High Resolution Linkage Mapping And Genome Sequencing For Chlamys Farreri

The scallop Chlamys farreri (Jones et Preston1904) is a commercially essentialbivalve species in China. Here, we develop a high-throughput SNP discovery technology,construct a high resolution linkage map and identify QTLs of important traits forChlamys farreri. Besides, genome characterization of Chlamys farreri is also observed bywhole genome sequencing, and the comprehensive platform is established by integratinggenome sequences with linkage map and physical map. Building up such integrativegenomic resources and platforms will facilitate future genetic, genomic and breedingstudies on Chlamys farreri.1Establishment of high-throughput SNP genotyping technology for Chlamys farreriTo develop a high-throughput SNP discovery method for scallop, the optimizationsof2b-RAD technology include choice of proper restriction enzymes and selective bases,barcodes design and validation. A novel statistical framework for de novo codominantand dominant genotyping is established and integrated in the package called RADtyping.The superb performances of RADtyping in achieving remarkably high genotypingaccuracy are also demonstrated. Based on simulation data, repetitive sites can beefficiently filtered out since the high-quality reference sites reconstructed for genotypingalmost exclusively derived from unique genomic regions. For codominant genotyping,genotyping accuracy can reach97%at the sequencing depth of20x, while for dominantgenotyping,98%can be achieved at a much lower sequencing depth. For large-scalelinkage mapping studies, a minimal sequencing depth of20x for both parents andprogeny should meet the desired level of genotyping accuracy. Measuring genotypingconsistency between replicate datasets reveals96%for codominant markers and99%fordominant markers. Genotyping accuracy evaluation by amplicon (Sanger) sequencing ofeight codominant and eight dominant markers from RADtyping suggests96%and97%for the codominant and dominant markers, respectively. 2A high density linkage map construction and QTL mapping for Chlamys farreriA high-resolution linkage map is constructed for Chlamys farreri with a marker densitythat has, to our knowledge, never been achieved in any other molluscs.The consensusmap contains3,806markers, and spans1543.4cM with an average marker interval of0.41cM. Genome coverage of the consensus map is nearly complete and reached99.5%.The average recombination rate across all the linkage groups is approximately1.3cM/Mb, and623markers reside in or close to genes. Two growth-related QTLs aredetected on LG1and LG3which can explain11.4%and16.9%of the total phenotypicvariation, respectively. Association analysis reveals a similar distribution pattern acrossall linkage groups as in QTL mapping analysis and supports QTL mapping results ingeneral. One marker named f68558is found to be located in the intron region of atranscription factor gene, PROP1(Homeobox protein prophet of Pit-1). It has been shownthat PROP1can regulate the production of growth hormone, a peptide that simulatesgrowth, cell reproduction and regeneration in animals. While for sex, a highly significantQTL is finely mapped on LG11with the confidence interval of0.37cM. Associationanalysis reveals27sex-related markers with high statistical significance (P <1E-6) andall of them fall into the narrow QTL region identified by the QTL mapping analysis. Onesex-related marker named f93422is located in the coding region of a transcription factorgene, ZNFX1(NFX1-type zinc finger-containing1), which has been shown to be tightlylinked with AMHR2(anti-Mullerian hormone receptor type II), the sex-determinationgene in the tiger pufferfish, Takifugu rubripes. Those candidate genes identified fromthese QTL regions represent important targets for further evaluation.3Genome sequencing of Chlamys farreri and integrating with the linkage mapGenome sequencing is performed for a single Chlamys farreri individual bysequencing pair-end and mate-pair DNA libraries with different insert sizes based on theIllumina HiSeq2000platform. According to a highly heterozygous of bivalves, thestrategy for genome sequence assembly adopts first separating two sets of heterozygtecontigs, then assembling respectively and finally integrating. In total,427Gb ofsequencing data, equivalent to448x genome coverage, are produced. K-mer analysisreveals remarkably high heterozygosity (1~1.4%) in the Chlamys farreri genome. Assembly of these data produces143,162scaffolds (N50=528.6Kb) with total length of805Mb and192,003contigs (N50=21.5Kb) with total length of777Mb.93%oftranscriptome sequences can match to genomic scaffolds, suggesting that the majority ofgenes are present in our reference genome dataset.62.7%genome scaffolds has beenfunctionally annotated through the de novo preidiction method and homology-basedcomparison against public databases. Aiding by the high density linkage map,2,923scaffolds are ordered and oriented. Physical map is also integrated with genomesequences and linkage map, thus establishing a comprehensive genomic resource andplatform for Chlamys farreri.