Actinidia arguta has become popular with consumers recently because of its edible and colorful fruit skin. The 3D spatial organization of its genome plays a key role in the formation of various biological traits. However, the function of 3D genome reorganization during fruit skin color formation is poorly understood in A. arguta. In this study we constructed the 3D genome of the red-skinned A. arguta cultivar ‘Zhonghongbei’ (ZHB) and the green-skinned cultivar ‘Zhonglvbei’ (ZLB), and performed chromatin structure comparisons between them at compartment, topologically associating domain (TAD), and loop levels. Global compartment comparisons at whole 3D genome level between red-skinned and green-skinned A. arguta showed that A–B compartment transition specifically occurred in chromosome 7 and chromosome 16, based on which all genes within 3 Mb upstream and downstream of A–B compartment transition were retrieved to construct a four-way Venn diagram, which showed that AaCBP60B-like, encoding calmodulin-binding protein 60 B-like, is the key candidate gene negatively correlating with fruit color. Exogenous calcium chloride treatments enhancing AaCBP60B-like expression to repress anthocyanin biosynthesis proved a negative role of AaCBP60B-like in anthocyanin biosynthesis. Overexpression and virus-induced gene silencing assays of AaCBP60B-like revealed the inhibition of anthocyanin biosynthesis derived from differential expression of AaCBP60B-like resulting from a 346-bp InDel variation located at the AaCBP60B-like promoter resulting in activity differences in red- and green-skinned A. arguta. ATAC-seq results proved that the 346-bp InDel variation affects 3D genome organization. Our study provides the first 3D chromosome organization in red- and green-skinned A. arguta, based on which a candidate gene, AaCBP60B-like, involved in anthocyanin regulation is identified.

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