) compared with CB suppression of PB I which resulted in the majority of cells produced being tetraploid (35.7%). Microsatellite (mSAT) analysis of these putative gynogen spat revealed a greater level of homozygosity in their genome relative to normal diploid controls; however the presence of null alleles and a lack of genetic variability at some mSAT loci reduced the power of these tools to fully distinguish parentage and validate the production of meiotic or mitotic gynogens. In a direct comparison of 6-DMAP and CB to restore the diploid state by suppression of 1st Cleavage, CB provided a greater percentage of diploid cells (28.4%) compared with 6-DMAP (23.7%) and significantly lower subsequent mortality of larvae. However using 6-DMAP to suppress PB I and II, survival and percentage of diploid cells produced was comparable to that obtained for similar trials using CB. The use of eggs obtained from recessive gold phenotype females crossed with inactivated sperm from homozygous wild type males was investigated to help elucidate the parentage of putative meiotic and mitotic gynogens. Survival of these putative gynogens was lower than for wild type female progeny however gold offspring were produced in both forms of gynogen induction and were not found in corresponding diploid controls. In summary, effective sperm deactivation for subsequent haploid gynogen production can be achieved by exposure to UV radiation for 5 minutes at energy levels ranging between 600 and 1000 μWcm⁻²s⁻¹. Restoring the diploid state of zygotes for meiotic gynogen production can be achieved through the addition of Cytochalasin B or 6-dimethylaminopurine targeting Polar body II suppression, however Cytochalasin B is recommended over 6-dimethylaminopurine for the production of putative mitotic gynogens through 1st cleavage suppression. Mitotic gynogens may be particular useful in the production of genetically identical lines, as they have the potential to be homozygous at every locus