【作者】 齐建建；

【导师】 黄三文；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2014， 博士

【摘要】 黄瓜是世界上重要的蔬菜作物之一，也是我国保护地栽培面积第一大蔬菜作物，为我国农村经济发展和蔬菜周年供应做出了重大贡献，因此黄瓜的基础理论研究和育种实践也一直受到人们的重视。作为世界上第一个被成功测序的蔬菜作物，黄瓜基因组的完成为后续的分子育种、功能基因组学和比较基因组学研究奠定了基础。通过将传统的遗传定位和基因组学相结合，黄瓜中果实长度和果实苦味等重要的农艺性状基因陆续被克隆或定位。本实验室通过对世界范围内3342份黄瓜资源进行SSR指纹图谱分析，对黄瓜种质资源的遗传变异获得了初步了解，并成功构建了一份由115份资源构成的核心种质库。拟将115份黄瓜核心种质资源进行全基因组重测序，以获得该作物最全面的遗传变异形式。以此为基础，全面分析黄瓜的遗传多样性、进化历程和人工驯化等最基本的生物学命题，较全面的解析黄瓜基因组水平的变异形式，以期为黄瓜生物学研究、基因克隆以及遗传改良提供理论指导。主要结果如下：1.通过23对高多态性的SSR标记，对收集于全世界的3342份黄瓜种质资源进行了筛选，进而获得了115份核心种质资源，包括30份印度黄瓜、19份西双版纳黄瓜、29份欧美黄瓜和37份东亚黄瓜，上述核心库可代表黄瓜全部遗传变异的77.2%以上。2.对115份核心种质材料进行18.3×的深度测序，鉴定了约330万个SNP、33万个Indel和594个PAVs等结构变异位点，构建了完整的黄瓜变异组图谱。从四个群体中随机挑选400个SNPs，经PCR和Sanger测序验证，确定预测SNP的准确性为98.9%。3.依据SNP位点信息，进行了系统发生树的重构和群体结构分类，将核心种质资源分为四个亚群，即印度黄瓜、西双版纳黄瓜、欧美黄瓜和东亚黄瓜，其中印度黄瓜是栽培黄瓜的祖先类型。在四个亚群的核苷酸多样性（π）中，印度黄瓜为4.48×10-3，西双版纳黄瓜为1.06×10-3，欧亚黄瓜1.85×10-3和东亚黄瓜1.03×10-3，表明印度黄瓜的多样性明显高于其它栽培黄瓜。通过Haploview软件计算LD decay，发现印度黄瓜的LD decay明显快于栽培黄瓜，这可能与栽培黄瓜的长期驯化选择有关。通过dadi分析确定栽培黄瓜群体属于单起源，是2700多年前从印度黄瓜驯化而来。4.通过计算群体间固定指数（FST），发现各类栽培黄瓜间具有明显的差异性。在不同衍生群体分化中发现了500多个受选择的基因，为研究不同群体特有的性状和黄瓜适应不同区域环境的机理提供了重要数据支撑。利用群体分化，分析西双版纳黄瓜和其他黄瓜群体间高度分化的异义突变SNP，在β-胡萝卜素基因ore的定位区间找到一个关键的异义突变SNP。分别构建该SNP所在基因Csa3G183920突变型和野生型与pAC-BETA的原核共表达体系，证明Csa3G183920就是ore的候选基因，并将其命名为CsaBCH1。5.黄瓜πW/πC的比值为1.96，说明黄瓜在驯化过程中经历了较为狭窄的瓶颈效应。通过计算πW/πC比值和XP-CLR得分，预测了黄瓜基因组内112个受驯化选择的区域。以重要农艺性状果实长度和果实苦味为例，利用9110Gt和9930构建的F2和RIL群体对其进行了精细定位，将Bt定位于5号染色体前端442kb的区域内，其中包含67个预测基因，且Bt位于驯化区域内部；利用SNPBi6-1和SSR00004两个标记将Bi定位于6号染色体前端300kb的区域，但该区间内未见栽培黄瓜多样性出现降低。此外，通过野生黄瓜hardwickii和栽培黄瓜新泰密刺杂交产生的F2和IL群体，还粗定位了5个果实长度相关的QTLs，这些位点全部和预测的驯化选择区域重合。上述结果充分证明，112个驯化区域可作为栽培黄瓜重要性状基因的候选区。更多还原

【Abstract】 Cucumber is one of the most important vegetables in the globe, with China contributes to morethan half of the world’s annual yield. However, limited knowledge of important agronomic genes ishampering the development of new varieties. The success of genome sequencing of cucumber line9930had greatly enhanced the functional genomics and molecular breeding of cucumber. After fingerprinting3342cucumber lines collected worldwide, we built a core collection consisting of115lines.With deep re-sequencing of the core set, a high density cucumber genomic variation map wasconstructed, based on which we performed analysis on cucumber domestication, populationdifferentiation and linkage disequilibrium, data obtained in this study can provide insights into thebiology and genetic improvement of this important fruit crop. Results as follows:1. Employing23highly polymorphic SSR markers, a cucumber core collection was extracted from3342accessions of universal distribution. The core collection, which captures77.2%of the totalgenetic diversity, can be divided into4geographic groups: Indian(30accessions),Xishuangbanna(19), Eurasian(29) and Eastasian(37).2. With18.3×deep resequcing of the core collection, a map of cucumber genome variation whichencompasses3,305,010SNPs,336,081small insertions and deletions and594PAVs wassuccessfully constructed. The accuracy of the predicted SNPs was estimated to be98.9%.3. Model-based analyses of population structure and phylogenetic reconstruction using25,228four-fold degenerate-site SNPs support the notion that the core collection can be divided into fourgroups. The basal nature of the Indian group compared to the other three groups is supported byits significantly higher nucleotide diversity π, fast decay of linkage disequilibrium (LD) measuredby r2and high percentage of private SNPs and Indels. dadi proposed that the three cultivatedgroups are monophyletic and domesticated separately from their respective ancestral types. TheEastasian cucumbers were brought to China by diplomat Zhang Qian2,700years ago.4. As shown by FST, the three cultivated groups diverge substantially. We further identified~500geneswhich were differently selected during cucumber differentiation, a phenomenon possibly caused bydifferential selection to adapt to their respective local environments. There were43highlydifferentiated nonsynonymous SNPs fixed between the Xishuangbanna group and the other3groups (FST=1), but only one resided within the physical interval(within gene Csa3G183920) thatspans the ore locus. Co-expression of wild-type and mutant the specific gene with pAC-BETAshowed that Csa3G183920defines the ore locus, and designated CsaBCH1in this study.5. Cucumbers must have undergone severe bottleneck during domestication, as shown by itsπW/πC(1.96). Genomic regions with the largest reduction in diversity within cultivated groups andextreme divergence in allele frequency between wild and cultivated groups can be regarded as selective sweeps. A total of112such regions were identified, with7of which overlapped withpreviously mapped QTLs(4concerning fruit length). There is no diversity in the cultivated groupsin the region to which Bt was mapped, a strong signature of a selective sweep during domestication,implying that Bt is responsible for the domestication trait of non-bitter fruit. Selected sweeps can becandidate regions harboring important agronomic traits.更多还原