Genomic basis of reticulate evolution and metabolic rewiring driving carotenoid diversity in Citrinae

The extreme phenotypic diversity in fruit coloration across the Citrinae subtribe poses a fascinating evolutionary puzzle. While carotenoids are essential for plant ecological interactions and human nutrition, the genomic basis underlying their vast metabolic variation in the context of complex species hybridization remains largely elusive. Here, we integrated whole-genome resequencing of 80 representative Citrinae accessions with high-resolution metabolic and transcriptional profiling to elucidate the evolutionary and genetic architectures of citrus fruit coloration. Population genomic modeling demonstrated that pervasive interspecific hybridization and multi-stage introgression from primary progenitors (C. reticulata, C. maxima, and C. medica) profoundly reshaped the mosaic metabolic landscapes of modern hybrid taxa (C. sinensis, C. aurantium, and C. limon). We uncovered a remarkable tissue-specific decoupling of metabolic flux: CitLCYB2 acts as the primary bottleneck limiting β-branch flux in the flavedo (peel), coupled with massive CitNCED2-mediated degradation in pummelos, whereas CitPSY1 serves as the core rate-limiting enzyme dictating source-driven accumulation in the pulp. Transgenic validation in vivo using a woody plant callus system directly confirmed the pivotal role of CitPSY1, yielding a > 10-fold surge in carotenoid content. Furthermore, by integrating high-resolution local ancestry inference with demography-robust genome-wide scans (CLR, iHS, and XP-CLR), we elucidated the divergent evolutionary trajectories of CitPSY1. We revealed that the ancestral mandarin lineage (C. reticulata) experienced a strong, targeted regulatory selective sweep to rapidly fix a highly active promoter haplotype for intense pigmentation, whereas pummelos and citrons retained basal metabolic capacities shaped predominantly by natural demographic history. Our findings provide a comprehensive paradigm for how historical gene flow and divergent selection orchestrate secondary metabolism in woody perennials, offering premier genetic targets for the nutritional biofortification of commercial fruits.