Comprehensive genomic and haplotype analysis of the phytocyanin gene family reveals evolutionary expansion and functional diversification in Setaria italica.

key messageThis study provides the first comprehensive genome-wide analysis of the phytocyanin gene family in Setaria italica ,identifying 63 SiPC genes that play critical roles in abiotic stress responses through complex regulatory networks involving transcription factors and miRNAs. Importantly, haplotype analysis revealed that natural genetic variationsin SiPC genes, particularly SiUC13 and SiUC23 ,are significantly associated with agronomic traits, off ering valuable targets for marker-assisted breeding to enhance stress tolerance and yield in cereals. Phytocyanins (PCs) are endogenous plant blue copper proteins that often occur as chimeras with arabinogalactan proteins (AGPs), playing crucial roles in electron transport. However, the origin and expansion of this gene family in plants remain poorly understood. In this study, we conducted a comprehensive bioinformatics analysis of the Setaria italica genome and identified 63 SiPC genes. Based on phylogenetic relationships and motif composition, the SiPC family was categorized into three subclasses: SiENODLs (32), SiUCs (28), and SiSCLs (3). Structural prediction revealed that 46 SiPCs encode glycosylphosphatidylinositol-anchored (GPI) proteins, while a majority (57) are chimeric AGPs. Gene duplication analysis suggested that chromosomal segmental duplications primarily drive SiPC family expansion, especially in the SiENODLs subfamily. In addition, promoter and co-expression analyses revealed that the SiPC gene is functionally linked to redox-active proteins and a conserved copper-binding domain, potentially playing a role in various stress responses, possibly being co-regulated by the TF-miR families. Moreover, expression profiling across vegetative, reproductive, and stress stages demonstrated strong developmental regulation. Finally, population analysis showed high haplotype diversity and SNP variation, with only a few SiPC genes showing significant phenotypic associations. This study provides a comprehensive overview of SiPCs, laying the foundation for future functional research and offering new insights into the molecular mechanisms underlying abiotic stress tolerance in millet.