三角帆蚌紫色珍珠质形成关键基因图位克隆和功能鉴定

Color is one of the most important pearl quality traits. Purple pearls, which produce in Hyriopsis cumingii, are best in freshwater pearl quality. Pearl color is related mainly to the shell nacre color of the donor mussel. The whole-genome sequencing of H.cumingii has completed and QTL on purple nacre has identified on high-density linkage map. In this study, QTL-seq based on whole gonome resequenceing will be employed for fine QTL mapping. SNP profiles among the purple and white-nacre bulks constructed from F2 mussels and two parents were identified. Then, linkage disequilibrium analysis will be employed for further minilizing QTL region. Genes located in fine QTLs and sequences of their regulatory regions are cloned on the base of genome sequences and gene annotations of H.cumingii. Next, gene and protein differential expressions in individuals with different traits are estimated for identifying candidate genes associated with purple nacre formation. The key gene will be determinated by deeply gene function analysis in controlling purple nacre formation. Finally, the variants of the key genes associated with purple nacre formation, which affects gene expression and nacre color, are identified. The study will benefit for understanding the genetic mechanism of purple nacre or pearl formation, and supply effective genes and molecular markers in molecular breeding of H.cumingii for improving purple nacre traits.

颜色是评价珍珠质量的重要指标之一，三角帆蚌所产紫色淡水珍珠质量最好，珍珠颜色与供片贝珍珠质颜色显著相关。本申请课题在完成三角帆蚌全基因测定和基于高密度遗传连锁图谱定位紫色珍珠质性状QTL基础上，采用基于全基因组重测序的混合分组分析法（QTL-seq），检测F2群体正态分布两端2个极异表型个体DNA混合样本和2个亲本DNA样本，实现QTL精细定位，然后，采用连锁不平衡分析法，进一步聚焦QTL区域；接下来，图位克隆聚焦QTL区域序列，根据基因组注释，获得功能基因及其调控区；分析上述基因在不同性状个体的RNA和蛋白质水平表达差异，筛选紫色珍珠质形成候选基因；进一步验证候选基因功能，深入分析其致色机制，获得紫色珍珠质形成关键基因，并鉴定调控关键基因表达和紫色珍珠质性状的因果变异位点。本课题成果可为解析紫色珍珠质或珍珠形成的遗传机制奠定理论基础，为紫色品系三角帆蚌分子育种提供有效的基因和分子标记。

颜色是影响珍珠质量和价值的重要指标之一，三角帆蚌所产紫色淡水珍珠稀少，价格高，解析紫色性状形成分子机制具有重要育种价值。本项目完成三角帆蚌基因组染色体水平组装，将总长2.04Gb scaffold（60.4%）挂载到19个连琐群上，采用GWAS分析筛选紫色性状连锁SNP标记，与前期获得的QTL联合分析，进行紫色性状基因图位克隆；根据基因功能注释和KEGG分类，获取紫色性状候选基因110个，比较候选基因在白色与紫色三角帆蚌样品外套膜、肝胰腺组织表达差异情况，鉴定白色、紫色三角帆蚌外泌体差异表达蛋白，综合鉴定紫色性状相关基因；克隆三角帆蚌HcStAR-like、溶血磷脂酰胆碱酰基转移酶1HcLPCAT1、环磷酸腺苷反应元件结合蛋白HcCreb、蛋白激酶C、尿卟啉原III合成酶HcUros、糖原合成激酶-3β（GSK3β）共6个珍珠颜色形成关键基因，通过组织差异表达、原位杂交、RNAi干扰等分析，阐明了上述基因在胡萝卜素、黑色素或卟啉代谢中具有重要调控作用，显著影响三角帆蚌珍珠颜色形成；发现三角帆蚌HcLPCAT1基因6个与珍珠颜色显著相关的SNP位点，H1和H2两种单倍型为紫色群体优势单倍型，HcPKC基因T1单倍型为白色群体优势单倍型，Hc-GSK3β基因C+185A位点的CA基因型在紫色蚌优势基因型。研究结果为解析三角帆蚌紫色性状形成分子机制奠定基础，为壳色育种提供了分子标记。