Comparative Analysis of Chloroplast Genome Structure and Phylogenetic Relationships Between a Typical Eucalypt Hybrid and Two Purebred Species

Background The chloroplast genome serves a dual function as both a cytoplasmic marker and a functional contributor in hybrid species. A comparative study of chloroplast genomes in a eucalypt hybrid­ Eucalyptus urophylla × Eucalyptus grandis ( E. urograndis ) and pure species ( E. urophylla and E. grandis ) can provide a theoretical foundation for understanding forest evolution and genetic, it can offer certain technical support for advancements in forest ecological conservation, and biotechnological development. Results By employing next-generation sequencing technology, bioinformatics analysis, and other methods, we conducted whole-genome sequencing of the chloroplasts in the hybrids E. urograndis and the pure species E. urophylla . Our findings revealed that the E. urograndis (160,201 bp) had an intermediate chloroplast genome size between that of E. urophylla (160,283 bp) and E. grandis (160,137 bp). Significant differences was evident in gene composition, IR region expansion, and SSR site distribution. In particular, trnK ^UUU , trnT ^GGU , psaB-psaA , ndhJ - ndhK , and rpl22 - rps19 - rpl2 were identified as significantly different regions. These regions can serve as potential barcode candidates in subsequent studies for species identification, allowing evaluation of their application potential in interspecific discrimination. A set of 14–16 optimal codons was identified in Eucalyptus , including GCA, CCA, UAA, GUU, ACA, UCA, CUU, GGU, CAA, UGU, AAU, AGU, GAA, ACU, UUG, and AAA, establishing a crucial foundation for the subsequent optimization of exogenous sequences based on codon usage patterns. This strategy provides essential technical support for advancing research in genetic engineering, trait improvement, and species conservation of Eucalyptus plants. Phylogenetic reconstruction confirmed that the eucalypt hybrid formed a monophyletic clade with the pure species E. urophylla . Conclusions The present study provides theoretical insights into the Structural variations in the chloroplast genome and evolutionary during eucalypt, while enhancing our understanding of interspecific diversity in chloroplast genomes.It also provides a theoretical foundation for sequencing the organelle genomes of eucalypt species, studying variations and evolution in organelle genomes, and developing molecular marker-assisted breeding strategies.