Abstract

The carob tree (Ceratonia siliqua L., Fabaceae) is an ecologically and economically significant species of the Mediterranean basin, yet its cytogenetic and genomic organisation have remained largely uncharacterised. Here we present the first comprehensive molecular cytogenetic characterisation of C. siliqua, combining conventional karyotyping, fluorochrome banding, fluorescence in situ hybridisation (FISH), and flow cytometric genome size estimation across 29 accessions spanning the Mediterranean basin. A uniform diploid chromosome number of 2n = 24 was confirmed across all 14 populations examined, including Algerian, French, and Lebanese accessions, with no karyotypic variation detected regardless of geographic origin, altitude, or cultivation status. Karyotype analysis revealed a bimodal chromosome set comprising two large metacentric pairs and ten smaller pairs, with an intermediate asymmetry class (AsI = 60.09; R = 2.71). Physical mapping of ribosomal RNA gene families by FISH identified three chromosome pairs bearing 35S rDNA loci, all co-localising with GC-rich CMA3-positive heterochromatin at satellite regions, and a single 5S rDNA locus at the telomeric region of a distinct chromosome pair, corresponding to the S-type arrangement. DAPI staining revealed an additional class of AT-rich constitutive heterochromatin at centromeric positions, compositionally and positionally independent of the rDNA arrays. Genome size was strikingly conserved across all 23 accessions assessed (2C = 1.10–1.23 pg; overall mean 1.14 ± 0.03 pg), with no significant variation attributable to geographic origin, altitude along an Algerian gradient (162–950 m a.s.l.), cultivation status, or sex. The near-identical genome sizes recorded in female and male individuals provide no cytometric evidence for heteromorphic sex chromosomes in this dioecious species. These results establish a stable genomic and cytogenetic baseline for C. siliqua across its Mediterranean range and provide a reference framework for future comparative cytogenetics, molecular marker development, and breeding programmes targeting this increasingly valued climate-resilient crop.

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