A number of cis-regulatory elements (CREs) conserved during evolution have been found to be responsible for phenotypic novelty and variation. Cucurbit crops such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo) and squash (Cucurbita maxima) develop fruits from an inferior ovary and share some similar biological processes during fruit development. Whether conserved regulatory sequences play critical roles in fruit development of cucurbit crops remains to be explored. In six well-studied cucurbit species, we identified 392,438 conserved non-coding sequences (CNSs), including 82,756 that are specific to cucurbits, by comparative genomics. Genome-wide profiling of accessible chromatin regions (ACRs) and gene expression patterns mapped 20,865–43,204 ACRs and their potential target genes for two fruit tissues at two key developmental stages in six cucurbits. Integrated analysis of CNSs and ACRs revealed 4,431 syntenic orthologous CNSs, including 1,687 cucurbit-specific CNSs that overlap with ACRs that are present in all six cucurbit crops and that may regulate the expression of 757 adjacent orthologous genes. CRISPR mutations targeting two CNSs present in the 1,687 cucurbit-specific sequences resulted in substantially altered fruit shape and gene expression patterns of adjacent NAC1 (NAM, ATAF1/2 and CUC2) and EXT-like (EXTENSIN-like) genes, validating the regulatory roles of these CNSs in fruit development. These results not only provide a number of target CREs for cucurbit crop improvement, but also provide insight into the roles of CREs in plant biology and during evolution.

中文翻译：

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葫芦科作物早期果实发育的保守顺式调控元件的鉴定和功能表征

人们发现，进化过程中保守的许多顺式调控元件（CRE）与表型的新颖性和变异有关。黄瓜 (Cucumis sativus)、西瓜 (Citrullus lanatus)、甜瓜 (Cucumis melo) 和南瓜 (Cucurbita maxima) 等葫芦类作物从下位子房发育果实，在果实发育过程中具有一些相似的生物过程。保守的调控序列是否在葫芦科作物的果实发育中发挥关键作用仍有待探索。通过比较基因组学，我们在六个经过充分研究的葫芦科植物中鉴定出了 392,438 个保守的非编码序列 (CNS)，其中包括 82,756 个葫芦科植物特有的序列。对可及染色质区域 (ACR) 和基因表达模式进行全基因组分析，绘制了 6 个葫芦科植物处于两个关键发育阶段的两个果实组织的 20,865-43,204 个 ACR 及其潜在靶基因。CNS 和 ACR 的综合分析揭示了 4,431 个同线直系同源 CNS，其中包括 1,687 个葫芦科特异性 CNS，它们与所有六种葫芦科作物中存在的 ACR 重叠，并且可能调节 757 个相邻直系同源基因的表达。针对 1,687 个葫芦科特异性序列中存在的两个 CNS 的 CRISPR 突变导致果实形状和相邻 NAC1（NAM、ATAF1/2 和 CUC2）和 EXT 样（EXTENSIN 样）基因的基因表达模式发生显着改变，验证了调控作用这些中枢神经系统在水果发育中的作用。这些结果不仅为葫芦科作物改良提供了许多目标CRE，而且还提供了对CRE在植物生物学和进化过程中的作用的深入了解。