生菜叶色不仅是重要感官品质，产生紫色的花青素还有重要的保健功能。生菜叶色受多位点调控。本项目在前期克隆的4个紫色调控基因基础上，新发现了3个生菜紫色调控位点（PLL5-7）。利用前期成功应用的BSA+RNA-seq遗传研究方法，已经确定紫色调控基因PLL5为UV-B信号传导途径中的负调控基因；PLL6基因编码MYB类转录因子；PLL7定位于1号染色体。本项目将研究PLL5在UV-B诱导的花青素合成途径中的作用；PLL6启动子对基因表达的影响；精细定位并克隆紫色调控基因PLL7，分析其作用机理。本课题还将分析生菜中所有7个叶色控制基因互作关系；通过基因差异表达分析及花青素相关基因的表达网络，挑选10个左右基因进行CRISPR/Cas9敲除，从而深入解析生菜叶色的调控网络。通过杂交创建7个位点不同组合、不同叶色、不同花青素的生菜类型。

Lettuce (Lactuca sativa) is one of the most important vegetables worldwide, and its leaf color is an important agronomic trait. Purple lettuces contain abundant anthocyanins, which provide health benefit to consumers. The purple color in lettuce is controlled by multiple loci, including both qualitative and quantitative loci. In this proposed study, we identified three new loci controlling leaf color in lettuce. BSA in combination with RNA-seq is used to clone corresponding genes. One of them, PLL5, was fine mapped and a candidate gene encodes a WD40-repeat protein that is phylogenetically and structurally related to the RUP1/RUP2 protein in Arabidopsis, which interacts directly with UVR8 as potent repressors of UV-B signaling. We are going to analyze the molecular mechanism of PLL5 involved in UV-B-induced anthocyanin biosynthesis. We have also genetically mapped and cloned the second gene, PLL6, which encoded a MYB transcription factor. We will genetically map and clone the third gene, PLL7, a positive regulator in anthocyanin biosynthesis. This study will provide insights on the genetics controlling leaf color and the mechanism for anthocyanin biosynthesis in lettuce. Above three genes will be combined with the four genes cloned previously to investigate the genetic network underlying the anthocyanin accumulation in lettuce, using the gene expression network as a reference. We will generate lettuce genotypes with diverse intensity of leaf color and diverse types of anthocyanins though various combination of the 7 genes controlling anthocyanin accumulation.