A haplotype-resolved T2T genome assembly of Indigofera pseudotinctoria reveals the genetic basis of flavonoid biosynthesis in Chinese Indigo

Indigofera pseudotinctoria, commonly known as Chinese Indigo, is a multifunctional leguminous shrub widely distributed in East Asia, valued for its medicinal, ecological, and forage importance, and naturally adapted to drought and nutrient-poor soils. However, the absence of genomic resources has hindered insights into the genetic basis of its bioactive metabolites and adaptive traits. Here, we present a haplotype-resolved, telomere-to-telomere (T2T) reference genome of I. pseudotinctoria, which represents the first complete genome within the genus Indigofera. By integrating PacBio HiFi sequencing, Oxford Nanopore ultra-long reads, and Hi-C chromatin conformation sequencing technologies, we successfully constructed two gap-free haplotypes assemblies (647.38 Mb and 657.52 Mb), characterized by high BUSCO completeness and QV scores, and fully captured all telomeric sequences. Comparative analysis between haplotypes revealed extensive synteny but notable structural heterozygosity but notable structural heterozygosity. Approximately 46% of structural variations overlap with genic or regulatory regions, leading to allele-specific expression divergence. Between the two haplotypes, we identified 2,607 and 2,331 haplotype-specific genes, reflecting a complementary functional specialization in defense and repair versus metabolism and growth. Integrative transcriptomic and metabolomic profiling across six tissues reconstructed the flavonoid biosynthetic network and identified MYB, bHLH, NAC, WRKY, and ERF transcription factors regulating five key pharmacologically active flavonoids (Calycosin, Butein, Sulfuretin, Chrysoeriol, and Genistin). These results collectively uncover the haplotype-specific regulatory and structural basis of Indigofera pseudotinctoria and establish a high-quality genomic framework for evolutionary, medicinal, and metabolic engineering studies in leguminous plants, and provide a framework for molecular breeding and bioactive compound discovery in medicinal legumes.