长牡蛎壳色突变体橙色性状的成因机制研究

长牡蛎（Crassostrea gigas）是世界上养殖范围最广、产量最大的海洋贝类，在我国海产贝类养殖业中占有十分重要的地位。本项目以长牡蛎橙色壳色突变体为研究素材，利用液相色谱-质谱联用法确定壳色素类型和组成；通过与野生个体构建双列杂交家系，查明橙色壳色性状的遗传模式；利用高通量测序技术进行连锁和关联分析，初定位橙色壳色QTL候选区间，通过进一步筛选重组子以及加密候选区间分子标记，实现橙色壳色QTL精细定位；利用高通量转录组分析，筛查、克隆突变体橙色壳色相关候选基因及其非编码调控RNA，查明候选基因的时空表达特性；利用RNA干扰、反义吗啉环寡核苷酸链干扰和CRISPR/Cas9靶向基因编辑技术，阐明橙色壳色形成关键基因的功能及调控网络，从而揭示长牡蛎橙色壳色突变体形成的分子机制。研究结果对于长牡蛎壳色性状分子机制研究以及橙色壳色优良品种培育具有重要的理论和应用意义。

As a commercially important marine bivalves, the Pacific oyster (Crassostrea gigas) is a major global aquaculture species, ranking first in the production among all aquaculture shellfish species. In this study, the pigment type and composition in the orange-shell mutant of C. gigas will be identified and quantified using high-performance liquid chromatograph-mass spectrometer analysis. To determine the inheritance pattern of the orange coloration in C. gigas, diallel crosses will be performed between the oysters with orange and wild type shell colors. A combined Linkage and association mapping approach will be conducted using next generation sequencing to initially localize the candidate QTL of the orange-shell trait, which are further fine mapped by screening of recombination events and densifying makers within candidate interval. The candidate genes and non-coding RNA involved in orange-shell color will be screened and cloned by transcriptome sequencing analysis, and the temporal and spatial expression characteristics of the candidate genes will be examined. RNA interference, morpholino antisense oligonucleotide interference and CRISPR/Cas9-targeted gene editing technology will be performed to elucidate the function and regulatory network of the key genes which regulate the formation of orange shell color, revealing the genetic mechanism of the orange trait of the shell color mutation in C. gigas. This work has important theoretical and practical significance for the studies on the molecular mechanisms of shell color traits and the breeding program of an outstanding strain of C. gigas with orange-shell trait.