Functional deciphering of the fibrillin gene family in foxtail millet reveals a non-synonymous SNP associated with carotenoid accumulation

Background Carotenoids are essential phytonutrients for human health and plant adaptation. In plastids, carotenoid storage and turnover occur in plastoglobules, whose core structural components are Fibrillin (FBN) proteins. The FBN family remains uncharacterized in foxtail millet ( Setaria italica ), a nutritious C4 cereal whose grain carotenoid content strongly correlates with color. The genetic basis of carotenoid variation in foxtail millet is largely unknown. Results We identified 12 SiFBN genes in foxtail millet, which were phylogenetically classified into 11 subfamilies. Expression analysis revealed three major patterns: constitutive ( SiFBN1b ), development-dependent ( SiFBN6 , SiFBN10 ), and differentially expressed between varieties ( SiFBN3 ). SiFBN3 was significantly upregulated in the high-carotenoid yellow-grain variety JG21 compared to JG25, though haplotype analysis in a 152-accession panel revealed no sequence polymorphism in this gene. Furthermore, a candidate-gene association analysis in a 152-accession panel identified a natural polymorphism in SiFBN10 (A/T at position 40085873) associated with carotenoid content. The T allele (Hap2, frequency 30.3%) was strongly associated with high carotenoid accumulation across two independent years, whereas the A allele (Hap1, 69.7%) linked to low content. Both SiFBN3 and SiFBN10 were experimentally confirmed to localize to chloroplasts. Conclusions This study suggests that carotenoid accumulation in foxtail millet grains involves multiple regulatory layers. SiFBN3 shows differential expression between the yellow-grain variety JG21 and the white-grain variety JG25, though the genetic basis of this difference remains to be elucidated given the absence of sequence polymorphism in the 152-accession panel. In addition, a natural polymorphism in SiFBN10 is associated with carotenoid content variation in the natural population and may contribute to phenotypic differences among accessions.