Table_2_Association Study of the 5′UTR Intron of the FAD2-2 Gene With Oleic and Linoleic Acid Content in Olea europaea L..docx

Cultivated olive (Olea europaea L. subsp. europaea var. europaea) is the most ancient and spread tree crop in the Mediterranean basin. An important quality trait for the extra virgin olive oil is the fatty acid composition. In particular, a high content of oleic acid and low of linoleic, linolenic, and palmitic acid is considered very relevant in the health properties of the olive oil. The oleate desaturase enzyme encoding-gene (FAD2-2) is the main responsible for the linoleic acid content in the olive fruit mesocarp and, therefore, in the olive oil revealing to be the most important candidate gene for the linoleic acid biosynthesis. In this study, an in silico and structural analysis of the 5′UTR intron of the FAD2-2 gene was conducted with the aim to explore the natural sequence variability and its role in the gene expression regulation. In order to identify functional allele variants, the 5′UTR intron was isolated and partially sequenced in 97 olive cultivars. The sequence analysis allowed to find a 117-bp insertion including two long duplications never found before in FAD2-2 genes in olive and the existence of many intron-mediated enhancement (IME) elements. The sequence polymorphism analysis led to detect 39 SNPs. The candidate gene association study conducted for oleic and linoleic acids content revealed seven SNPs and one indel significantly associated able to explain a phenotypic variation ranging from 7% to 16% among the years. Our study highlighted new structural variants within the FAD2-2 gene in olive, putatively involved in the regulation mechanisms of gene expression associated with the variation of the content of oleic and linoleic acid.

栽培油橄榄（Olea europaea L. subsp. europaea var. europaea）是地中海盆地最古老且分布最广的木本作物。特级初榨橄榄油的重要品质性状之一为脂肪酸组成，其中高油酸、低亚油酸、亚麻酸及棕榈酸含量的橄榄油，其保健特性被认为极具优势。编码油酸去饱和酶的基因（FAD2-2）是调控橄榄果实中果皮乃至橄榄油中亚油酸含量的核心基因，因此也是亚油酸生物合成途径中最重要的候选基因。本研究对FAD2-2基因的5′非翻译区内含子（5′UTR intron）开展了计算机模拟（in silico）与结构分析，旨在探究其天然序列变异及其在基因表达调控中的作用。为鉴定功能性等位基因变异，本研究对97个橄榄栽培品种的5′非翻译区内含子进行了分离与部分测序。序列分析结果显示，存在一段117 bp的插入序列，其包含两处长片段重复单元——该结构此前在橄榄的FAD2-2基因中从未被报道过——同时还检测到大量内含子介导的增强效应（intron-mediated enhancement, IME）元件。序列多态性分析共检测到39个单核苷酸多态性（SNPs）。针对油酸与亚油酸含量开展的候选基因关联分析显示，7个单核苷酸多态性位点与1个插入缺失标记（indel）和表型性状显著相关，这些位点在不同年份间可解释7%至16%的表型变异。本研究揭示了橄榄FAD2-2基因中新的结构变异，这些变异可能参与调控与油酸及亚油酸含量变异相关的基因表达机制。