The downstream molecular network of FaMYB44.1, a key factor in strawberry quality regulation, was investigated by DNA affinity purification sequencing

Strawberry (<italic>Fragaria × ananassa</italic> Duch.) is a small berry characterized by its bright color and sweet-sour flavor. With the global rise in living standards, the strawberry industry has experienced rapid growth, and enhancing the comprehensive quality of the fruit has become a focal point in current strawberry research. Previous studies often concentrated on improving single quality indices, with a notable lack of regulatory factors for enhancing overall strawberry fruit quality. In this study, transgenic strawberries with inhibited <italic>FaMYB44.1</italic> expression were developed using instantaneous transformation technology. Further analysis of related quality indices revealed that FaMYB44.1 is a MYB transcription factor that inhibits fruit ripening. TRV<italic>-FaMYB44.1</italic> transgenic strawberry fruits exhibited significantly increased anthocyanin content and markedly decreased organic acid levels. DAP-seq analysis demonstrated that FaMYB44.1 binds to multiple promoters, untranslated regions (UTRs), exons, and introns within the genome. The associated target genes are enriched in various primary and secondary metabolic pathways, including carbohydrate metabolism, citric acid cycle (TCA cycle), flavonoid biosynthesis, and flavonoid and flavanol biosynthesis. Motif analysis identified DREME (AAGTA) as the binding motif of the strawberry FaMYB44.1 transcription factor. This research confirms <italic>FaMYB44.1</italic>'s involvement in regulating diverse metabolic processes, contributing to the improvement of overall strawberry fruit quality, and provides a valuable gene target for molecular breeding endeavors.