Genomic resources and genetic dissection of complex quantitative traits in the silver-lipped pearl oyster, Pinctada maxima
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Pearl oysters are not only farmed for their gemstone quality pearls worldwide, but are also becoming important model organisms for investigating genetic mechanisms of biomineralisation and bivalve evolution. However, despite their economic and scientific significance, limited genomic resources are available for this important group of bivalves, hampering investigations aiming to identify genes that regulate important pearl quality traits and unique biological characteristics (i.e. biomineralisation). The silver-lipped pearl oyster, Pinctada maxima, is one species where there is interest in understanding genes that regulate commercially important pearl traits, but presently there is a dearth of genomic information. Recently, the market for the highly valuable South Sea pearl produced by P. maxima has expanded, creating strong interest in stock improvement to increase the production of high quality South Sea pearls. Molecular stock improvement techniques such as marker assisted selection (MAS) have great potential in accelerating selective breeding programs for animal production industries as they bypass limitations that have so far restricted the success of phenotypic breeding programs. Genetic breeding programs not only require accurate phenotypes, but they also need established high density genomic resources including genome-wide molecular markers and dense genomic maps, of which none are available for any pearl oyster species. These resources allow the dissection of complex phenotypic traits of commercial importance and the identification of genetic variation and marker associations that disproportionately influence variation in these traits, both of which are integral before genetic breeding programs can be established. Once marker associations to traits are identified, stock populations can be screened for negative or beneficial genetic variants, which can either be avoided or selected. The overarching objective of this thesis is to utilise recent advances in genome sequencing and genotyping technologies to develop genome-wide genomic resources necessary for incorporating genetic selection breeding programs into the Pinctada maxima pearling industry. Specifically, I describe the large scale sequencing and annotation of the P. maxima transcriptome, the development of thousands of genomewide microsatellite and single nucleotide polymorphism (SNP) loci, the construction of the first moderate-density genetic linkage map for a pearl oyster species, and the identification of preliminary quantitative trait loci (QTL) and marker associations to two commercially important groups of pearl oyster traits; oyster shell growth and pearl quality. Firstly, the large-scale sequencing (~1.3 million sequences) of the P. maxima mantle transcriptome allowed the subsequent development and validation of two large suites of type I genome-wide molecular markers; SNPs, and microsatellites. This thesis describes molecular marker development, along with the assembly (96,794 contigs) and sequence annotation of the mantle transcriptome database. The SNP discovery effort resulted in the de novo identification of 172,625 SNPs, of which 9,108 were identified as high-value (MAF ≥ 0.15, read depth ≥ 8). Validation of 2,782 of these SNPs using Illumina iSelect Infinium genotyping technology returned some of the highest assay conversion (86.6%) and validation (59.9%; mean MAF 0.28) rates observed in aquaculture species to date. In addition, results show that subsets of the SNP loci will have widespread use as parentage and genetic diversity markers across species within Pinctada, which may potentially compliment the use of current suites of microsatellite genetic tools. Likewise, a total of 2,322 unique microsatellite loci were identified throughout the transcriptome database, with 360 shown to be polymorphic in silico using allelic motif variation. The mantle transcriptome database and molecular marker suites presented here form a strong foundation for genome mapping studies and have been pivotal to research investigating the biological mechanisms behind biomineralisation, quantitative trait loci mapping and association analysis as demonstrated within this thesis. Secondly, this thesis describes the construction of a dense genetic linkage map for P. maxima, including investigations into segregation distortions, family-specific heterogeneity and sex-specific recombination rates. The construction of a moderate- to high-density genetic linkage map for P. maxima is not only essential for mapping QTL and unraveling the genomic architecture of complex pearl quality traits, but also provides indispensable information on the genome structure of pearl oysters. A total of 335 oysters from eight full-sib families (six phase known and two phase unknown) were genotyped over 2,782 genome-wide SNPs. Of the 2,782 SNPs, 1,189 were informative and incorporated into linkage map construction. The final linkage map consisted of 887 SNPs in 14 linkage groups and spans a total genetic distance of 831.7 centimorgans (cM). The average marker interval (excluding intervals of 0 cM) was 2 cM and the map covers an estimated 96% of the P. maxima genome. Assessments of sex-specific recombination revealed mild heterochiasmy, but pronounced localised differences between male and female recombination throughout the linkage groups, whereby male recombination was suppressed near the centromeres compared to female recombination, but inflated towards telomeric regions. Numerous candidate genes for nacre biomineralisation were also localised providing some of the first positional information for these genes. Finally, the genetic architecture of complex oyster growth (i.e. shell height, length, width and weight) and pearl quality traits (pearl size, weight, surface complexion and colour) were explored using genome-wide association tests and QTL approaches. This thesis documents, for the first time, QTL and genetic associations to these commercially important pearl oyster growth and pearl quality traits and provides important insights into the genetic control of these traits. A total of 16 QTL and 32 genetic associations were detected for all oyster shell growth and pearl quality traits that explained from 27.4% to 46.1% of the phenotypic variation of a trait per family. The majority of QTL and marker associations were detected for the traits oyster shell width and pearl colour. This study confirms previous quantitative genetic studies providing conclusive evidence that oyster growth and pearl quality traits have a low to moderate additive genetic component and are complex and polygenic in nature. As a body of work, this thesis presents the most comprehensive genomic resources produced for any pearl oyster to date and presents the first quantitative genetic dissection of performance traits within P. maxima. Such work is pivotal to enabling the incorporation of genetic breeding programs within the P. maxima pearling industry and for investigating broader biological questions including the dynamic process of biological-mediated biomineralisation and bivalve evolution.
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