Characterization of the complete chloroplast genomes of three grapefruit (Citrus × paradisi Macfad.) cultivars

Chloroplast genomes (plastomes) are valuable resources for understanding evolutionary relationships and tracing maternal lineages. Citrus is a genus shaped by extensive hybridization, introgression, and complex domestication histories, which complicate phylogenetic signal in the nuclear genome. Here, we present complete, high-confidence chloroplast genome assemblies for three red-fleshed grapefruit cultivars, Rio Red (RR), Ruby Red (RU), and Texas Red (TX), assembled from PacBio HiFi whole-genome sequencing data without dedicated organellar enrichment. All three plastomes were recovered as single-contig circular molecules of 160,185 bp exhibiting the canonical angiosperm quadripartite structure with identical partition sizes: a large single-copy region (LSC) of 87,790 bp, a small single-copy region (SSC) of 18,395 bp, and two inverted repeats (IRs) of 27,000 bp each. Annotation identified 113 unique genes per plastome, comprising 79 protein-coding genes, 30 tRNAs, and 4 rRNAs, including the two-intron genes clpP1 and pafI and the trans-spliced rps12 . All three cultivars share fully identical plastome architectures, gene content, codon-usage profiles, and repeat landscapes dominated by short A/T-rich mononucleotide cpSSRs, confirming a common maternal plastid haplotype. IR boundary organization was conserved, with only minor 2–4 bp variation at the SSC/IRb junction in Texas Red. Pairwise whole-plastome comparisons using MUMmer4 confirmed zero sequence variation among cultivars and between each cultivar and pummelo, while comparisons against sweet orange revealed 131–137 variant sites. A maximum-likelihood phylogeny placed all three cultivars in a strongly supported clade sister to pummelo.