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Article|15 Feb 2024|OPEN
Hypermethylation in the promoter regions of flavonoid pathway genes is associated with skin color fading during ‘Daihong’ apple fruit development 
Jihua Xu1,2 ,† , Lan Xiong2 ,† , Jia-Long Yao3 , Peilei Zhao2 , Shenghui Jiang2 , Xiaohong Sun1,2 , Chaohua Dong1,2 , Hongyuan Jiang1 , Xinyue Xu1 , Yugang Zhang,2,4 ,
1College of Life Sciences, Key Laboratory of Plant Biotechnology of Shandong Province, Qingdao Agricultural University, Qingdao, 266109, China
2College of Horticulture, Engineering Laboratory of Genetic Improvement of Horticultural Crops of Shandong Province, Qingdao Agricultural University, Qingdao, 266109, China
3The New Zealand Institute for Plant and Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand
4Academy of Dongying Efficient Agricultural Technology and Industry on Saline and Alkaline Land in Collaboration with Qingdao Agricultural University, Dongying, 257300, China
*Corresponding author. E-mail:
Both authors contributed equally to the study.

Horticulture Research 11,
Article number: uhae031 (2024)
Views: 757

Received: 21 Sep 2023
Accepted: 23 Jan 2024
Published online: 15 Feb 2024


Apple fruit skin color fading is not well understood although the molecular mechanism of skin color formation is well known. The red-fleshed apple cultivar ‘Daihong’ (DH) exhibited fading skin color during fruit development despite having a heterozygous R6 allele but lacking Red-TE for red fruit skin. In this study, transcriptomic analysis revealed the expression level of MdMYB10 increased with fruit development whereas reduced expression levels of MdMYBPA1MdCHSMdANSMdUFGTMdLAR, and MdANR were observed, consistent with decreased levels of chalcone, anthocyanin, catechin, epicatechin, and procyanidin B2. Whole-genome bisulfite sequencing (WGBS) indicated a global gain in cytosine methylation levels and increased methylation in 5′ and 3′ flanking regions of genes and transposable elements (TEs), and in TE bodies in all CG, CHG and CHH contexts, especially the mCHH context, during fruit development. The increased DNA methylation was attributed to reduced expression levels of DNA demethylase genes, including MdDME1MdROS1, and MdROS2. Association analysis revealed a significant negative correlation between promoter methylation levels of MdCHSMdCHIMdMYBPA1, and their respective transcript levels, as well as a negative correlation between promoter methylation levels of MdCHSMdCHIMdANR, and MdFLS, and the content of chalcones, naringenin-7-glucoside, epicatechin, and quercetin. Treatment with the DNA demethylation agent 5-aza-2′-deoxycytidine verified the negative correlation between DNA methylation and gene expression within the flavonoid pathway. These findings suggest that hypermethylation in promoter regions of genes of the flavonoid biosynthesis pathway is associated with the reduction of gene expression and flavonoid content, and fruit skin color fading during DH apple development.