Haplotype-resolved chromosomal-level genome assembly reveals regulatory variations in mulberry fruit anthocyanin content

Abstract

Understanding the intricate regulatory mechanisms underlying the anthocyanin content (AC) in fruits and vegetables is crucial for advanced biotechnological customization. In this study, we generated high-quality haplotype-resolved genome assemblies for two mulberry cultivars: the high-AC ‘Zhongsang5801’ (ZS5801) and the low-AC ‘Zhenzhubai’ (ZZB). Additionally, we conducted a comprehensive analysis of genes associated with AC production. Through genome-wide association studies (GWAS) on 112 mulberry fruits, we identified MaVHAG3, which encodes a vacuolar-type H⁺-ATPase G3 subunit, as a key gene linked to purple pigmentation. To gain deeper insights into the genetic and molecular processes underlying high AC, we compared the genomes of ZS5801 and ZZB, along with fruit transcriptome data across five developmental stages, and quantified the accumulation of metabolic substances. Compared to ZZB, ZS5801 exhibited significantly more differentially expressed genes (DEGs) related to anthocyanin metabolism and higher levels of anthocyanins and flavonoids. Comparative analyses revealed expansions and contractions in the flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) genes, resulting in altered carbon flow. Co-expression analysis demonstrated that ZS5801 displayed more significant alterations in genes involved in late-stage AC regulation compared to ZZB, particularly during the phase stage. In summary, our findings provide valuable insights into the regulation of mulberry fruit AC, offering genetic resources to enhance cultivars with higher AC traits.

Acknowledgements

This research received support from the Earmarked Fund for CARS (CARS-18-ZJ0201), the Chongqing Modern Agricultural Industry Technology System (CQMAITS202311), and the Hainan Province Science and Technology Special Fund (ZDYF2022SHFZ319). We extend our gratitude to Professor Xingtan Zhang from the Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences, for invaluable comments and suggestions on the manuscript. We also acknowledge the valuable assistance in data analysis provided by Professor Yu Jiang at Northwest A&F University. Furthermore, we appreciate the contributions of Professor Feng Jiao at Northwest A&F University for sharing phenotypic information on germplasm resources and Professor Chunmei Li at Huazhong Agricultural University for providing mulberry fruit metabolism data. We sincerely thank ANNOROAD for its sequencing support and the Hefei Advanced Computing Center for providing computing resources.

Data availability

The original resequencing reads HiFi data, Hi-C data, RNA-seq data, and Iso-seq data generated during this study have been deposited in the Genome Sequence Archive (GSA) [114] at the National Genomics Data Center (NGDC), Beijing Institute of Genomics (BIG), Chinese Academy of Sciences (CAS) / China National Center for Bioinformation (CNCB) [115] under the accession number CRA011833. These data are publicly accessible at https://ngdc.cncb.ac.cn/gsa. Additionally, the genome assemblies and gene annotations have been deposited in the Genome Warehouse [116] at the NGDC, BIG, CAS / CNCB, with the following accession numbers: GWHDOOQ00000000 (ZS5801_Hap1), GWHDOOP00000000 (ZS5801_Hap2), GWHDOOT00000000 (ZS5801_Monoploid), GWHDOOM00000000 (ZZB_Hap1), GWHDOOO00000000 (ZZB_Hap2), and GWHDOON00000000 (ZZB_Monoploid). These resources are publicly accessible at https://ngdc.cncb.ac.cn/gwh.

Conflict of interest statement

The authors declare no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

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