Multi‐omics dissection of steviol glycoside synthesis reveals haplotype‐linked specialization of UGT76G genes in Stevia rebaudiana

Steviol glycosides (SGs), intensely sweet diterpenoids found in Stevia rebaudiana (stevia), exhibit natural variation in composition that influences taste quality and commercial value. However, the genetic and cellular mechanisms underlying this variation remain poorly understood. We aimed to uncover how haplotype-level diversity and cell-type-specific gene expression contribute to SG profile diversity in stevia. We generated a chromosome-scale reference genome and conducted integrative multi-omics analyses combining haplotype-resolved population genomics, single-nucleus RNA sequencing, and imaging mass spectrometry. These approaches were applied to a diverse panel of breeding lines and a segregating population to associate genetic variants with SG composition and expression patterns. We identified a physically linked cluster of UGT76G glycosyltransferase genes whose structural and regulatory polymorphisms drive major differences in SG composition. Restricted expression of UGT91D4 to specific subsets of mesophyll and epidermal cells further constrained the biosynthesis of high-value SGs, such as rebaudioside D and rebaudioside M. Functional divergence among UGT76G paralogs was supported by sequence, expression, and structural modeling data. Our findings define a multi-allelic and spatially coordinated regulatory architecture underlying SG biosynthesis in stevia. These insights provide a foundation for haplotype-guided breeding and metabolic engineering strategies aimed at improving stevia sweetness quality and yield.