Genetic contribution to pearl formation

The silver, Pinctada maxima, and black-lip, Pinctada margaritifera, pearl oysters are two commercially important species which produce high-value "South Sea" pearls. Although pearl culture techniques were developed in the early 1950's and have been continually refined, a large proportion of the pearl harvest (~60%) from these two species still fails to be categorised as "gem" quality. The pearling industry stands to benefit substantially from improvements in pearl quality, as it is the proportion of "gem" quality pearls that largely contributes to the profitability of the industry. Despite research into innovative husbandry, nuclei implantation, and optimum grow-out environments, the industry has not seen dramatic increases in the proportion of high quality pearls from harvests. There is, however, the potential for genetic approaches to increase the proportion of "gem" quality pearls produced through selective breeding. Before targeted breeding programs can be developed though, there needs to be a strong understanding of the genetic basis of traits and this is currently lacking for pearls. To date, little research has focussed on the genetic processes behind pearl formation, a complex process potentially involving the genetic contribution from two individual oysters (host and donor oyster). Given the complexity of pearl production and the potential genetic contribution from two oyster genomes, without a clear understanding of the role of each oyster in the pearl biomineralisation process targeted selection cannot be effective. This thesis defines the respective roles of the host and donor oysters in pearl formation, by first examining their phenotypic contribution to pearl quality traits followed by a fine scale examination of their molecular contribution to the pearl biomineralisation process.\ud \ud Prior to genetic improvement of pearl quality through selective breeding, the respective roles the donor and host oysters play in the determination of pearl quality traits must first be defined. Current pearl culture techniques do not permit differentiation between the host and donor oyster pearl phenotypes due to con-specifics being used as the host and donor oyster (allografts). One possible way to provide information on the contribution from the host and donor to pearl traits is by adopting a novel approach of using mantle grafts originating from one pearl oyster species implanted into a second recipient species that is closely related and characteristically has a different pearl phenotype (termed a xenograft). For the first time, this thesis definitively demonstrates the contribution from the donor and host oysters to pearl phenotypic traits through xenotransplantation of two closely related yet distinctly different pearl producing species, P. maxima and P. margaritifera. The results conclusively revealed that the donor oyster is the main contributor to pearl quality. In particular, pearl colour and size were strongly influenced by the donor oyster species used as xenografts. P. maxima donors produced larger, silver colour based pearls, whilst, P. margaritifera xenografts produced smaller, black colour based pearls. Through the novel approach of producing xenografted pearl oysters, this study demonstrates the potential of xenografts as a means to improve pearl quality traits such as pearl size, and highlights the role that donor oysters have in the realisation of pearl growth, colour and surface complexion.\ud \ud In light of phenotypically detecting the donor oyster as the main contributor to pearl quality traits, the next logical question is what is happening at the molecular level in regard to the expression of biomineralisation related proteins that govern pearl formation. Whilst, studies have shown that genomic DNA from a mantle allograft remains present in the pearl sac at the time of pearl harvest, what remains unclear is whether biomineralisation genes from the donor mantle allograft are transcriptionally active and contribute to pearl formation. One of the biggest impediments in determining whether the donor or host cells are transcriptionally active for biomineralisation genes in the pearl sac is discriminating between the gene products of the two potentially contributing oysters. Currently there is insufficient information on levels of intra-specific polymorphisms in putative biomineralisation genes to characterise gene products that may be derived from the host/donor oysters. This thesis took a powerful and novel approach in determining if the donor oyster cells remain transcriptionally active in the pearl sac, by xenografting two species of pearl oyster, P. maxima and P. margaritifera, which contain species-diagnostic gene differences. Diagnostic PCR tests revealed that donor oyster cells not only remained present in the pearl sac at the time of pearl harvest, but were found for the first time to be transcriptionally active in the expression of two biomineralisation genes, N44 and N66. These results support that the donor oyster is an important contributor to the biomineralisation process in pearl culture.\ud \ud To further elaborate on what is happening at the molecular level in regard to the expression of biomineralisation related proteins that govern pearl formation, the pearl sac transcriptome of P. maxima and P. margaritifera was examined through high through-put RNA sequencing (Illumina GAII). Allografted and xenografted pearl sacs from two pearl oyster species with unique genomes, P. maxima and P. margaritifera, were produced. Putative molluscan biomineralisation-related genes identified within the sequenced allografted pearl sacs of both P. maxima and P. margaritifera revealed 19 biomineralisation genes similarly expressed in both species. This is the largest proportion of genes linked to the process of biomineralisation within the pearl sac to date. Based on the presence/absence of species diagnostic gene transcripts within xenografted pearl sacs, all genes examined were found to be expressed by the species used as the donor oyster. In one individual it also found that the host was expressing Linkine. These results convincingly show for the first time that not only is the donor mantle tissue transcriptionally active, it is primarily responsible for the expression of biomineralisation genes in the pearl sac.\ud \ud Outcomes of this thesis have provided a substantial advancement in the understanding of cultured pearl formation. By understanding the importance of the donor oyster to pearl formation and quality, this research provides grounds for a donor specific selective breeding program based on pearl growth, colour and surface complexion. Now that the major genes potentially involved in pearl biomineralisation have been identified in this thesis and the donor established as the main contributor to the expression of these genes, the next step is to identify the specific function of these genes that lead to different pearl quality traits. This will ensure pearl quality traits are not only selected upon based purely on phenotype, but an understanding of the molecular mechanisms underlying pearl traits to achieve maximum genetic gains. This thesis not only provides a solid foundation for elucidating the biological process of pearl formation in general, but it also provides valuable information that can be directly utilised for selective breeding programs in the cultured pearl industry to improve pearl quality