华贵栉孔扇贝“南澳金贝”类胡萝卜素含量的QTL精细定位及分子解析

Recently years, exploring genetic rules of important economic traits and clarifying their gene functions in cultured shellfish have become a hot topic in the field of shellfish genetic breeding and genomics study. Carotenoids are important antioxidants and play important roles in animal and human health, which are also importantly economic traits. Many shellfish are rich in carotenoids, but the information on molecular mechanism of their accumulating carotenoids from their diets has been scarcely. “Nan’ao Golden Scallop” and brown strain is separately a new variety and a new strain both bred from the same cultured stock of noble scallop Chlamys nobilis using selection breeding by our project group in Shantou University. The “Nan’ao Golden Scallop” is not only of golden color in shell, adductor, mantle, intestinal tract and blood but also contains significantly higher in total carotenoids content (TCC) in its tissues than the brown stain with brown shell, white adductor and white mantle. In the present project, the mapping populations for analyzing QTL on TCC will be firstly produced by crossing between the “Nan’ao Golden Scallop” and the brown strain. Meanwhile, verifying populations for identifying the key genes will be separately produced using the “Nan’ao Golden Scallop” and the brown strain by successive inbreeding of ful-sibling within the “Nan’ao Golden Scallop” and the brown strain. Then, a high-density genetic mapping on TCC will be constructed using 2b-RAD (Type IIB endonucleases restriction site-associated DNA) method. Next, based on this mapping, pQTL (phenotyping Quantitative Trait Loci) of TCC will be analyzed, and some candidate genes and regulation loci (genes) will be discovered. Then, eQTL (expression Quantitative Trait Loci) will be analyzed using expression profile data, some eQTL hotspots will be confirmed, and some candidate genes and regulation loci (genes) related to TCC will be identified. Using RNAi (RNA interference) or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) /Cas9 and gene overexpression, some key genes and regulation loci (genes) related to carotenoids accumulation will be further identified and their functions will be revealed. At last, a regulatory network will be constructed and the molecular mechanisms underlying the effective accumulation of carotenoids in the “Nan’ao Golden Scallop” will be clarified. The results will not only deeply reveal mechanisms of the “Nan’ao Golden Scallop” accumulating in carotenoids and enrich theory of shellfish genetics and breeding, but also provide a guide for the genetic improvement of the “Nan’ao Golden Scallop” in the future. Therefore, this project has significant values both in theory and practice.

对养殖贝类重要经济性状遗传规律的探索和功能基因的解析是近年来贝类遗传育种和基因组学领域关注的热点。类胡萝卜素是重要的经济性状，许多贝类富含类胡萝卜素，但其如何积累的分子机制却鲜为人知。本项目拟以总类胡萝卜素含量(TCC)差异显著的华贵栉孔扇贝“南澳金贝”和褐色品系为亲本，采用杂交和近交分别产生作图群体和验证群体；构建闭壳肌TCC的高密度图谱，进行pQTL分析，挖掘出相关基因和调控位点(基因)，结合表达谱数据进行eQTL分析，确定eQTL热点，挖掘候选关键基因及调控位点(基因)；然后用RNAi或CRISPR/Cas9和基因过表达等技术验证关键基因及调控位点(基因)的功能，构建“南澳金贝”积累类胡萝卜素的调控网络，最终阐明其高效积累类胡萝卜素的分子机制。研究成果可深入解析“南澳金贝”积累类胡萝卜素的机制，丰富贝类遗传育种学理论，为“南澳金贝”进一步遗传改良提供指导，具有重要理论意义和应用价值。

类胡萝卜素是重要的经济性状，许多贝类富含类胡萝卜素，但其如何积累的分子机制却鲜为人知。本项目以总类胡萝卜素含量（TCC）差异显著的华贵栉孔扇贝“南澳金贝”和褐色品系为亲本，首先构建了F2作图群体和验证群体和高密度遗传图谱，同时构建了全基因组图谱。然后通过对壳色、生长、性别和类胡萝卜素含量等性状的QTL定位分析，定位到两个与壳色相关的QTL，其中位于LG09上的位点对表型贡献率为94.51%；在LG09和LG14上定位到2个与闭壳肌TCC（总类胡萝卜素含量）相关的QTL，表型贡献率分别为15.77%和28.83%，壳色与闭壳肌TCC存在强连锁关系；定位到5个与类胡萝卜素含量呈显著正相关的基因：SRB-like-3、己糖激酶（HK）、锌指蛋白转录因子（ZFP）、E3泛素连接酶（E3 ubiquitin-protein ligase）、细胞核复合蛋白2同源物（NOC2L）等基因，1个与类胡萝卜素降解相关的基因：类胡萝卜素双加氧酶（CnCCD）基因。最后通过比较基因组分析发现SRB-like-3发生了倒位现象，导致SRB-like-3这个基因座在“南澳金贝”基因组中发生显著的结构变异，即在“南澳金贝”中内含子显著扩张，基因长度显著增大，且启动子也发生了显著变异，这表明SRB-like-3为决定“南澳金贝”高效富集类胡萝卜素的关键基因。依托本项目发表学术论文15篇（其中SCI收录论文14篇，含中科院1区8篇、2区5篇、3区1篇）、荣获科研奖项4项、培养毕业博士生4人、硕士生11人，成果丰硕。本项目初步阐明了“南澳金贝”高效积累类胡萝卜素的分子机制，具有重要的科学意义；同时培育的华贵栉孔扇贝“汕大黄金贝”新品系有望育成国家级水产新品种，应用前景广阔。