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

Comparative Analysis of Chloroplast Genome Structure and Phylogenetic Relationships Between a Typical Eucalypt Hybrid and Two Purebred Species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Analysis of Chloroplast Genome Structure and Phylogenetic Relationships Between a Typical Eucalypt Hybrid and Two Purebred Species Gupeng Yi, Guo Liu, Jianzhong Luo, Wanhong Lu, Yan Lin, Ying Cheng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9082114/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. eucalypt chloroplast genome comparative analysis codon usage phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Highlights 1. 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). 2. Analysis of SSRs in the chloroplast genomes of 12 eucalypt species revealed a variation ranging from 68 SSRs in E. grandis to 115 in C. citriodora . 3. The psbL , psbN , infA , and pbf1 genes show differences among the three eucalypt species. 4. A set of 14-16 optimal codons was identified in eucalypt species, including GCA, CCA, UAA, GUU, ACA, UCA, CUU, GGU, CAA, UGU, AAU, AGU, GAA, ACU, UUG, and AAA, with a predominance of codons ending in A or U. This finding provides a key foundation for optimizing exogenous sequences based on codon usage patterns. 5. Consistent with established taxonomic frameworks, two Angophora species, three Corymbia species, and 29 eucalypt specie formed monophyletic clades with bootstrap support values exceeding 80%. 1. Introduction Eucalypt, which includes the genera Eucalyptus , Angophora , and Corymbia in the Myrtaceae family, is a tree species known for its rapid growth, short rotation period, drought resistance, and broad adaptability. It is widely utilized in pulp and papermaking, plywood production, and solid wood processing, making it a key timber species in southern China’s rapidly expanding plantations. According to the latest data, China’s eucalypt plantation area has increased to 5.93 million hectares, with an annual wood production of about 40 million cubic meters[ 1 ]accounting for more than one-third of the country’s total timber output. eucalypt plantations are critical for ensuring China’s wood security, boosting economic growth, and promoting rural revitalization. Eucalyptus urophylla × Eucalyptus grandis ( E. urograndis ), a typical eucalypt hybrid, is produced by crossing E. grandis (father) with E. urophylla (mother). Initially created in the late 1980s at Guangxi Dongmen Forest Farm, it has since emerged as a key artificial forest species nationwide, owing to its robust adaptability, rapid growth, short growth cycle, ease of vegetative propagation, and favorable trunk morphology. The chloroplast genomes of higher plants exhibit a highly conserved quadripartite structure, in which two inverted repeat sequences (IR) partition the entire circular chloroplast genome into a large single-copy region (LSC) and a small single-copy region (SSC). The chloroplast genome (cpDNA) [ 2 – 4 ], characterized by its structural conservation, low recombination rate, and typical maternal inheritance pattern, serves as an ideal model for investigating species evolution and hybridization. Although the NCBI database currently includes cpDNA data for 73 eucalypt species ( https://ngdc.cncb.ac.cn/cgir/genome?term=eucalyptus , accessed on March 28 2025) and phylogenetic studies have confirmed a deep divergence between Eucalyptus and Corymbia lineages[ 5 ], research in this area remains limited compared to other plant systems. Notably, Research on specific nuclear genes or functional traits of important economic eucalypt species such as Eucalyptus grandis and Eucalyptus regia has been extensively conducted. However, systematic comparative genomics and evolutionary analysis data at the complete chloroplast genome level remain significantly lacking. Therefore, this work presents the first comparative framework for analyzing the chloroplast genomes of the hybrid species E. urograndis and the pure species E. urophylla, E. grandis . By conducting a comprehensive analysis of genome architecture, simple sequence repeats (SSRs),synonymous codon usage preference, optimal codon, inverted repeat (IR) boundary variations, nucleotide diversity, and phylogenetic relationships among E. urophylla , E. grandis and E. urograndis , this research aims to clarify the distinctive characteristics and evolutionary patterns of their chloroplast genomes. The results of this work will serve as a scientific basis for the conservation and sustainable utilization of these genetic resources. Furthermore, it provides a theoretical foundation for future studies, including organelle genome sequencing of other eucalypt species, in-depth exploration of organelle genome variations and evolutionary relationships, and the development of molecular marker-assisted breeding strategies. 2. Methods 2.1 Plant materials We obtained improved varieties of E. urograndis (No. Gui S-SC-EUG-001-2009) from the Guangxi State-owned Dongmen Forest Farm (107°51′52″E, 22°19′53″N) and E. urophylla from the Jijia Town, Leizhou City, Guangdong Province (109°47′44″E, 20°56′15″N). For both species, healthy mature leaves were collected from the southern and northern sides of three trees exhibiting uniform growth and favorable conditions. These leaves were combined into a single composite sample for analysis. The same sampling protocol was applied to E. urograndis (3-year-old trees) and E. urophylla (6-year-old trees). The collected leaf samples were submerged in liquid nitrogen immediately for rapid freezing and stored at -80°C for future analysis. 2.2 DNA extraction, library construction, and sequencing Total DNA from leaf samples of the two eucalypt species was extracted using the DNAsecure Plant kit (TIANGEN, Beijing, China). Following a quality assessment of the samples, we constructed the library using the MGIEasy Fast Enzyme-Cutting Library Preparation procedures. The NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA) was used to determine DNA concentrations and quality. Trimmomatic software[ 6 ] was used to control the raw reads received from sequencing using the default parameter setting. This was followed by the filtration of the adaptor and low-quality sequences to obtain high-quality clean reads. 2.3 Chloroplast genome splicing, annotation, and physical mapping The splicing process was divided into three stages: initial splicing, contig screening, and extension, filling, and re-splicing. The limited data (2 × 150 bp, 10 G) prompted the use of GetOrganelle v1.7.7.1 software, which demonstrated superior performance for circular genome splicing. The chloroplast genomes were annotated using CPGAVAS2 software[ 7 ], and the physical maps of the chloroplast genomes of E. urograndis and E. urophylla were created by Organellar Genome DRAW online software[ 8 ]. 2.4 Repeat sequence analysis The SSRs of the chloroplast genome were analyzed by MISA software[ 9 ] using the following parameters: mononucleotides ≥ 10, dinucleotides ≥ 5, trinucleotides ≥ 4, tetranucleotides ≥ 3, pentanucleotides ≥ 3, and hexanucleotides ≥ 3. The minimum spacing between two SSRs was fixed at 100 bp. The dispersed repeats in the chloroplast genome were detected using REPuter software ( http://bibiserv.techfak.uni-bielefeld.de/reputer )[ 10 ] Forward (F), palindrome (P), reverse (R), and complementary long repeats (C) were detected with a repeat length of more than 30 bp and a Hamming distance of 3. 2.5 Relative Usage of Synonymous Codons To improve the accuracy of subsequent codon preference analysis, the codon usage of three chloroplast protein-coding genes from eucalypt species was screened using MEGA 7.0.26 software based on the following criteria: length > 300 bp, start codon ATG, stop codons TGA, TAA, or TAG, and nucleotide count as a multiple of three. Ultimately, 45, 48, and 44 CDS sequences were selected for subsequent data analysis, respectively. Using the online platform EMBOSS Explorer ( https://www.bioinformatics.nl/emboss-explorer/ ), the nucleotide composition percentages of the three eucalypt chloroplast genomes were calculated[ 11 ], along with the GC content at the first, second, and third codon positions (denoted as GC1, GC2, and GC3, respectively). Meanwhile, using Rstudio software, genes were ranked from high to low based on their ENC values, and the top and bottom 10% of genes were selected as the high-expression and low-expression libraries, respectively. Codons with ΔRSCU > 0.08 within each library were then identified as high-expression codons based on the genes' RSCU values. Codons meeting both ΔRSCU > 0.08 and RSCU > 1 were defined as optimal codons[ 12 ]. Finally, the corresponding results were visualized through plotting. 2.6 Genome comparison and analysis, IR border and divergence analyses Geneious Prime v2021.1.1[ 13 ] was used to determine the chloroplast genome length, GC level, and LSC, SSC, and IRs. It was also used to estimate the number and classification of genes. Chloroplast genomes from the 10 eucalypt species were examined using the IRscope online software[ 14 ] to identify shrinkage or expansion at the boundaries. MAFFT v.7.450 was used to carry out multiple sequence alignment of E. urograndis and its parental chloroplast genome sequences [ 15 ]. DNAPARS v.6.0 [ 16 ]was used to calculate nucleotide polymorphisms (π values) and identify mutation genes and hotspots. The step size and search window length were set at 200 bp and 600 bp, respectively, and the results were plotted in Excel. 2.7 Phylogenetic tree analysis Table S1 lists species and accessions used in phylogenetic analysis. Thirty-four species were selected to represent major clades, including main sections and series with Eucalyptus and related taxa . The samples contained 18 Eucalyptus species belonging to the subgenus Symphyomyrtus , representing nine sections within that subgenus, five species of subgenus Eucalyptus that represented four sections, one species of subgenus Alveolata ( E. microcorys ), one species of subgenus Cuboidea ( E. tenuipes ), one species of subgenus Eudesmia ( E. erythrocorys ), and one species of subgenus Idiogenes ( E. cloeziana ). In addition, we also included two Angophora species, three Corymbia species, and three outgroup species, including Arabidopsis thaliana and Populus trichocarpa . We utilized MAFFT v7.313 to align the complete chloroplast genome sequences, followed by trimAI to remove indels[ 17 ] Maximum likelihood (ML) was used to assess the phylogenetic relationships. Meanwhile, ML reconstruction was performed with IQ-TREE[ 18 ] using ModelFinder’s best-fit nucleotide substitution model[ 19 ]. Ten thousand ultrafast bootstrap replications were used to evaluate branch support for the ML tree [ 20 ]. 3. Results 3.1 Structural and functional characteristics of three Eucalyptus chloroplast genomes The chloroplast genomes of E. urophylla and E. urograndis were sequenced and assembled, whereas the chloroplast genome of E. grandis was retrieved from NCBI (Reference sequence: NC_014570.1). The assembled genomes of the three species were similar in size, ranging from 160,137 bp for E. grandis to 161,283 bp for E. urophylla (Fig. 1 ). All three chloroplast genomes were AT-rich (GC level = 36.86–36.89%, Table 1 ) and had a quadripartite structure similar to many angiosperms, with two inverted repeats (IR) region copies (52780–52798 bp), one large single-copy region (LSC, 88876–88987 bp), and one small single-copy region (SSC, 18481–18498 bp) (Table 1 ). The GC level in each region differed between the three chloroplast genomes with 42.7%, 30.4%-30.5%, and 34.7%-34.8% variance in IR, SSC, and LSC regions, respectively (Table 1 ). Table 1 The complete chloroplast genome features for three Eucalyptus species Species Size/bp Number of genes LSC(bp) /GC SSC(bp) /GC IRs(bp) /GC Total GC% PCGs/(bp) rRNA /(bp) tRNA /(bp) E. urograndis 160201 127 34.7% 30.4% 42.7% 36.86% 83/78016 8/9056 36/2736 E. grandis 160137 127 34.8% 30.5% 42.7% 36.89% 83/91066 8/9050 36/2745 E. urophylla 160283 129 34.7% 30.4% 42.7% 36.86% 85/85394 8/9050 36/2736 Notes:LSC, large single-copy region; SSC, small single-copy region; IRs, two inverted repeats; PCGs, Protein-coding genes. 3.2 Dispersed repeat and SSR analysis The E. urophylla chloroplast genome included 102 qualified SSRs, comprising 79 mononucleotide SSRs, 4 dinucleotide SSRs, 4 trinucleotide SSRs, and 15 tetranucleotide SSRs (Table S2 ). The chloroplast genome of E. grandis exhibited fewer SSRs (68), which included 48 mononucleotide SSRs, 2 dinucleotide SSRs, 4 trinucleotide SSRs, and 14 tetranucleotide SSRs (Fig. 2 ). The hybrid E. urograndis exhibited identical SSR types and numbers as E. urophylla . SSRs were predominantly distributed in the LSC region of all three species, with fewer in the IRs and SSC regions. Mononucleotide SSRs accounted for the highest proportion, while dinucleotide, trinucleotide, and tetranucleotide SSRs were relatively less common. Furthermore, SSRs in the chloroplast genomes of all three Eucalyptus species included predominantly adenine (A) and thymine (T). E. urophylla possessed 4 dinucleotide SSRs with A/T and T/C motifs, 4 trinucleotide SSRs with ATT/TAA motifs, and 15 tetranucleotide SSRs rich in A and T. E. grandis contained 2 dinucleotide SSRs (A/T and T/C motifs), 5 trinucleotide SSRs (ATT/TAA motifs), and 13 A/T-rich tetranucleotide SSRs. The hybrid E. urograndis shared the same nucleotide repeat composition as E. urophylla . Dispersed repeats are a type of repeat sequence interspersed throughout the genome. They are classified into four types: forward, palindromic, reverse, and complementary. E. urophylla comprised 31 dispersed repeats, including 16 forward and 15 palindromic (Fig. 3 ). The longest forward repeat spanned 518 bp (0.32% of the genome), while the longest palindromic repeat measured 26,923 bp (16.81%). E. grandis shared the same number of dispersed repeats (31) as E. urophylla , with the longest forward repeat at 524 bp (0.32%) and the longest palindromic repeat at 26,838 bp (16.80%). The hybrid E. urograndis included 32 dispersed repeats, comprising 17 forward and 15 palindromic repeats. The longest palindromic repeat was 26,923 bp long, accounting for 16.80% of the genome. However, no reverse or complement repeats were observed in the three Eucalyptus species (Fig. 3 ). 3.3 Comparative analysis of SSR among eucalypt species Analysis of SSRs in the chloroplast genomes of 12 eucalypt species revealed a variation ranging from 68 SSRs in E. grandis to 115 in C. citriodora (Fig. 4 A). The SSR composition included 48 to 87 mononucleotide repeats, 1 to 4 dinucleotide repeats, 3 to 8 trinucleotide repeats, 4 to 17 tetranucleotide repeats, 0 to 1 pentanucleotide repeats, and 0 to 1 hexanucleotide repeats. These SSRs were predominantly located in the LSC region of the chloroplast genomes. Tetranucleotide repeats were generally more abundant than trinucleotide repeats across the 12 species, whereas pentanucleotide and hexanucleotide repeats were relatively rare. E. pauciflora exhibited the highest proportion of mononucleotide repeats (81.74%). Four species— E. urograndis , E. urophylla , E. nitens , and E. robusta —shared an identical SSR composition, each containing 79 mononucleotide repeats, along with 4 dinucleotide and 4 trinucleotide repeats. C. citriodora was the most distinctive species, uniquely possessing both pentanucleotide and hexanucleotide repeats among the 12 examined species, whereas all other species contained only the other four repeat types. Dispersed repeats represent a distinct category of repetitive sequences, distinguished by their scattered distribution throughout the genome. These repeats are classified into four types: Forward (F), Palindromic (P), Reverse (R), and Complement (C). An analysis of dispersed repeats within the chloroplast genomes of 12 eucalypt species (Fig. 4 B) demonstrated that forward and palindromic repeats were predominat, whereas complement repeats were the least prevalent. Only C. citriodora and E. nitens exhibited all four repeat types (F, P, R, C) in their chloroplast genomes; the remaining ten species lacked complement repeats. E. globulus displayed a notable disparity in repeat counts, with seven more forward repeats than reverse repeats. In other species, the numbers of forward and palindromic repeats were generally comparable. Complement repeats remained consistently rare across all 12 species, occurring exclusively in C. citriodora and E. nitens . 3.4 Analysis of Relative Synonymous Codon Usage (RSCU) The total average GC1, GC2, and GC3 values of the chloroplast genome CDS codons of E. urograndis , E. urophylla , and E. grandis are 38.4%, with individual averages of 47.14%, 38.67%, and 29.387%, respectively, showing a pattern of GC1 > GC2 > GC3. Using an ENC value of 45 as the threshold for bias classification, the overall average ENC values for 45, 48, and 44 CDS codons in the chloroplast genomes of the three eucalypt species range from 48.83 to 49.41, all exceeding 45(Table 2 ). This indicates a relatively weak codon usage bias in the chloroplast genomes of these three species. Table 2 Genomic features of chloroplast genomes of three Eucalyptus plant species. Parameters E. urograndis E. urophylla E. grandis CDS number (before processing) 83 85 83 CDS number (after processing) 45 48 44 L_aa 51687 58938 44406 GC1(%) 47.54 46.02 47.86 GC2(%) 38.9 38.03 39.08 GC3(%) 29.56 28.74 29.86 ENc 49.41 48.83 49.36 Average GC(%) 38.67 37.6 38.93 Note: L_aa means the total number of amino acids; GC1, GC2 and GC3 indicate the GC content at the first, second and third codon positions. Based on the analysis criteria of RSCU values (where RSCU > 1 and > 1.5 represent relatively high and high-frequency codons, respectively), the chloroplast genomes of the three Eucalyptus species exhibit similar codon usage preferences, with their RSCU values for all encoded amino acids primarily distributed in the range of 0–3 (Fig. 5 ). E. urograndis , E. urophylla , and E. grandis each have 30 high-frequency codons with RSCU values greater than 1, accounting for 46.87% of the total codons. Among these high-frequency codons, only one ends with G, while the rest end with A or U. The results indicate that the codon usage in the chloroplast genomes of the three Eucalyptus species tends to favor A or U endings. Additionally, in the chloroplast genomes of the three ucalypt species, the codon UUA for leucine (Leu) acts as an overlapping codon and is used more frequently than other codons. Specifically, the RSCU values are 1.91 for E. urograndis , 1.98 for E. urophylla , and 1.91 for E. grandis. 3.5 Analysis of Optimal Codons In E.urograndis , 14 optimal codons were identified with the highest ΔRSCU value of 1.243 and an average ΔRSCU value of 0.535 (Fig. 6 ). For E. urophylla , the highest number of optimal codons was observed, totaling 16, along with a maximum ΔRSCU value of 1.343 and an average ΔRSCU value of 0.417. In E. grandis , 14 optimal codons were identified, matching the highest ΔRSCU value observed in E. urograndis , and an average ΔRSCU value of 0.446. Across the three Eucalyptus species, a total of 14–16 optimal codons were identified, including GCA, CCA, UAA, GUU, ACA, UCA, CUU, GGU, CAA, UGU, AAU, AGU, GAA, ACU, UUG, and AAA( Fig. 7 ). 3.6 IR border and divergence analyses In the chloroplast genomes of the ten eucalypt species (Fig. 8 ), variations were observed in the positions of the rpl22 , rps19 , and rpl2 genes at the IR boundaries, as well as in their gene lengths. The rpl22 and rps19 genes were located at the IR/LSC boundary (JLA) in E. urophylla , positioned to the right of the junction, with rps19 located 1 bp from the boundary. In E. grandis , these genes were found at the LSC/IRb boundary (JLB), positioned to the left of the junction, with rps19 located 2 bp from the boundary. E. urograndis , both genes were located at the SSC/IRA boundary (JSA), positioned to the left of the junction, with rps19 located 2 bp from the boundary. The rpl2 gene was located to the right of the JSA junction in E. urograndis but was not detected in E. grandis . In the remaining eight species, rpl2 consistently appeared to the right of the IR/LSC boundary (JLA). The ndhF gene was located 187 bp to the right of the JLA junction in E. urograndis . It was located 187 bp and 94 bp to the right of the JSB junction in E. urophylla and C. citriodora , respectively, but was absent in the other seven species. To identify sequence variation sites within the three Eucalyptus chloroplast genomes, we calculated nucleotide diversity (Pi) values (range, 0-0.34889; average, 0.21225) (Table S3, Fig. 9 ). We detected five highly divergent regions ( trnK UUU , trnT GGU , psaB-psaA , ndhJ-ndhK , and rpl22-rps19-rpl2 ), with nucleotide diversity exceeding 0.34. The trnT GGU gene exhibited the most significant divergence, the highest Pi value (0.34889), and is located in the LSC region. These highly variable regions could be candidate molecular biomarkers for phylogenetic reconstruction in eucalypt species. 3.7 Phylogenomic analysis We constructed a ML phylogenetic tree using chloroplast genomes of E. urophylla , E. grandis , E. urograndis , and 29 published eucalypt species, with A. thaliana and P. trichocarpa as outgroups (Fig. 10 ). The results revealed that the outgroups ( A. thaliana and P. trichocarpa ) differed significantly from the 32 eucalypt species. Consistent with established taxonomic frameworks, two Angophora species, three Corymbia species, and 29 eucalypt specie formed monophyletic clades with bootstrap support values exceeding 80%. The 29 eucalypt species were classified into six subgenera, with chloroplast genome sequences effectively separating most except Eucalyptus queenslandica . This is consistent with previous studies by Liu et al[ 21 ] and Steane et al[ 22 ], which found a close genetic relationship between the subgenus Eucalyptus and Monocalyptus . this work found that the two eucalypt hybrids ( E. sideroxylon × E. melliodora and E. urograndis ) were genetically similar to their maternal parents, which is consistent with the maternal inheritance pattern outlined in eucalypt chloroplast genomes. Within the subgenus Symphyomyrtus , E. camaldulensis from Exsertaria exhibited closer genetic proximity to species from Transversaria and section Globulus . This finding provides a theoretical foundation for using interspecific hybridization among these three divisions to develop superior hybrids in breeding programs. However, the genetic similarity among species from various sections within the same subgenus differs from the most recent published classification of the Eucalyptus genus [ 23 ]. For example, within the subgenus Eucalyptus , E. regnans , E. elata , and E. baxteri are classified under the section Eucalyptus , yet in this work, E. regnans exhibited a closer genetic affinity to E. marginata (section Longistylus ) than to its section members. This discrepancy may reflect the limitations of chloroplast genomes-inherited maternally and highly conserved-in resolving fine-scale taxonomic relationships [ 24 ]. 4. Discussion The elucidation of chloroplast genomes lays the groundwork for in-depth research into chloroplast functions, intracellular gene transfer, species diversity, plant resource conservation, and genetic breeding [ 25 ]. Our study sequenced, assembled, and annotated the chloroplast genomes of E. urograndis (a widely used hybrid in Chinese eucalypt plantations) and the pure species E. urophylla , the complete chloroplast genome data were collected and compared to the published E. grandis chloroplast genome(NC_014570.1). E. urophylla , E. grandis , and E. urograndis exhibited the typical angiosperm quadripartite structure in their chloroplast genomes, with sizes ranging from 160,137 bp to 160,283 bp. This finding is consistent with the results of Bayly et al., which reported minimal size variation among chloroplast genomes in 39 eucalypt species [ 5 ]. E. urograndis , E. grandis , and E. urophylla chloroplast genomes comprised 127, 127, and 129 genes (Table 1 ), respectively, including 83, 83, and 85 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. These findings are consistent with previously reported eucalypt chloroplast genomes[ 26 – 28 ], demonstrating a conserved chloroplast genome organization across the genus. The hybrid E. urograndis was more closely related to E. urophylla . The psbL gene was detected in the chloroplast genomes of E. urograndis and E. grandis but not in E. urophylla . The psbN gene is retained in E. urophylla and E. grandis , but lost in E. urograndis , most likely due to evolutionary gene loss. This is consistent with the findings of Zhang et al.[ 29 ] who demonstrated a high conservation of psbN in angiosperms. Due to their distinct life-history strategies, parasitic plants may lose or modify certain chloroplast genes (including psbN ) [ 30 ],potentially reflecting ecological adaptations. This work found variations in the psbL , psbN , infA , and pbf1 genes between E. urograndis , E. urophylla and E. grandis . These discrepancies may arise from structural variations in the chloroplast genome or from adaptive evolutionary processes. Alternatively, chloroplast genome of the hybrid could have been inherited from an as-yet uncharacterized parental lineage.These factors may operate independently or in combination, leading to subtle. Future research will focus on expanding the sample set and validating these genomic regions. SSRs, which are highly variable, drive chloroplast genome rearrangement and expansion. this work revealed 68 to 102 SSRs in the three Eucalyptus chloroplast genomes (Fig. 2 ). Notably, the hybrid E. urograndis harbored one additional forward repeat compared to other two eucalypt, with the longest forward repeats varying by 48 and 42 bp (Fig. 3 ), perhaps due to IR contraction during evolution. Mononucleotide SSRs dominated the repeat composition, providing a foundation for interspecies genomic variation studies in eucalypt. Under environmental stress[ 31 ], SSR dynamics may alter gene expression and influence adaptive capacity. Significant differences in SSR abundance and distribution patterns between E. urograndis , E. urophylla , and E. grandis demonstrate their potential as molecular biomarkers for species identification and phylogenetic reconstruction in eucalypt. The chloroplast genomes of the three Eucalyptus species exhibit weak codon usage bias, and their preference pattern aligns with that of most plant chloroplasts, showing a significant tendency to use codons ending with A or U. This finding is consistent with previous studies on Cupressaceae [ 32 ], Theaceae [ 33 ], and Davidia involucrata [ 34 ]. Moreover, a total of 14–16 optimal codons were identified across the three Eucalyptus species, including GCA, CCA, UAA, GUU, ACA, UCA, CUU, GGU, CAA, UGU, AAU, AGU, GAA, ACU, UUG, and AAA, with a predominance of codons ending in A or U (Fig. 7 ). This pattern shows a high degree of similarity to the codon preferences observed in Euphorbia esula [ 35 ] and the Hevea genus [ 36 ]. Studies have demonstrated that codon usage bias can serve as an effective molecular marker for distinguishing closely related species, such as bamboos[ 37 ]. The results of this study provide a strong reference for the accurate classification and identification of eucalypt species, aiding in the recognition of their genetic diversity and characteristic differences. Additionally, leveraging their codon usage patterns to optimize exogenous sequences can effectively enhance gene expression efficiency, offering technical support for subsequent research on genetic engineering improvements and species conservation in the genus Eucalyptus . IR region contraction and expansion in plant chloroplast genomes are common phenomena that determine genome size variation across species[ 38 ]. We observed that E. urograndis had a shorter LSC region than E. urophylla and E. grandis , with LSC variances accounting for the majority of the differences in genome size (Fig. 5 ). It is hypothesized that variations in the LSC region length are primarily responsible for the observed differences in the chloroplast genome size of E. urograndis . Studies indicate that longer IR regions offer additional repetitive sequences[ 39 ], improve genome stability, and lower the chances of structural rearrangements[ 40 ]. Our results demonstrate that E. urograndis has a modest IR expansion compared to other two species of eucalypt, which may confer increased genomic stability and adaptability under environmental stress. The contraction-expansion divergence at IR boundaries in three species of Eucalyptu s likely contributes to chloroplast genome size variance, which may affect overall genome architecture and function. These findings are consistent with observations in Paeonia species, where alterations in the LSC/IRb boundary positions correlate with evolutionary divergence[ 41 ]. Minor boundary variations may thus serve as evolutionary markers for chloroplast genome differentiation[ 42 ]. We discovered five highly divergent regions between the chloroplast genomes of eucalypt hybrid and pure species, predominantly located in the LSC region. The nucleotide diversity of IR regions was significantly lower than the SSC regions (Fig. 7 ), consistent with prior findings[ 43 ]. Zhang et al.[ 44 ] conducted a similar study, comparing entire chloroplast genomes in 24 Myrtales species using MVISTA and variable locus proportions across 114 non-coding regions and 74 coding genes with DnaSP. The study demonstrated that IR regions are less divergent than SC regions in Asteraceae and Myrtales. Variations were also observed in genes near the IR/SSC boundaries, such as rpl22 , rps19 , and rpl2 , consistent with the results of the IRscope boundary analysis. Previous studies have shown that the chloroplast genome can be used for species identification and phylogenetic reconstruction [ 45 – 47 ]. Our findings reveal significant divergence in the chloroplast genomes of Angophora , Corymbia , and Eucalyptus , allowing for the unambiguous distinction between the three genera. However, chloroplast genomes cannot discriminate between species in the subgenera Eucalyptus and Monocalyptus , which share closer genetic proximity. However, chloroplast genomes continue to be effective in elucidating phylogenetic relationships for other subgenera. As the largest Eucalyptus subgenus, Symphyomyrtus exhibits close genetic distances across its sections, allowing for interspecific hybridization between section members to produce high-performance hybrids and clonal variants. For instance, hybrids derived from crosses between sections Latoangulata ( E. urophylla , E. grandis , E. pellita ), Exsertaria ( E. camaldulensis , E. tereticornis ), and Maidenaria ( E. dunnii , E. globulus ) have demonstrated superior heterosis in plantation applications [ 6 ][ 48 – 51 ]. this study also revealed close genetic relationships among the sections Globulus, Exsertaria , and Transversaria . The tree species within these three sections may exhibit higher hybrid compatibility, which could facilitate the successful production of hybrid offspring. However, this inference, based on genomic similarity, requires further functional validation through direct hybridization experiments. 5. Conclusions As previously mentioned, eucalypt species have highly conserved boundaries and similar flanking genes but with slightly different expansion degrees. The only variations detected were in genome size, GC level, repetitive sequences, and IR boundary. In conclusion, we propose five hypervariable regions, trnK UUU , trnT GGU , psaB-psaA , ndhJ-ndhK , and rpl22-rps19-rpl2 , as candidate molecular biomarkers for identifying E. urograndis . A set of 14–16 optimal codons was identified in eucalypt species, 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 eucalypt plants. These findings enrich the chloroplast genomic database for eucalypt species and provide a theoretical foundation for reconstructing their phylogenetic relationships. this study increases our understanding of interspecific diversity in chloroplast genomes between eucalypt species . Abbreviations A Adenine C Complementary F Forward IR Inverted repeats JLA IRA/LSC JLB LSC/IRb JSA SSC/IRA JSB IRB/LSC LSC Large single-copy region ML Maximum likelihood P Palindromic PCGs Protein-coding genes Pi Nucleotide diversity R Reverse SSC Small single-copy region SSRs Simple sequence repeats T thymine. Declarations Acknowledgments Not applicable. Funding This research was funded by the Fundamental Research Funds of CAF (Grant No. CAFYBB2023MB034) and the National Key R&D Program of China (Grant No. 2022YFD2200203 and 2023YFD2201001). Availability of data and materials The datasets supporting the results of this article are available on NCBI, https://www.ncbi.nlm.nih.gov/ (accession number: PV464070 and OL804288). Authors’ contributions GupengYi: Writing-original draft, Methodology, Investigation, Formal analysis, Data curation. Wanhong Lu and Yan Lin: Software, Methodology, Investigation. Jianzhong Luo and Guo Liu: Writing-review and editing, Supervision, Funding acquisition, Conceptualization. Ying Cheng and Zhijiao Song: Visualization, Validation, Methodology, Investigation, Conceptualization. Supplementary information Table S1. 34 species and accessions used for phylogenetic analysis; Table S2 .SSR information in the chloroplast genomes of tewlve eucalypt species; Table S3. Sliding window test of Pi in the Hybrid tail E.urograndis , E.urophylla and E.grandis chloroplast genomes. Ethics approval and consent to participate The appropriate permissions were obtained for all materials used in this work. We complied with all relevant institutional, national, and international guidelines and legislation. The Guangxi State-owned Dongmen Forest Farm (Chongzuo, Guangxi Zhuang Autonomous Region, China) and Leizhou Forestry Bureau (Zhanjiang, Guangdong Province, China) gave permission for the leaves of E. urograndis and E. urophylla to be collected for the study. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Liu T, Xie YJ. Analysis and prospects of the rapid development of Eucalyptus plantations in China. Eucalypt Sci Technol. 2020;37(4):38–47. Maheswari P, Kunhikannan C, Yasodha R. Chloroplast genome analysis of Angiosperms and phylogenetic relationships among Lamiaceae members with particular reference to teak ( Tectona grandis L.f). J Biosci. 2021;46:43. https://doi.org/10.1007/s12038-021-00166-2 . Zhang J, Yuan Q, Wei Y, Liu H, Zheng X, Wang Y, Liu H. Evolutionary analysis of chloroplast genomes of Sect.Isika (Lonicera)species, Nematotinus plants. Chin Tradit Herb Drugs. 2024;55(9):3085–97. Yan A, Zhu D. Application of chloroplast genome in phylogenetics and genetic engineering. Chin J Cell Biology. 2004;26:153–6. Bayly MJ, Rigault P, Spokevicius A, Ladiges PY, Ades PK, Anderson C, Bossinger G, Merchant A, Udovicic F, Woodrow IE, Tibbits J. Chloroplast genome analysis of Australian eucalypts - Eucalyptus , Corymbia , Angophora , Allosyncarpia and Stockwellia (Myrtaceae). Mol Phylogenet Evol. 2013;69(3):704–16. https://doi.org/10.1016/j.ympev.2013.07.006 . Guo Y, Yao B, Yuan M, Li J. The complete chloroplast genome and phylogenetic analysis of Astragalus scaberrimus Bunge 1833. Mitochondrial DNA Part B. 2021;6(12):3364–6. https://doi.org/10.1080/23802359.2021.1997108 . Shi L, Chen H, Jiang M, Wang L, Wu X, Huang L, Liu C. CPGAVAS2, an integrated plastome sequence annotator and analyzer. Nucleic Acids Res. 2019;47(W1):W65–73. https://doi.org/10.1093/nar/gkz345 . Zhu B, Gao Z, Qian F, Yang X, Lv X, Cai M. The complete chloroplast genome of a purple Ethiopian rape (Brassica carinata: Brassicaceae) from Guizhou Province, China and its phylogenetic analysis. Mitochondrial DNA Part B. 2021;6:1821–3. https://doi.org/10.1080/23802359.2021.1926365 . Wang W, Yu H, Wang J, Lei W, Gao J, Qiu X, Wang J. The complete chloroplast genome sequences of the medicinal plant Forsythia suspensa (Oleaceae). Int J Mol Sci. 2017;18(11):2288. https://doi.org/10.3390/ijms18112288 . Kurtz S, Choudhuri JV, Ohlebusch E, Schleiermacher C, Stoye J, Giegerich R. REPuter: the manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res. 2001;29(22):4633–42. https://doi.org/10.1093/nar/29.22.4633 . Rice P, Longden I, Bleasby A. EMBOSS: the European molecular biology open software suite. Trends Genet. 2000;16(6):276–7. Wu P, Xiao W, Luo Y, Xiong Z, Chen X, He J, Sha A, Gui M, Li Q. Comprehensive analysis of codon bias in 13 Ganoderm a mitochondrial genomes. Fron-tiersin Microbiol. 2023;14:1170790. https://doi.org/10.3389/fmicb.2023.1170790 . Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28(12):1647–9. https://doi.org/10.1093/bioinformatics/bts199 . Amiryousefi A, Hyvönen J, Poczai P. IRscope: an online program to visualize the junction sites of chloroplast genomes. Bioinformatics. 2018;34(17):3030–1. https://doi.org/10.1093/bioinformatics/bty220 . Katoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2017;20(4):1160–6. https://doi.org/10.1093/bib/bbx108 . Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, Sánchez-Gracia A. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol. 2017;34:3299–302. https://doi.org/10.1093/molbev/msx248 . Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25(15):1972–3. https://doi.org/10.1093/bioinformatics/btp348 . Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32(1):268–74. https://doi.org/10.1093/molbev/msu300 . Kalyaanamoorthy S, Minh BQ, Wong TKF, Von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods. 2017;14(6):587–9. https://doi.org/10.1038/nmeth.4285 . Minh BQ, Nguyen MAT, Von Haeseler A. Ultrafast approximation for phylogenetic bootstrap. Mol Biol Evol. 2013;30(5):1188–95. https://doi.org/10.1093/molbev/mst024 . Liu G, Arnold RJ, Xie Y, Wu Z. Genetic relationships among 40 species of Eucalyptus based on simple sequence repeat markers. J Trop For Sci. 2018;30(3):402–14. https://www.jstor.org/stable/26512525 . Steane DA, Nicolle D, McKinnon GE, Vaillancourt RE, Potts BM. Higher-level relationships among the eucalypts are resolved by ITS-sequence data. Aust Syst Bot. 2002;15(1):49–62. https://doi.org/10.1071/SB00039 . Nicolle D. Classification of the eucalypts, genus Eucalyptus.Version 7[EB/OL].(2025-4-16). http://www.dn.com.au/Classification-Of-The-Eucalypts.pdf Zhang L, Morales-Briones DF, Li Y, Zhang G, Zhang T, Huang CH, Ma H. Phylogenomics insights into gene evolution, rapid species diversification, and morphological innovation of the apple tribe (Maleae, Rosaceae). New Phytol. 2023;240(5):2102–20. https://doi.org/10.1111/nph.19175 . Liu C, Tang L, Han L. Characterization of the chloroplast genome of Lindera setchuenensis and phylogenetics of the genus Lindera. Scientia Silvae Sinicae. 2021;57(7):167–74. 10.11707/j.1001-7488.20211217 . http://www.linyekexue.net/EN/ . Wang WW, Schalamun M, Morales-Suarez A, Kainer D, Schwessinger B, Lanfear R. Assembly of chloroplast genomes with long- and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case. BMC Genomics. 2018;19(1):977. https://doi.org/10.1186/s12864-018-5348-8 . Gao Y, Zhang J, Tang H, Liu N, Li G, Yue D. The characteristics of the complete chloroplast genome for Eucalyptus robusta (Myrtaceae). Mitochondrial DNA Part B. 2021;6(12):3517–8. https://doi.org/10.1080/23802359.2021.2005491 . Steane DA. Complete nucleotide sequence of the chloroplast genome from the Tasmanian blue gum, Eucalyptus globulus (Myrtaceae). DNA Res. 2005;12(3):215–20. https://doi.org/10.1093/dnares/dsi006 . Zhang H, Huang T, Zhou Q, Sheng Q, Zhu Z. Complete chloroplast genomes and phylogenetic relationships of Bougainvillea spectabilis and Bougainvillea glabra (Nyctaginaceae). Int J Mol Sci. 2024;24(16):13044. https://doi.org/10.3390/ijms241713044 . Gao L, Su Y, Wang T. Plastid genome sequencing, comparative genomics, and phylogenomics: Current status and prospects. J Syst Evol. 2010;48(1):77–93. https://doi.org/10.1111/j.1759-6831.2010.00071.x . Daniell H, Lin CS, Yu M, Chang WJ. Chloroplast genomes: Diversity, evolution, and applications in genetic engineering. Genome Biol. 2016;17(1):134. https://doi.org/10.1186/s13059-016-1004-2 . Huang SQ, Zhang QG, Ye ZL, Qin XH, Wang QS, Lin XQ, Zheng ZW, Lin WF, Zou XX. Codon bias analysis of chloroplast genomes of 5 Cupressaceae plants. J Fujian Agric Forestry Univ (Natural Sci Edition). 2024;53(2):214–20. Wang Z, Cai Q, Wang Y, Li M, Wang C, Wang Z, Jiao C, Xu C, Wang H, Zhang Z. Comparative analysis of codon bias in the chloroplast genomes of theaceae species. Front Genet. 2022;13:824610. https://doi.org/10.3389/fgene.2022.824610 . Luo YJ, Wang R, Zhao RF, Lu XX, Yin GK, Deng ZJ. Analysis of synonymous codon usage bias in the chloroplast genome of Davidia involucrata. J Beijing Forestry Univ. 2024;46(3):8–16. Wang Z, Xu B, Li B, Zhou Q, Wang G, Jiang X, Wang C, Xu Z. Comparative analysis of codon usage patterns in chloroplast genomes of six Euphorbiaceae species. 2020;8:e8251. https://doi.org/10.7717/peerj.8251 Yang Y, Liu X, He L, Li Z, Yuan B, Fang F, Wang X. Comparative chloroplast genomics and codon usage bias analysis in Hevea Genus. Genes. 2025;16(2):201. https://doi.org/10.3390/genes16020201 . Kelchner SA, Group BP. Higher level phylogenetic relationships within the bamboos (Poaceae: Bambusoideae) based on five plastid markers. Mol Phylogenet Evol. 2013;67(2):404–13. Huang H, Shi C, Liu Y, Mao S, Gao L. Thirteen Camellia chloroplast genome sequences determined by high-throughput sequencing: Genome structure and phylogenetic relationships. BMC Evol Biol. 2014;14(1):151. https://doi.org/10.1186/1471-2148-14-151 . Jiang Z, Chen H, Bao H, Dai Y. Chloroplast genome characteristics and molecular marker development of Pennisetum. J Zhejiang A&F Univ. 2025;42(2):365–72. Wang Y, Wang S, Liu Y, Yuan Q, Sun J, Guo L. Chloroplast genome variation and phylogenetic relationships of Atractylodes species. BMC Genomics. 2021;22(1):103. https://doi.org/10.1186/s12864-021-07394-8 . Zhou X, Zhang K, Peng Z, Sun S, Ya H, Zhang Y, Chen Y. Comparative Analysis of Chloroplast Genome Characteristics between Paeonia jishanensis and Other Five Species of Paeonia. CABI Digit Libr. 2020;56(4):82–8. Li Q, Yan N, Song Q, Guo J. Complete chloroplast genome sequence and characteristics analysis of Morus multicaulis. Chin Bull Bot. 2018;l53(1):94–103. 10.11983/CBB16247 . https://www.chinbullbotany.com/EN/ . Sun J, Wang Y, Garran TA, Qiao P, Wang M, Yuan Q, Guo L, Huang L. Heterogeneous genetic diversity estimation of a promising domestication medicinal motherwort Leonurus cardiaca based on chloroplast genome resources. Front Genet. 2021;12:721022. https://doi.org/10.3389/fgene.2021.721022 . Zhang X, Landis JB, Wang H, Zhu Z, Wang H. Comparative analysis of chloroplast genome structure and molecular dating in Myrtales. BMC Plant Biol. 2021;21(1):219. https://doi.org/10.1186/s12870-021-02985-9 . Li C, Lu J, Li Y, Zhu J, Zhang L. Comparative morphology of the leaf epidermis in Lobelia (Lobelioideae) from China. Microsc Res Tech. 2017;80(7):763–78. https://doi.org/10.1002/jemt.22862 . Ding Y, Bi G, Hu S, Zhou MY, Li H, Xia Z. Chloroplast genome characteristics and phylogenetic analysis of different flower color variation types in safflower ( Carthamus tinctorius ). Chin Traditional Herb Drugs. 2023;54(1):262–71. Yu B, Sun Y, Liu X, Huang L, Xu Y, Zhao C. Complete chloroplast genome sequence and phylogenetic analysis of Camellia fraterna. Mitochondrial DNA Part B. 2020;5(3):3840–2. https://doi.org/10.1080/23802359.2020.1841576 . Li G, Xu J, Wang W, Wu S, Zhu Y, Wang YS, Pan J, Guo HY, Shi TY. Study on heterosis estimation and genetic analysis of Eucalyptus hybrids in cold area. J Nanjing Forestry Univ (Natural Sci Edition). 2017;41(4):55–63. http://njlydxxb.periodicals.net.cn/default.html . Mo JY, Lan J, Luo JZ, Wu MFn, Peng ZB. Growth characteristics of hybrids between Eucalyptus urophylla and Section Exsertaria species. GuangXi Plant. 2021;41(4):631–9. Luo JZ, Xie YJ, Cao JG, Lu WH, Ren SQ. Genetic variation in growth and wind resistance of two-year-old Eucalyptus hybrids. Acta Prataculturae Sinica. 2009;18(3):91–7. Weng QJ, Lai QX, Li FG, Zhou CP, Li JW, Li M, Gan SM. Genetic analysis on early growth and cold tolerance of Eucalyptus urophylla × E. dunnii hybrids. J Nanjing Forestry Univ (Natural Sci Edition). 2015;39(5):33–8. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTables.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9082114","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608453742,"identity":"2a7b6d13-e79d-48ca-9ee3-c94b8abcc9e9","order_by":0,"name":"Gupeng Yi","email":"","orcid":"","institution":"Chinese Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Gupeng","middleName":"","lastName":"Yi","suffix":""},{"id":608453743,"identity":"9b06102f-68dc-4a76-91d7-cc95401e00b6","order_by":1,"name":"Guo Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYBACPmYog5+Z+fADorSwwbRItrOlGRCnBcYwOM+jIEGcFnYe49c8FXfsjQ/zMBgw1NhEE+EwHjNrnjPPmM0O8x54wHAsLbeBGC3GvG2H2cwO8yUYMDYcJl4Lj3Ezj4EEsVqMHwO1SBgwE6+FrYxxzplnBhKHgYGcQIxf+PkPb/7wBhhi/P2HDz/4UGNDWAvIImB0HIAwE4hQDgLMH+BaRsEoGAWjYBRgAwCgCjUIhppDyAAAAABJRU5ErkJggg==","orcid":"","institution":"Chinese Academy of Forestry","correspondingAuthor":true,"prefix":"","firstName":"Guo","middleName":"","lastName":"Liu","suffix":""},{"id":608453744,"identity":"6d86a229-ae1c-41cd-875e-4f401edbc4d1","order_by":2,"name":"Jianzhong Luo","email":"","orcid":"","institution":"Chinese Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Jianzhong","middleName":"","lastName":"Luo","suffix":""},{"id":608453745,"identity":"9216a621-bf21-4f0f-b4ae-aa24d41eedef","order_by":3,"name":"Wanhong Lu","email":"","orcid":"","institution":"Chinese Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Wanhong","middleName":"","lastName":"Lu","suffix":""},{"id":608453746,"identity":"9985d201-1f4d-4277-93b5-b3d7374c0ac6","order_by":4,"name":"Yan Lin","email":"","orcid":"","institution":"Chinese Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Lin","suffix":""},{"id":608453747,"identity":"4fabbdec-074b-41db-8227-ae079b2ff7a8","order_by":5,"name":"Ying Cheng","email":"","orcid":"","institution":"Chinese Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Cheng","suffix":""},{"id":608453748,"identity":"4b4ce40f-fe84-48c6-85ef-4ab57283b5e8","order_by":6,"name":"Zhijiao Song","email":"","orcid":"","institution":"Baoshan University","correspondingAuthor":false,"prefix":"","firstName":"Zhijiao","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2026-03-10 09:38:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9082114/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9082114/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104963000,"identity":"30e248b6-6f77-4750-b14b-fac45d1fc4d7","added_by":"auto","created_at":"2026-03-19 09:20:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":733168,"visible":true,"origin":"","legend":"\u003cp\u003eChloroplastic genomes structure of \u003cem\u003eE. urophylla\u003c/em\u003e,\u003cem\u003e E. grandis\u003c/em\u003e and \u003cem\u003eE. urograndis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/cd8f4d43368b24bad18cec53.png"},{"id":105035323,"identity":"a8cf16f7-f316-42e8-9700-ad2570ab9934","added_by":"auto","created_at":"2026-03-20 07:25:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":174216,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of SSRs and repeated sequences in the chloroplast genomes of three \u003cem\u003eEucalyptus\u003c/em\u003especies. Notes: Mono, Mononucleotide SSRs; Di, Dinucleotide SSRs; Tri, Trinucleotide SSRs; Tetre, Tetranucleotide SSRs; LSC, Large single-copy region; IR, Inverted repeats; SSC, Small single-copy region.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/b47e1a4361c0f0ad56afb343.png"},{"id":105034955,"identity":"ae78d17c-7f53-4d7e-91e4-c2b22fc3c1ae","added_by":"auto","created_at":"2026-03-20 07:24:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":162111,"visible":true,"origin":"","legend":"\u003cp\u003eScattered repeating sequences in three \u003cem\u003eEucalyptus\u003c/em\u003especies. Notes: F, Forward sequence; P, Palindromic sequence.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/8cfe2fb6bdfbc07c57c65f71.png"},{"id":105035016,"identity":"5e58b047-9837-4d9b-9cd6-38edc915e1b5","added_by":"auto","created_at":"2026-03-20 07:25:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":244545,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Analysis of SSRs and repeated sequences in the chloroplast genomes of 12 eucalypt species.\u003cstrong\u003e B. \u003c/strong\u003eScattered repeating sequences in 12 eucalypt species. Notes: F, Forward sequence; P, Palindromic sequence.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/f699955e90dc099dca93fce4.png"},{"id":104963004,"identity":"172e20d6-5154-44b9-84cd-386cf44f1fff","added_by":"auto","created_at":"2026-03-19 09:20:33","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":123825,"visible":true,"origin":"","legend":"\u003cp\u003eUsage of codon preferences in three \u003cem\u003eEucalyptus\u003c/em\u003e species. Notes: A, \u003cem\u003eE. urograndis\u003c/em\u003e; B, \u003cem\u003eE. urophylla\u003c/em\u003e; C, \u003cem\u003eE. grandis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/fae837f4f2c86066e6800272.jpeg"},{"id":104963009,"identity":"4ed64c36-171e-4278-a7a6-93c11ba1384c","added_by":"auto","created_at":"2026-03-19 09:20:34","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59845,"visible":true,"origin":"","legend":"\u003cp\u003eRanking of ΔRSCU values for each amino acid in the three \u003cem\u003eEucalyptus\u003c/em\u003e species. Notes: A, \u003cem\u003eE. urograndis\u003c/em\u003e; B, \u003cem\u003eE. urophylla\u003c/em\u003e; C, \u003cem\u003eE. grandis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/dacfc13ec722f5fd0dfebbd5.jpeg"},{"id":105034909,"identity":"b32aa914-5e37-468c-9ed7-6fe208f0378d","added_by":"auto","created_at":"2026-03-20 07:24:46","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":52877,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of optimal codons for each amino acid in the three \u003cem\u003eEucalyptus\u003c/em\u003e species. Note: A, \u003cem\u003eE. urograndis\u003c/em\u003e; B, \u003cem\u003eE. urophylla\u003c/em\u003e; C, \u003cem\u003eE. grandis\u003c/em\u003e. The horizontal axis indicates the low-expression library preference region, and the vertical axis indicates the high-expression library preference region. Yellow dots represent optimal codons, the red dashed line indicates the threshold of ΔRSCU \u0026gt; 0.08, and the blue dashed line indicates RSCU \u0026gt; 1.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/aa40dc665575a8eb1ddc7e24.jpeg"},{"id":104963007,"identity":"184fceb9-3f6f-4636-8767-0a2d392fae30","added_by":"auto","created_at":"2026-03-19 09:20:33","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":148565,"visible":true,"origin":"","legend":"\u003cp\u003eDivergence in boundary regions of chloroplast genomes among 10 eucalypt species. JLB,LSC/IRb; JSB, IRb/SSC; JSA, SSC/IRa; JLA, IRa/LSC. The light blue/yellow/light green regions correspond to the LSC/IRa and IRb/SSC regions, respectively.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/494be91b88b55f0e142fbb86.jpeg"},{"id":104963012,"identity":"b3bbc302-e354-4250-a794-ce560ca2db2c","added_by":"auto","created_at":"2026-03-19 09:20:40","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":365754,"visible":true,"origin":"","legend":"\u003cp\u003eSliding window test of Pi in the Hybrid tail \u003cem\u003eE.urograndis\u003c/em\u003e, \u003cem\u003eE.urophylla\u003c/em\u003e and \u003cem\u003eE.grandis\u003c/em\u003echloroplast genomes.Window length: 600 bp; step size: 200 bp. X-axis: the position of the midpoint of a window. Y-axis:nucleotide diversity of each window. LSC, IRA, SSC, and IRB represent the four regions of chloroplast genome structure.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/08a524f1b6c47143d0a0753b.png"},{"id":105035367,"identity":"28ad0718-c416-47c6-af18-ef6cee69ed15","added_by":"auto","created_at":"2026-03-20 07:25:57","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":734648,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of 32 eucalypt species and two outgroup species based on the complete chloroplast genome data. Numbers near the nodes are bootstrap percentages.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/f4f6636d6f30ae67bec54c29.png"},{"id":106923160,"identity":"01802377-32aa-42c9-9584-2824daf22f27","added_by":"auto","created_at":"2026-04-14 20:39:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4009720,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/84bc3928-3b29-4e04-bfba-9eca17c1ee39.pdf"},{"id":104963005,"identity":"37d85d44-54b5-442b-afa8-dd81fd933c7b","added_by":"auto","created_at":"2026-03-19 09:20:33","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":40402,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9082114/v1/993fc1b6880f0bd6ccadbe55.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Analysis of Chloroplast Genome Structure and Phylogenetic Relationships Between a Typical Eucalypt Hybrid and Two Purebred Species","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. The \u003cem\u003eE. urograndis\u003c/em\u003e (160,201 bp) had an intermediate chloroplast genome size between that of \u003cem\u003eE. urophylla\u003c/em\u003e (160,283 bp) and \u003cem\u003eE. grandis\u003c/em\u003e (160,137 bp).\u003c/p\u003e\n\u003cp\u003e2. Analysis of SSRs in the chloroplast genomes of 12 eucalypt species revealed a variation ranging from 68 SSRs in \u003cem\u003eE. grandis\u003c/em\u003e to 115 in \u003cem\u003eC. citriodora\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. The \u003cem\u003epsbL\u003c/em\u003e, \u003cem\u003epsbN\u003c/em\u003e, \u003cem\u003einfA\u003c/em\u003e, and \u003cem\u003epbf1\u003c/em\u003e genes show differences among the three eucalypt species.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4. A set of 14-16 optimal codons was identified in eucalypt species, including GCA, CCA, UAA, GUU, ACA, UCA, CUU, GGU, CAA, UGU, AAU, AGU, GAA, ACU, UUG, and AAA, with a predominance of codons ending in A or U. This finding provides a key foundation for optimizing exogenous sequences based on codon usage patterns.\u003c/p\u003e\n\u003cp\u003e5.\u0026nbsp;Consistent with established taxonomic frameworks, two \u003cem\u003eAngophora\u003c/em\u003e species, three\u003cem\u003e\u0026nbsp;Corymbia\u003c/em\u003e species, and 29\u0026nbsp;eucalypt\u003cem\u003e\u0026nbsp;\u003c/em\u003especie formed monophyletic clades with bootstrap support values exceeding 80%.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eEucalypt, which includes the genera \u003cem\u003eEucalyptus\u003c/em\u003e, \u003cem\u003eAngophora\u003c/em\u003e, and \u003cem\u003eCorymbia\u003c/em\u003e in the Myrtaceae family, is a tree species known for its rapid growth, short rotation period, drought resistance, and broad adaptability. It is widely utilized in pulp and papermaking, plywood production, and solid wood processing, making it a key timber species in southern China\u0026rsquo;s rapidly expanding plantations. According to the latest data, China\u0026rsquo;s eucalypt plantation area has increased to 5.93\u0026nbsp;million hectares, with an annual wood production of about 40\u0026nbsp;million cubic meters[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]accounting for more than one-third of the country\u0026rsquo;s total timber output. eucalypt plantations are critical for ensuring China\u0026rsquo;s wood security, boosting economic growth, and promoting rural revitalization. \u003cem\u003eEucalyptus urophylla\u003c/em\u003e \u0026times; \u003cem\u003eEucalyptus grandis\u003c/em\u003e (\u003cem\u003eE. urograndis\u003c/em\u003e), a typical eucalypt hybrid, is produced by crossing \u003cem\u003eE. grandis\u003c/em\u003e (father) with \u003cem\u003eE. urophylla\u003c/em\u003e (mother). Initially created in the late 1980s at Guangxi Dongmen Forest Farm, it has since emerged as a key artificial forest species nationwide, owing to its robust adaptability, rapid growth, short growth cycle, ease of vegetative propagation, and favorable trunk morphology.\u003c/p\u003e \u003cp\u003eThe chloroplast genomes of higher plants exhibit a highly conserved quadripartite structure, in which two inverted repeat sequences (IR) partition the entire circular chloroplast genome into a large single-copy region (LSC) and a small single-copy region (SSC). The chloroplast genome (cpDNA) [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], characterized by its structural conservation, low recombination rate, and typical maternal inheritance pattern, serves as an ideal model for investigating species evolution and hybridization. Although the NCBI database currently includes cpDNA data for 73 eucalypt species (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/cgir/genome?term=eucalyptus\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/cgir/genome?term=eucalyptus\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on March 28 2025) and phylogenetic studies have confirmed a deep divergence between \u003cem\u003eEucalyptus\u003c/em\u003e and \u003cem\u003eCorymbia\u003c/em\u003e lineages[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], research in this area remains limited compared to other plant systems. Notably, Research on specific nuclear genes or functional traits of important economic eucalypt species such as \u003cem\u003eEucalyptus grandis\u003c/em\u003e and \u003cem\u003eEucalyptus regia\u003c/em\u003e has been extensively conducted. However, systematic comparative genomics and evolutionary analysis data at the complete chloroplast genome level remain significantly lacking. Therefore, this work presents the first comparative framework for analyzing the chloroplast genomes of the hybrid species \u003cem\u003eE. urograndis\u003c/em\u003e and the pure species \u003cem\u003eE. urophylla, E. grandis\u003c/em\u003e. By conducting a comprehensive analysis of genome architecture, simple sequence repeats (SSRs),synonymous codon usage preference, optimal codon, inverted repeat (IR) boundary variations, nucleotide diversity, and phylogenetic relationships among \u003cem\u003eE. urophylla\u003c/em\u003e, \u003cem\u003eE. grandis\u003c/em\u003e and \u003cem\u003eE. urograndis\u003c/em\u003e, this research aims to clarify the distinctive characteristics and evolutionary patterns of their chloroplast genomes. The results of this work will serve as a scientific basis for the conservation and sustainable utilization of these genetic resources. Furthermore, it provides a theoretical foundation for future studies, including organelle genome sequencing of other eucalypt species, in-depth exploration of organelle genome variations and evolutionary relationships, and the development of molecular marker-assisted breeding strategies.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant materials\u003c/h2\u003e \u003cp\u003eWe obtained improved varieties of \u003cem\u003eE. urograndis\u003c/em\u003e (No. Gui S-SC-EUG-001-2009) from the Guangxi State-owned Dongmen Forest Farm (107\u0026deg;51\u0026prime;52\u0026Prime;E, 22\u0026deg;19\u0026prime;53\u0026Prime;N) and \u003cem\u003eE. urophylla\u003c/em\u003e from the Jijia Town, Leizhou City, Guangdong Province (109\u0026deg;47\u0026prime;44\u0026Prime;E, 20\u0026deg;56\u0026prime;15\u0026Prime;N). For both species, healthy mature leaves were collected from the southern and northern sides of three trees exhibiting uniform growth and favorable conditions. These leaves were combined into a single composite sample for analysis. The same sampling protocol was applied to \u003cem\u003eE. urograndis\u003c/em\u003e (3-year-old trees) and \u003cem\u003eE. urophylla\u003c/em\u003e (6-year-old trees). The collected leaf samples were submerged in liquid nitrogen immediately for rapid freezing and stored at -80\u0026deg;C for future analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 DNA extraction, library construction, and sequencing\u003c/h2\u003e \u003cp\u003eTotal DNA from leaf samples of the two eucalypt species was extracted using the DNAsecure Plant kit (TIANGEN, Beijing, China). Following a quality assessment of the samples, we constructed the library using the MGIEasy Fast Enzyme-Cutting Library Preparation procedures. The NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA) was used to determine DNA concentrations and quality. Trimmomatic software[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] was used to control the raw reads received from sequencing using the default parameter setting. This was followed by the filtration of the adaptor and low-quality sequences to obtain high-quality clean reads.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Chloroplast genome splicing, annotation, and physical mapping\u003c/h2\u003e \u003cp\u003eThe splicing process was divided into three stages: initial splicing, contig screening, and extension, filling, and re-splicing. The limited data (2 \u0026times; 150 bp, 10 G) prompted the use of GetOrganelle v1.7.7.1 software, which demonstrated superior performance for circular genome splicing. The chloroplast genomes were annotated using CPGAVAS2 software[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and the physical maps of the chloroplast genomes of \u003cem\u003eE. urograndis\u003c/em\u003e and \u003cem\u003eE. urophylla\u003c/em\u003e were created by Organellar Genome DRAW online software[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Repeat sequence analysis\u003c/h2\u003e \u003cp\u003eThe SSRs of the chloroplast genome were analyzed by MISA software[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] using the following parameters: mononucleotides\u0026thinsp;\u0026ge;\u0026thinsp;10, dinucleotides\u0026thinsp;\u0026ge;\u0026thinsp;5, trinucleotides\u0026thinsp;\u0026ge;\u0026thinsp;4, tetranucleotides\u0026thinsp;\u0026ge;\u0026thinsp;3, pentanucleotides\u0026thinsp;\u0026ge;\u0026thinsp;3, and hexanucleotides\u0026thinsp;\u0026ge;\u0026thinsp;3. The minimum spacing between two SSRs was fixed at 100 bp. The dispersed repeats in the chloroplast genome were detected using REPuter software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bibiserv.techfak.uni-bielefeld.de/reputer\u003c/span\u003e\u003cspan address=\"http://bibiserv.techfak.uni-bielefeld.de/reputer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Forward (F), palindrome (P), reverse (R), and complementary long repeats (C) were detected with a repeat length of more than 30 bp and a Hamming distance of 3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Relative Usage of Synonymous Codons\u003c/h2\u003e \u003cp\u003eTo improve the accuracy of subsequent codon preference analysis, the codon usage of three chloroplast protein-coding genes from eucalypt species was screened using MEGA 7.0.26 software based on the following criteria: length\u0026thinsp;\u0026gt;\u0026thinsp;300 bp, start codon ATG, stop codons TGA, TAA, or TAG, and nucleotide count as a multiple of three. Ultimately, 45, 48, and 44 CDS sequences were selected for subsequent data analysis, respectively. Using the online platform EMBOSS Explorer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.nl/emboss-explorer/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.nl/emboss-explorer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the nucleotide composition percentages of the three eucalypt chloroplast genomes were calculated[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], along with the GC content at the first, second, and third codon positions (denoted as GC1, GC2, and GC3, respectively). Meanwhile, using Rstudio software, genes were ranked from high to low based on their ENC values, and the top and bottom 10% of genes were selected as the high-expression and low-expression libraries, respectively. Codons with ΔRSCU\u0026thinsp;\u0026gt;\u0026thinsp;0.08 within each library were then identified as high-expression codons based on the genes' RSCU values. Codons meeting both ΔRSCU\u0026thinsp;\u0026gt;\u0026thinsp;0.08 and RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1 were defined as optimal codons[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Finally, the corresponding results were visualized through plotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Genome comparison and analysis, IR border and divergence analyses\u003c/h2\u003e \u003cp\u003eGeneious Prime v2021.1.1[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] was used to determine the chloroplast genome length, GC level, and LSC, SSC, and IRs. It was also used to estimate the number and classification of genes. Chloroplast genomes from the 10 eucalypt species were examined using the IRscope online software[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] to identify shrinkage or expansion at the boundaries. MAFFT v.7.450 was used to carry out multiple sequence alignment of \u003cem\u003eE. urograndis\u003c/em\u003e and its parental chloroplast genome sequences [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. DNAPARS v.6.0 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]was used to calculate nucleotide polymorphisms (π values) and identify mutation genes and hotspots. The step size and search window length were set at 200 bp and 600 bp, respectively, and the results were plotted in Excel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Phylogenetic tree analysis\u003c/h2\u003e \u003cp\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e lists species and accessions used in phylogenetic analysis. Thirty-four species were selected to represent major clades, including main sections and series with \u003cem\u003eEucalyptus\u003c/em\u003e and related \u003cem\u003etaxa\u003c/em\u003e. The samples contained 18 \u003cem\u003eEucalyptus\u003c/em\u003e species belonging to the subgenus \u003cem\u003eSymphyomyrtus\u003c/em\u003e, representing nine sections within that subgenus, five species of subgenus \u003cem\u003eEucalyptus\u003c/em\u003e that represented four sections, one species of subgenus \u003cem\u003eAlveolata\u003c/em\u003e (\u003cem\u003eE. microcorys\u003c/em\u003e), one species of subgenus Cuboidea (\u003cem\u003eE. tenuipes\u003c/em\u003e), one species of subgenus \u003cem\u003eEudesmia\u003c/em\u003e (\u003cem\u003eE. erythrocorys\u003c/em\u003e), and one species of subgenus \u003cem\u003eIdiogenes\u003c/em\u003e (\u003cem\u003eE. cloeziana\u003c/em\u003e). In addition, we also included two \u003cem\u003eAngophora\u003c/em\u003e species, three \u003cem\u003eCorymbia\u003c/em\u003e species, and three outgroup species, including \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003ePopulus trichocarpa\u003c/em\u003e. We utilized MAFFT v7.313 to align the complete chloroplast genome sequences, followed by trimAI to remove indels[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] Maximum likelihood (ML) was used to assess the phylogenetic relationships. Meanwhile, ML reconstruction was performed with IQ-TREE[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] using ModelFinder\u0026rsquo;s best-fit nucleotide substitution model[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Ten thousand ultrafast bootstrap replications were used to evaluate branch support for the ML tree [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Structural and functional characteristics of three Eucalyptus chloroplast genomes\u003c/h2\u003e \u003cp\u003eThe chloroplast genomes of \u003cem\u003eE. urophylla\u003c/em\u003e and \u003cem\u003eE. urograndis\u003c/em\u003e were sequenced and assembled, whereas the chloroplast genome of \u003cem\u003eE. grandis\u003c/em\u003e was retrieved from NCBI (Reference sequence: NC_014570.1). The assembled genomes of the three species were similar in size, ranging from 160,137 bp for \u003cem\u003eE. grandis\u003c/em\u003e to 161,283 bp for \u003cem\u003eE. urophylla\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll three chloroplast genomes were AT-rich (GC level\u0026thinsp;=\u0026thinsp;36.86\u0026ndash;36.89%, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and had a quadripartite structure similar to many angiosperms, with two inverted repeats (IR) region copies (52780\u0026ndash;52798 bp), one large single-copy region (LSC, 88876\u0026ndash;88987 bp), and one small single-copy region (SSC, 18481\u0026ndash;18498 bp) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The GC level in each region differed between the three chloroplast genomes with 42.7%, 30.4%-30.5%, and 34.7%-34.8% variance in IR, SSC, and LSC regions, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe complete chloroplast genome features for three \u003cem\u003eEucalyptus\u003c/em\u003e species\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSize/bp\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLSC(bp)\u003c/p\u003e \u003cp\u003e/GC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSSC(bp)\u003c/p\u003e \u003cp\u003e/GC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIRs(bp)\u003c/p\u003e \u003cp\u003e/GC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTotal GC%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePCGs/(bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003erRNA\u003c/p\u003e \u003cp\u003e/(bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003etRNA\u003c/p\u003e \u003cp\u003e/(bp)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. urograndis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e160201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e36.86%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e83/78016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8/9056\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e36/2736\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. grandis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e160137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e36.89%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e83/91066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8/9050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e36/2745\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. urophylla\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e160283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e36.86%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e85/85394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8/9050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e36/2736\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003eNotes:LSC, large single-copy region; SSC, small single-copy region; IRs, two inverted repeats; PCGs, Protein-coding genes.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Dispersed repeat and SSR analysis\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eE. urophylla\u003c/em\u003e chloroplast genome included 102 qualified SSRs, comprising 79 mononucleotide SSRs, 4 dinucleotide SSRs, 4 trinucleotide SSRs, and 15 tetranucleotide SSRs (Table \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS2\u003c/span\u003e). The chloroplast genome of \u003cem\u003eE. grandis\u003c/em\u003e exhibited fewer SSRs (68), which included 48 mononucleotide SSRs, 2 dinucleotide SSRs, 4 trinucleotide SSRs, and 14 tetranucleotide SSRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The hybrid \u003cem\u003eE. urograndis\u003c/em\u003e exhibited identical SSR types and numbers as \u003cem\u003eE. urophylla\u003c/em\u003e. SSRs were predominantly distributed in the LSC region of all three species, with fewer in the IRs and SSC regions. Mononucleotide SSRs accounted for the highest proportion, while dinucleotide, trinucleotide, and tetranucleotide SSRs were relatively less common. Furthermore, SSRs in the chloroplast genomes of all three \u003cem\u003eEucalyptus\u003c/em\u003e species included predominantly adenine (A) and thymine (T). \u003cem\u003eE. urophylla\u003c/em\u003e possessed 4 dinucleotide SSRs with A/T and T/C motifs, 4 trinucleotide SSRs with ATT/TAA motifs, and 15 tetranucleotide SSRs rich in A and T. \u003cem\u003eE. grandis\u003c/em\u003e contained 2 dinucleotide SSRs (A/T and T/C motifs), 5 trinucleotide SSRs (ATT/TAA motifs), and 13 A/T-rich tetranucleotide SSRs. The hybrid \u003cem\u003eE. urograndis\u003c/em\u003e shared the same nucleotide repeat composition as \u003cem\u003eE. urophylla\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDispersed repeats are a type of repeat sequence interspersed throughout the genome. They are classified into four types: forward, palindromic, reverse, and complementary. \u003cem\u003eE. urophylla\u003c/em\u003e comprised 31 dispersed repeats, including 16 forward and 15 palindromic (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The longest forward repeat spanned 518 bp (0.32% of the genome), while the longest palindromic repeat measured 26,923 bp (16.81%). \u003cem\u003eE. grandis\u003c/em\u003e shared the same number of dispersed repeats (31) as \u003cem\u003eE. urophylla\u003c/em\u003e, with the longest forward repeat at 524 bp (0.32%) and the longest palindromic repeat at 26,838 bp (16.80%). The hybrid \u003cem\u003eE. urograndis\u003c/em\u003e included 32 dispersed repeats, comprising 17 forward and 15 palindromic repeats. The longest palindromic repeat was 26,923 bp long, accounting for 16.80% of the genome. However, no reverse or complement repeats were observed in the three \u003cem\u003eEucalyptus\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Comparative analysis of SSR among eucalypt species\u003c/h2\u003e \u003cp\u003eAnalysis of SSRs in the chloroplast genomes of 12 eucalypt species revealed a variation ranging from 68 SSRs in \u003cem\u003eE. grandis\u003c/em\u003e to 115 in \u003cem\u003eC. citriodora\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The SSR composition included 48 to 87 mononucleotide repeats, 1 to 4 dinucleotide repeats, 3 to 8 trinucleotide repeats, 4 to 17 tetranucleotide repeats, 0 to 1 pentanucleotide repeats, and 0 to 1 hexanucleotide repeats. These SSRs were predominantly located in the LSC region of the chloroplast genomes. Tetranucleotide repeats were generally more abundant than trinucleotide repeats across the 12 species, whereas pentanucleotide and hexanucleotide repeats were relatively rare. \u003cem\u003eE. pauciflora\u003c/em\u003e exhibited the highest proportion of mononucleotide repeats (81.74%). Four species\u0026mdash;\u003cem\u003eE. urograndis\u003c/em\u003e, \u003cem\u003eE. urophylla\u003c/em\u003e, \u003cem\u003eE. nitens\u003c/em\u003e, and \u003cem\u003eE. robusta\u003c/em\u003e\u0026mdash;shared an identical SSR composition, each containing 79 mononucleotide repeats, along with 4 dinucleotide and 4 trinucleotide repeats. \u003cem\u003eC. citriodora\u003c/em\u003e was the most distinctive species, uniquely possessing both pentanucleotide and hexanucleotide repeats among the 12 examined species, whereas all other species contained only the other four repeat types.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDispersed repeats represent a distinct category of repetitive sequences, distinguished by their scattered distribution throughout the genome. These repeats are classified into four types: Forward (F), Palindromic (P), Reverse (R), and Complement (C). An analysis of dispersed repeats within the chloroplast genomes of 12 eucalypt species (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) demonstrated that forward and palindromic repeats were predominat, whereas complement repeats were the least prevalent. Only \u003cem\u003eC. citriodora\u003c/em\u003e and \u003cem\u003eE. nitens\u003c/em\u003e exhibited all four repeat types (F, P, R, C) in their chloroplast genomes; the remaining ten species lacked complement repeats. \u003cem\u003eE. globulus\u003c/em\u003e displayed a notable disparity in repeat counts, with seven more forward repeats than reverse repeats. In other species, the numbers of forward and palindromic repeats were generally comparable. Complement repeats remained consistently rare across all 12 species, occurring exclusively in \u003cem\u003eC. citriodora\u003c/em\u003e and \u003cem\u003eE. nitens\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Analysis of Relative Synonymous Codon Usage (RSCU)\u003c/h2\u003e \u003cp\u003eThe total average GC1, GC2, and GC3 values of the chloroplast genome CDS codons of \u003cem\u003eE. urograndis\u003c/em\u003e, \u003cem\u003eE. urophylla\u003c/em\u003e, and \u003cem\u003eE. grandis\u003c/em\u003e are 38.4%, with individual averages of 47.14%, 38.67%, and 29.387%, respectively, showing a pattern of GC1\u0026thinsp;\u0026gt;\u0026thinsp;GC2\u0026thinsp;\u0026gt;\u0026thinsp;GC3. Using an ENC value of 45 as the threshold for bias classification, the overall average ENC values for 45, 48, and 44 CDS codons in the chloroplast genomes of the three eucalypt species range from 48.83 to 49.41, all exceeding 45(Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This indicates a relatively weak codon usage bias in the chloroplast genomes of these three species.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenomic features of chloroplast genomes of three \u003cem\u003eEucalyptus\u003c/em\u003e plant species.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. urograndis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. urophylla\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eE. grandis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDS number\u003c/p\u003e \u003cp\u003e(before processing)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDS number\u003c/p\u003e \u003cp\u003e(after processing)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL_aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51687\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44406\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGC1(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGC2(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGC3(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eENc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage GC(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: L_aa means the total number of amino acids; GC1, GC2 and GC3 indicate the GC content at the first, second and third codon positions.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBased on the analysis criteria of RSCU values (where RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1 and \u0026gt;\u0026thinsp;1.5 represent relatively high and high-frequency codons, respectively), the chloroplast genomes of the three \u003cem\u003eEucalyptus\u003c/em\u003e species exhibit similar codon usage preferences, with their RSCU values for all encoded amino acids primarily distributed in the range of 0\u0026ndash;3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). \u003cem\u003eE. urograndis\u003c/em\u003e, \u003cem\u003eE. urophylla\u003c/em\u003e, and \u003cem\u003eE. grandis\u003c/em\u003e each have 30 high-frequency codons with RSCU values greater than 1, accounting for 46.87% of the total codons. Among these high-frequency codons, only one ends with G, while the rest end with A or U. The results indicate that the codon usage in the chloroplast genomes of the three \u003cem\u003eEucalyptus\u003c/em\u003e species tends to favor A or U endings. Additionally, in the chloroplast genomes of the three ucalypt species, the codon UUA for leucine (Leu) acts as an overlapping codon and is used more frequently than other codons. Specifically, the RSCU values are 1.91 for \u003cem\u003eE. urograndis\u003c/em\u003e, 1.98 for \u003cem\u003eE. urophylla\u003c/em\u003e, and 1.91 for \u003cem\u003eE. grandis.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Analysis of Optimal Codons\u003c/h2\u003e \u003cp\u003eIn \u003cem\u003eE.urograndis\u003c/em\u003e, 14 optimal codons were identified with the highest ΔRSCU value of 1.243 and an average ΔRSCU value of 0.535 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e). For \u003cem\u003eE. urophylla\u003c/em\u003e, the highest number of optimal codons was observed, totaling 16, along with a maximum ΔRSCU value of 1.343 and an average ΔRSCU value of 0.417. In \u003cem\u003eE. grandis\u003c/em\u003e, 14 optimal codons were identified, matching the highest ΔRSCU value observed in \u003cem\u003eE. urograndis\u003c/em\u003e, and an average ΔRSCU value of 0.446. Across the three \u003cem\u003eEucalyptus\u003c/em\u003e species, a total of 14\u0026ndash;16 optimal codons were identified, including GCA, CCA, UAA, GUU, ACA, UCA, CUU, GGU, CAA, UGU, AAU, AGU, GAA, ACU, UUG, and AAA( Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 IR border and divergence analyses\u003c/h2\u003e \u003cp\u003eIn the chloroplast genomes of the ten eucalypt species (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003e), variations were observed in the positions of the \u003cem\u003erpl22\u003c/em\u003e, \u003cem\u003erps19\u003c/em\u003e, and \u003cem\u003erpl2\u003c/em\u003e genes at the IR boundaries, as well as in their gene lengths. The \u003cem\u003erpl22\u003c/em\u003e and \u003cem\u003erps19\u003c/em\u003e genes were located at the IR/LSC boundary (JLA) in \u003cem\u003eE. urophylla\u003c/em\u003e, positioned to the right of the junction, with \u003cem\u003erps19\u003c/em\u003e located 1 bp from the boundary. In \u003cem\u003eE. grandis\u003c/em\u003e, these genes were found at the LSC/IRb boundary (JLB), positioned to the left of the junction, with \u003cem\u003erps19\u003c/em\u003e located 2 bp from the boundary. \u003cem\u003eE. urograndis\u003c/em\u003e, both genes were located at the SSC/IRA boundary (JSA), positioned to the left of the junction, with \u003cem\u003erps19\u003c/em\u003e located 2 bp from the boundary. The \u003cem\u003erpl2\u003c/em\u003e gene was located to the right of the JSA junction in \u003cem\u003eE. urograndis\u003c/em\u003e but was not detected in \u003cem\u003eE. grandis\u003c/em\u003e. In the remaining eight species, \u003cem\u003erpl2\u003c/em\u003e consistently appeared to the right of the IR/LSC boundary (JLA). The \u003cem\u003endhF\u003c/em\u003e gene was located 187 bp to the right of the JLA junction in \u003cem\u003eE. urograndis\u003c/em\u003e. It was located 187 bp and 94 bp to the right of the JSB junction in \u003cem\u003eE. urophylla\u003c/em\u003e and \u003cem\u003eC. citriodora\u003c/em\u003e, respectively, but was absent in the other seven species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify sequence variation sites within the three \u003cem\u003eEucalyptus\u003c/em\u003e chloroplast genomes, we calculated nucleotide diversity (Pi) values (range, 0-0.34889; average, 0.21225) (Table S3, Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e9\u003c/span\u003e). We detected five highly divergent regions (\u003cem\u003etrnK\u003c/em\u003e\u003csup\u003e\u003cem\u003eUUU\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnT\u003c/em\u003e\u003csup\u003e\u003cem\u003eGGU\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003epsaB-psaA\u003c/em\u003e, \u003cem\u003endhJ-ndhK\u003c/em\u003e, and \u003cem\u003erpl22-rps19-rpl2\u003c/em\u003e), with nucleotide diversity exceeding 0.34. The \u003cem\u003etrnT\u003c/em\u003e\u003csup\u003e\u003cem\u003eGGU\u003c/em\u003e\u003c/sup\u003e gene exhibited the most significant divergence, the highest Pi value (0.34889), and is located in the LSC region. These highly variable regions could be candidate molecular biomarkers for phylogenetic reconstruction in eucalypt species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Phylogenomic analysis\u003c/h2\u003e \u003cp\u003eWe constructed a ML phylogenetic tree using chloroplast genomes of \u003cem\u003eE. urophylla\u003c/em\u003e, \u003cem\u003eE. grandis\u003c/em\u003e, \u003cem\u003eE. urograndis\u003c/em\u003e, and 29 published eucalypt species, with \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eP. trichocarpa\u003c/em\u003e as outgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The results revealed that the outgroups (\u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eP. trichocarpa\u003c/em\u003e) differed significantly from the 32 eucalypt species. Consistent with established taxonomic frameworks, two \u003cem\u003eAngophora\u003c/em\u003e species, three \u003cem\u003eCorymbia\u003c/em\u003e species, and 29 eucalypt specie formed monophyletic clades with bootstrap support values exceeding 80%. The 29 eucalypt species were classified into six subgenera, with chloroplast genome sequences effectively separating most except \u003cem\u003eEucalyptus queenslandica\u003c/em\u003e. This is consistent with previous studies by Liu et al[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and Steane et al[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], which found a close genetic relationship between the subgenus \u003cem\u003eEucalyptus\u003c/em\u003e and \u003cem\u003eMonocalyptus\u003c/em\u003e. this work found that the two eucalypt hybrids (\u003cem\u003eE. sideroxylon \u0026times; E. melliodora\u003c/em\u003e and \u003cem\u003eE. urograndis\u003c/em\u003e) were genetically similar to their maternal parents, which is consistent with the maternal inheritance pattern outlined in eucalypt chloroplast genomes. Within the subgenus \u003cem\u003eSymphyomyrtus\u003c/em\u003e, \u003cem\u003eE. camaldulensis\u003c/em\u003e from \u003cem\u003eExsertaria\u003c/em\u003e exhibited closer genetic proximity to species from \u003cem\u003eTransversaria\u003c/em\u003e and section \u003cem\u003eGlobulus\u003c/em\u003e. This finding provides a theoretical foundation for using interspecific hybridization among these three divisions to develop superior hybrids in breeding programs. However, the genetic similarity among species from various sections within the same subgenus differs from the most recent published classification of the \u003cem\u003eEucalyptus\u003c/em\u003e genus [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For example, within the subgenus \u003cem\u003eEucalyptus\u003c/em\u003e, \u003cem\u003eE. regnans\u003c/em\u003e, \u003cem\u003eE. elata\u003c/em\u003e, and \u003cem\u003eE. baxteri\u003c/em\u003e are classified under the section \u003cem\u003eEucalyptus\u003c/em\u003e, yet in this work, \u003cem\u003eE. regnans\u003c/em\u003e exhibited a closer genetic affinity to \u003cem\u003eE. marginata\u003c/em\u003e (section \u003cem\u003eLongistylus\u003c/em\u003e) than to its section members. This discrepancy may reflect the limitations of chloroplast genomes-inherited maternally and highly conserved-in resolving fine-scale taxonomic relationships [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe elucidation of chloroplast genomes lays the groundwork for in-depth research into chloroplast functions, intracellular gene transfer, species diversity, plant resource conservation, and genetic breeding [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our study sequenced, assembled, and annotated the chloroplast genomes of \u003cem\u003eE. urograndis\u003c/em\u003e (a widely used hybrid in Chinese eucalypt plantations) and the pure species \u003cem\u003eE. urophylla\u003c/em\u003e, the complete chloroplast genome data were collected and compared to the published \u003cem\u003eE. grandis\u003c/em\u003e chloroplast genome(NC_014570.1). \u003cem\u003eE. urophylla\u003c/em\u003e, \u003cem\u003eE. grandis\u003c/em\u003e, and \u003cem\u003eE. urograndis\u003c/em\u003e exhibited the typical angiosperm quadripartite structure in their chloroplast genomes, with sizes ranging from 160,137 bp to 160,283 bp. This finding is consistent with the results of Bayly et al., which reported minimal size variation among chloroplast genomes in 39 eucalypt species [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cem\u003eE. urograndis\u003c/em\u003e, \u003cem\u003eE. grandis\u003c/em\u003e, and \u003cem\u003eE. urophylla\u003c/em\u003e chloroplast genomes comprised 127, 127, and 129 genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), respectively, including 83, 83, and 85 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. These findings are consistent with previously reported eucalypt chloroplast genomes[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], demonstrating a conserved chloroplast genome organization across the genus. The hybrid \u003cem\u003eE. urograndis\u003c/em\u003e was more closely related to \u003cem\u003eE. urophylla\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003epsbL\u003c/em\u003e gene was detected in the chloroplast genomes of \u003cem\u003eE. urograndis\u003c/em\u003e and \u003cem\u003eE. grandis\u003c/em\u003e but not in \u003cem\u003eE. urophylla\u003c/em\u003e. The \u003cem\u003epsbN\u003c/em\u003e gene is retained in \u003cem\u003eE. urophylla\u003c/em\u003e and \u003cem\u003eE. grandis\u003c/em\u003e, but lost in \u003cem\u003eE. urograndis\u003c/em\u003e, most likely due to evolutionary gene loss. This is consistent with the findings of Zhang et al.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] who demonstrated a high conservation of \u003cem\u003epsbN\u003c/em\u003e in angiosperms. Due to their distinct life-history strategies, parasitic plants may lose or modify certain chloroplast genes (including \u003cem\u003epsbN\u003c/em\u003e) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e],potentially reflecting ecological adaptations. This work found variations in the \u003cem\u003epsbL\u003c/em\u003e, \u003cem\u003epsbN\u003c/em\u003e, \u003cem\u003einfA\u003c/em\u003e, and \u003cem\u003epbf1\u003c/em\u003e genes between \u003cem\u003eE. urograndis\u003c/em\u003e, \u003cem\u003eE. urophylla\u003c/em\u003e and \u003cem\u003eE. grandis\u003c/em\u003e. These discrepancies may arise from structural variations in the chloroplast genome or from adaptive evolutionary processes. Alternatively, chloroplast genome of the hybrid could have been inherited from an as-yet uncharacterized parental lineage.These factors may operate independently or in combination, leading to subtle. Future research will focus on expanding the sample set and validating these genomic regions.\u003c/p\u003e \u003cp\u003eSSRs, which are highly variable, drive chloroplast genome rearrangement and expansion. this work revealed 68 to 102 SSRs in the three \u003cem\u003eEucalyptus\u003c/em\u003e chloroplast genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, the hybrid \u003cem\u003eE. urograndis\u003c/em\u003e harbored one additional forward repeat compared to other two eucalypt, with the longest forward repeats varying by 48 and 42 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e), perhaps due to IR contraction during evolution. Mononucleotide SSRs dominated the repeat composition, providing a foundation for interspecies genomic variation studies in eucalypt. Under environmental stress[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], SSR dynamics may alter gene expression and influence adaptive capacity. Significant differences in SSR abundance and distribution patterns between \u003cem\u003eE. urograndis\u003c/em\u003e, \u003cem\u003eE. urophylla\u003c/em\u003e, and \u003cem\u003eE. grandis\u003c/em\u003e demonstrate their potential as molecular biomarkers for species identification and phylogenetic reconstruction in eucalypt.\u003c/p\u003e \u003cp\u003eThe chloroplast genomes of the three \u003cem\u003eEucalyptus\u003c/em\u003e species exhibit weak codon usage bias, and their preference pattern aligns with that of most plant chloroplasts, showing a significant tendency to use codons ending with A or U. This finding is consistent with previous studies on Cupressaceae [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], Theaceae [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and \u003cem\u003eDavidia involucrata\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, a total of 14\u0026ndash;16 optimal codons were identified across the three \u003cem\u003eEucalyptus\u003c/em\u003e species, including GCA, CCA, UAA, GUU, ACA, UCA, CUU, GGU, CAA, UGU, AAU, AGU, GAA, ACU, UUG, and AAA, with a predominance of codons ending in A or U (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This pattern shows a high degree of similarity to the codon preferences observed in \u003cem\u003eEuphorbia esula\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and the \u003cem\u003eHevea genus\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Studies have demonstrated that codon usage bias can serve as an effective molecular marker for distinguishing closely related species, such as bamboos[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The results of this study provide a strong reference for the accurate classification and identification of eucalypt species, aiding in the recognition of their genetic diversity and characteristic differences. Additionally, leveraging their codon usage patterns to optimize exogenous sequences can effectively enhance gene expression efficiency, offering technical support for subsequent research on genetic engineering improvements and species conservation in the genus \u003cem\u003eEucalyptus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIR region contraction and expansion in plant chloroplast genomes are common phenomena that determine genome size variation across species[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We observed that \u003cem\u003eE. urograndis\u003c/em\u003e had a shorter LSC region than \u003cem\u003eE. urophylla\u003c/em\u003e and \u003cem\u003eE. grandis\u003c/em\u003e, with LSC variances accounting for the majority of the differences in genome size (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). It is hypothesized that variations in the LSC region length are primarily responsible for the observed differences in the chloroplast genome size of \u003cem\u003eE. urograndis\u003c/em\u003e. Studies indicate that longer IR regions offer additional repetitive sequences[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], improve genome stability, and lower the chances of structural rearrangements[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our results demonstrate that \u003cem\u003eE. urograndis\u003c/em\u003e has a modest IR expansion compared to other two species of eucalypt, which may confer increased genomic stability and adaptability under environmental stress. The contraction-expansion divergence at IR boundaries in three species of \u003cem\u003eEucalyptu\u003c/em\u003es likely contributes to chloroplast genome size variance, which may affect overall genome architecture and function. These findings are consistent with observations in \u003cem\u003ePaeonia\u003c/em\u003e species, where alterations in the LSC/IRb boundary positions correlate with evolutionary divergence[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Minor boundary variations may thus serve as evolutionary markers for chloroplast genome differentiation[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe discovered five highly divergent regions between the chloroplast genomes of eucalypt hybrid and pure species, predominantly located in the LSC region. The nucleotide diversity of IR regions was significantly lower than the SSC regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003e), consistent with prior findings[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Zhang et al.[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] conducted a similar study, comparing entire chloroplast genomes in 24 Myrtales species using MVISTA and variable locus proportions across 114 non-coding regions and 74 coding genes with DnaSP. The study demonstrated that IR regions are less divergent than SC regions in Asteraceae and Myrtales. Variations were also observed in genes near the IR/SSC boundaries, such as \u003cem\u003erpl22\u003c/em\u003e, \u003cem\u003erps19\u003c/em\u003e, and \u003cem\u003erpl2\u003c/em\u003e, consistent with the results of the IRscope boundary analysis.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that the chloroplast genome can be used for species identification and phylogenetic reconstruction [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Our findings reveal significant divergence in the chloroplast genomes of \u003cem\u003eAngophora\u003c/em\u003e, \u003cem\u003eCorymbia\u003c/em\u003e, and \u003cem\u003eEucalyptus\u003c/em\u003e, allowing for the unambiguous distinction between the three genera. However, chloroplast genomes cannot discriminate between species in the subgenera \u003cem\u003eEucalyptus\u003c/em\u003e and \u003cem\u003eMonocalyptus\u003c/em\u003e, which share closer genetic proximity. However, chloroplast genomes continue to be effective in elucidating phylogenetic relationships for other subgenera. As the largest \u003cem\u003eEucalyptus\u003c/em\u003e subgenus, \u003cem\u003eSymphyomyrtus\u003c/em\u003e exhibits close genetic distances across its sections, allowing for interspecific hybridization between section members to produce high-performance hybrids and clonal variants. For instance, hybrids derived from crosses between sections Latoangulata (\u003cem\u003eE. urophylla\u003c/em\u003e, \u003cem\u003eE. grandis\u003c/em\u003e, \u003cem\u003eE. pellita\u003c/em\u003e), Exsertaria (\u003cem\u003eE. camaldulensis\u003c/em\u003e, \u003cem\u003eE. tereticornis\u003c/em\u003e), and Maidenaria (\u003cem\u003eE. dunnii\u003c/em\u003e, \u003cem\u003eE. globulus\u003c/em\u003e) have demonstrated superior heterosis in plantation applications [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e][\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. this study also revealed close genetic relationships among the sections \u003cem\u003eGlobulus, Exsertaria\u003c/em\u003e, and \u003cem\u003eTransversaria\u003c/em\u003e. The tree species within these three sections may exhibit higher hybrid compatibility, which could facilitate the successful production of hybrid offspring. However, this inference, based on genomic similarity, requires further functional validation through direct hybridization experiments.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eAs previously mentioned, eucalypt species have highly conserved boundaries and similar flanking genes but with slightly different expansion degrees. The only variations detected were in genome size, GC level, repetitive sequences, and IR boundary. In conclusion, we propose five hypervariable regions, \u003cem\u003etrnK\u003c/em\u003e\u003csup\u003e\u003cem\u003eUUU\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnT\u003c/em\u003e\u003csup\u003e\u003cem\u003eGGU\u003c/em\u003e\u003c/sup\u003e ,\u003cem\u003epsaB-psaA\u003c/em\u003e, \u003cem\u003endhJ-ndhK\u003c/em\u003e, and \u003cem\u003erpl22-rps19-rpl2\u003c/em\u003e, as candidate molecular biomarkers for identifying \u003cem\u003eE. urograndis\u003c/em\u003e. A set of 14\u0026ndash;16 optimal codons was identified in eucalypt species, 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 eucalypt plants. These findings enrich the chloroplast genomic database for eucalypt species and provide a theoretical foundation for reconstructing their phylogenetic relationships. this study increases our understanding of interspecific diversity in chloroplast genomes between eucalypt species .\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdenine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eComplementary\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInverted repeats\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJLA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIRA/LSC\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJLB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLSC/IRb\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSSC/IRA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJSB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIRB/LSC\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLarge single-copy region\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eML\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMaximum likelihood\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePalindromic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein-coding genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNucleotide diversity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSmall single-copy region\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSSRs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSimple sequence repeats\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethymine.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Fundamental Research Funds of CAF (Grant No. CAFYBB2023MB034) and the National Key R\u0026amp;D Program of China (Grant No. 2022YFD2200203 and 2023YFD2201001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the results of this article are available on NCBI, https://www.ncbi.nlm.nih.gov/ (accession number: PV464070 and OL804288).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGupengYi:\u003c/strong\u003e Writing-original draft, Methodology, Investigation, Formal analysis, Data curation. \u003cstrong\u003eWanhong Lu\u003c/strong\u003e and \u003cstrong\u003eYan Lin:\u003c/strong\u003e Software, Methodology, Investigation. \u003cstrong\u003eJianzhong Luo\u003c/strong\u003e and \u003cstrong\u003eGuo Liu:\u003c/strong\u003e Writing-review and editing, Supervision, Funding acquisition, Conceptualization. \u003cstrong\u003eYing Cheng\u003c/strong\u003e and \u003cstrong\u003eZhijiao Song:\u003c/strong\u003e Visualization, Validation, Methodology, Investigation, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable S1. 34 species and accessions used for phylogenetic analysis; Table S2 .SSR information in the chloroplast genomes of tewlve eucalypt species;\u0026nbsp;Table S3. Sliding window test of Pi in the Hybrid tail \u003cem\u003eE.urograndis\u003c/em\u003e,\u0026nbsp;\u003cem\u003eE.urophylla\u003c/em\u003e and \u003cem\u003eE.grandis\u003c/em\u003e chloroplast genomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe appropriate permissions were obtained for all materials used in this work. We complied with all relevant institutional, national, and international guidelines and legislation. The Guangxi State-owned Dongmen Forest Farm (Chongzuo, Guangxi Zhuang Autonomous Region, China) and\u0026nbsp;Leizhou Forestry Bureau (Zhanjiang, Guangdong Province, China) gave permission for the leaves of \u003cem\u003eE. urograndis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eE. urophylla\u0026nbsp;\u003c/em\u003eto be collected for the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu T, Xie YJ. Analysis and prospects of the rapid development of Eucalyptus plantations in China. Eucalypt Sci Technol. 2020;37(4):38\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaheswari P, Kunhikannan C, Yasodha R. Chloroplast genome analysis of Angiosperms and phylogenetic relationships among Lamiaceae members with particular reference to teak (\u003cem\u003eTectona grandis\u003c/em\u003e L.f). J Biosci. 2021;46:43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12038-021-00166-2\u003c/span\u003e\u003cspan address=\"10.1007/s12038-021-00166-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Yuan Q, Wei Y, Liu H, Zheng X, Wang Y, Liu H. Evolutionary analysis of chloroplast genomes of \u003cem\u003eSect.Isika\u003c/em\u003e (Lonicera)species, Nematotinus plants. Chin Tradit Herb Drugs. 2024;55(9):3085\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan A, Zhu D. Application of chloroplast genome in phylogenetics and genetic engineering. Chin J Cell Biology. 2004;26:153\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBayly MJ, Rigault P, Spokevicius A, Ladiges PY, Ades PK, Anderson C, Bossinger G, Merchant A, Udovicic F, Woodrow IE, Tibbits J. Chloroplast genome analysis of Australian eucalypts - \u003cem\u003eEucalyptus\u003c/em\u003e, \u003cem\u003eCorymbia\u003c/em\u003e, \u003cem\u003eAngophora\u003c/em\u003e, \u003cem\u003eAllosyncarpia\u003c/em\u003e and \u003cem\u003eStockwellia\u003c/em\u003e (Myrtaceae). Mol Phylogenet Evol. 2013;69(3):704\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ympev.2013.07.006\u003c/span\u003e\u003cspan address=\"10.1016/j.ympev.2013.07.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo Y, Yao B, Yuan M, Li J. The complete chloroplast genome and phylogenetic analysis of Astragalus scaberrimus Bunge 1833. Mitochondrial DNA Part B. 2021;6(12):3364\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/23802359.2021.1997108\u003c/span\u003e\u003cspan address=\"10.1080/23802359.2021.1997108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi L, Chen H, Jiang M, Wang L, Wu X, Huang L, Liu C. CPGAVAS2, an integrated plastome sequence annotator and analyzer. Nucleic Acids Res. 2019;47(W1):W65\u0026ndash;73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkz345\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkz345\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu B, Gao Z, Qian F, Yang X, Lv X, Cai M. The complete chloroplast genome of a purple Ethiopian rape (Brassica carinata: Brassicaceae) from Guizhou Province, China and its phylogenetic analysis. Mitochondrial DNA Part B. 2021;6:1821\u0026ndash;3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/23802359.2021.1926365\u003c/span\u003e\u003cspan address=\"10.1080/23802359.2021.1926365\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang W, Yu H, Wang J, Lei W, Gao J, Qiu X, Wang J. The complete chloroplast genome sequences of the medicinal plant Forsythia suspensa (Oleaceae). Int J Mol Sci. 2017;18(11):2288. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms18112288\u003c/span\u003e\u003cspan address=\"10.3390/ijms18112288\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurtz S, Choudhuri JV, Ohlebusch E, Schleiermacher C, Stoye J, Giegerich R. REPuter: the manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res. 2001;29(22):4633\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/29.22.4633\u003c/span\u003e\u003cspan address=\"10.1093/nar/29.22.4633\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRice P, Longden I, Bleasby A. EMBOSS: the European molecular biology open software suite. Trends Genet. 2000;16(6):276\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu P, Xiao W, Luo Y, Xiong Z, Chen X, He J, Sha A, Gui M, Li Q. Comprehensive analysis of codon bias in 13 Ganoderm a mitochondrial genomes. Fron-tiersin Microbiol. 2023;14:1170790. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2023.1170790\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2023.1170790\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28(12):1647\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/bts199\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/bts199\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmiryousefi A, Hyv\u0026ouml;nen J, Poczai P. IRscope: an online program to visualize the junction sites of chloroplast genomes. Bioinformatics. 2018;34(17):3030\u0026ndash;1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/bty220\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/bty220\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2017;20(4):1160\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bib/bbx108\u003c/span\u003e\u003cspan address=\"10.1093/bib/bbx108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRozas J, Ferrer-Mata A, S\u0026aacute;nchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, S\u0026aacute;nchez-Gracia A. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol. 2017;34:3299\u0026ndash;302. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/msx248\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msx248\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCapella-Guti\u0026eacute;rrez S, Silla-Mart\u0026iacute;nez JM, Gabald\u0026oacute;n T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25(15):1972\u0026ndash;3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/btp348\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/btp348\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32(1):268\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/msu300\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msu300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalyaanamoorthy S, Minh BQ, Wong TKF, Von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods. 2017;14(6):587\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmeth.4285\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.4285\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinh BQ, Nguyen MAT, Von Haeseler A. Ultrafast approximation for phylogenetic bootstrap. Mol Biol Evol. 2013;30(5):1188\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/mst024\u003c/span\u003e\u003cspan address=\"10.1093/molbev/mst024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu G, Arnold RJ, Xie Y, Wu Z. Genetic relationships among 40 species of Eucalyptus based on simple sequence repeat markers. J Trop For Sci. 2018;30(3):402\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.jstor.org/stable/26512525\u003c/span\u003e\u003cspan address=\"https://www.jstor.org/stable/26512525\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteane DA, Nicolle D, McKinnon GE, Vaillancourt RE, Potts BM. Higher-level relationships among the eucalypts are resolved by ITS-sequence data. Aust Syst Bot. 2002;15(1):49\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1071/SB00039\u003c/span\u003e\u003cspan address=\"10.1071/SB00039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicolle D. Classification of the eucalypts, genus Eucalyptus.Version 7[EB/OL].(2025-4-16). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.dn.com.au/Classification-Of-The-Eucalypts.pdf\u003c/span\u003e\u003cspan address=\"http://www.dn.com.au/Classification-Of-The-Eucalypts.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Morales-Briones DF, Li Y, Zhang G, Zhang T, Huang CH, Ma H. Phylogenomics insights into gene evolution, rapid species diversification, and morphological innovation of the apple tribe (Maleae, Rosaceae). New Phytol. 2023;240(5):2102\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.19175\u003c/span\u003e\u003cspan address=\"10.1111/nph.19175\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu C, Tang L, Han L. Characterization of the chloroplast genome of Lindera setchuenensis and phylogenetics of the genus Lindera. Scientia Silvae Sinicae. 2021;57(7):167\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.11707/j.1001-7488.20211217\u003c/span\u003e\u003cspan address=\"10.11707/j.1001-7488.20211217\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.linyekexue.net/EN/\u003c/span\u003e\u003cspan address=\"http://www.linyekexue.net/EN/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang WW, Schalamun M, Morales-Suarez A, Kainer D, Schwessinger B, Lanfear R. Assembly of chloroplast genomes with long- and short-read data: a comparison of approaches using \u003cem\u003eEucalyptus pauciflora\u003c/em\u003e as a test case. BMC Genomics. 2018;19(1):977. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12864-018-5348-8\u003c/span\u003e\u003cspan address=\"10.1186/s12864-018-5348-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao Y, Zhang J, Tang H, Liu N, Li G, Yue D. The characteristics of the complete chloroplast genome for \u003cem\u003eEucalyptus robusta\u003c/em\u003e (Myrtaceae). Mitochondrial DNA Part B. 2021;6(12):3517\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/23802359.2021.2005491\u003c/span\u003e\u003cspan address=\"10.1080/23802359.2021.2005491\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteane DA. Complete nucleotide sequence of the chloroplast genome from the Tasmanian blue gum, \u003cem\u003eEucalyptus globulus\u003c/em\u003e (Myrtaceae). DNA Res. 2005;12(3):215\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/dnares/dsi006\u003c/span\u003e\u003cspan address=\"10.1093/dnares/dsi006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Huang T, Zhou Q, Sheng Q, Zhu Z. Complete chloroplast genomes and phylogenetic relationships of \u003cem\u003eBougainvillea spectabilis\u003c/em\u003e and \u003cem\u003eBougainvillea glabra\u003c/em\u003e (Nyctaginaceae). Int J Mol Sci. 2024;24(16):13044. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms241713044\u003c/span\u003e\u003cspan address=\"10.3390/ijms241713044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao L, Su Y, Wang T. Plastid genome sequencing, comparative genomics, and phylogenomics: Current status and prospects. J Syst Evol. 2010;48(1):77\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1759-6831.2010.00071.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1759-6831.2010.00071.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaniell H, Lin CS, Yu M, Chang WJ. Chloroplast genomes: Diversity, evolution, and applications in genetic engineering. Genome Biol. 2016;17(1):134. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13059-016-1004-2\u003c/span\u003e\u003cspan address=\"10.1186/s13059-016-1004-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang SQ, Zhang QG, Ye ZL, Qin XH, Wang QS, Lin XQ, Zheng ZW, Lin WF, Zou XX. Codon bias analysis of chloroplast genomes of 5 Cupressaceae plants. J Fujian Agric Forestry Univ (Natural Sci Edition). 2024;53(2):214\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Cai Q, Wang Y, Li M, Wang C, Wang Z, Jiao C, Xu C, Wang H, Zhang Z. Comparative analysis of codon bias in the chloroplast genomes of theaceae species. Front Genet. 2022;13:824610. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fgene.2022.824610\u003c/span\u003e\u003cspan address=\"10.3389/fgene.2022.824610\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo YJ, Wang R, Zhao RF, Lu XX, Yin GK, Deng ZJ. Analysis of synonymous codon usage bias in the chloroplast genome of Davidia involucrata. J Beijing Forestry Univ. 2024;46(3):8\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Xu B, Li B, Zhou Q, Wang G, Jiang X, Wang C, Xu Z. Comparative analysis of codon usage patterns in chloroplast genomes of six Euphorbiaceae species. 2020;8:e8251. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7717/peerj.8251\u003c/span\u003e\u003cspan address=\"10.7717/peerj.8251\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Liu X, He L, Li Z, Yuan B, Fang F, Wang X. Comparative chloroplast genomics and codon usage bias analysis in Hevea Genus. Genes. 2025;16(2):201. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/genes16020201\u003c/span\u003e\u003cspan address=\"10.3390/genes16020201\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKelchner SA, Group BP. Higher level phylogenetic relationships within the bamboos (Poaceae: Bambusoideae) based on five plastid markers. Mol Phylogenet Evol. 2013;67(2):404\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang H, Shi C, Liu Y, Mao S, Gao L. Thirteen Camellia chloroplast genome sequences determined by high-throughput sequencing: Genome structure and phylogenetic relationships. BMC Evol Biol. 2014;14(1):151. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1471-2148-14-151\u003c/span\u003e\u003cspan address=\"10.1186/1471-2148-14-151\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang Z, Chen H, Bao H, Dai Y. Chloroplast genome characteristics and molecular marker development of Pennisetum. J Zhejiang A\u0026amp;F Univ. 2025;42(2):365\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Wang S, Liu Y, Yuan Q, Sun J, Guo L. Chloroplast genome variation and phylogenetic relationships of Atractylodes species. BMC Genomics. 2021;22(1):103. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12864-021-07394-8\u003c/span\u003e\u003cspan address=\"10.1186/s12864-021-07394-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou X, Zhang K, Peng Z, Sun S, Ya H, Zhang Y, Chen Y. Comparative Analysis of Chloroplast Genome Characteristics between \u003cem\u003ePaeonia jishanensis\u003c/em\u003e and Other Five Species of Paeonia. CABI Digit Libr. 2020;56(4):82\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Yan N, Song Q, Guo J. Complete chloroplast genome sequence and characteristics analysis of Morus multicaulis. Chin Bull Bot. 2018;l53(1):94\u0026ndash;103. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.11983/CBB16247\u003c/span\u003e\u003cspan address=\"10.11983/CBB16247\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chinbullbotany.com/EN/\u003c/span\u003e\u003cspan address=\"https://www.chinbullbotany.com/EN/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun J, Wang Y, Garran TA, Qiao P, Wang M, Yuan Q, Guo L, Huang L. Heterogeneous genetic diversity estimation of a promising domestication medicinal motherwort Leonurus cardiaca based on chloroplast genome resources. Front Genet. 2021;12:721022. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fgene.2021.721022\u003c/span\u003e\u003cspan address=\"10.3389/fgene.2021.721022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Landis JB, Wang H, Zhu Z, Wang H. Comparative analysis of chloroplast genome structure and molecular dating in Myrtales. BMC Plant Biol. 2021;21(1):219. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12870-021-02985-9\u003c/span\u003e\u003cspan address=\"10.1186/s12870-021-02985-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Lu J, Li Y, Zhu J, Zhang L. Comparative morphology of the leaf epidermis in Lobelia (Lobelioideae) from China. Microsc Res Tech. 2017;80(7):763\u0026ndash;78. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jemt.22862\u003c/span\u003e\u003cspan address=\"10.1002/jemt.22862\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing Y, Bi G, Hu S, Zhou MY, Li H, Xia Z. Chloroplast genome characteristics and phylogenetic analysis of different flower color variation types in safflower (\u003cem\u003eCarthamus tinctorius\u003c/em\u003e). Chin Traditional Herb Drugs. 2023;54(1):262\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu B, Sun Y, Liu X, Huang L, Xu Y, Zhao C. Complete chloroplast genome sequence and phylogenetic analysis of Camellia fraterna. Mitochondrial DNA Part B. 2020;5(3):3840\u0026ndash;2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/23802359.2020.1841576\u003c/span\u003e\u003cspan address=\"10.1080/23802359.2020.1841576\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi G, Xu J, Wang W, Wu S, Zhu Y, Wang YS, Pan J, Guo HY, Shi TY. Study on heterosis estimation and genetic analysis of Eucalyptus hybrids in cold area. J Nanjing Forestry Univ (Natural Sci Edition). 2017;41(4):55\u0026ndash;63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://njlydxxb.periodicals.net.cn/default.html\u003c/span\u003e\u003cspan address=\"http://njlydxxb.periodicals.net.cn/default.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMo JY, Lan J, Luo JZ, Wu MFn, Peng ZB. Growth characteristics of hybrids between \u003cem\u003eEucalyptus urophylla\u003c/em\u003e and Section Exsertaria species. GuangXi Plant. 2021;41(4):631\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo JZ, Xie YJ, Cao JG, Lu WH, Ren SQ. Genetic variation in growth and wind resistance of two-year-old Eucalyptus hybrids. Acta Prataculturae Sinica. 2009;18(3):91\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeng QJ, Lai QX, Li FG, Zhou CP, Li JW, Li M, Gan SM. Genetic analysis on early growth and cold tolerance of \u003cem\u003eEucalyptus urophylla\u003c/em\u003e \u0026times; \u003cem\u003eE. dunnii\u003c/em\u003e hybrids. J Nanjing Forestry Univ (Natural Sci Edition). 2015;39(5):33\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"eucalypt, chloroplast genome, comparative analysis, codon usage, phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-9082114/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9082114/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe 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\u0026shy;\u003cem\u003eEucalyptus urophylla\u003c/em\u003e \u0026times; \u003cem\u003eEucalyptus grandis\u003c/em\u003e (\u003cem\u003eE. urograndis\u003c/em\u003e) and pure species (\u003cem\u003eE. urophylla\u003c/em\u003e and \u003cem\u003eE. grandis\u003c/em\u003e) 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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBy employing next-generation sequencing technology, bioinformatics analysis, and other methods, we conducted whole-genome sequencing of the chloroplasts in the hybrids \u003cem\u003eE. urograndis\u003c/em\u003e and the pure species \u003cem\u003eE. urophylla\u003c/em\u003e. Our findings revealed that the \u003cem\u003eE. urograndis\u003c/em\u003e (160,201 bp) had an intermediate chloroplast genome size between that of \u003cem\u003eE. urophylla\u003c/em\u003e (160,283 bp) and \u003cem\u003eE. grandis\u003c/em\u003e (160,137 bp). Significant differences was evident in gene composition, IR region expansion, and SSR site distribution. In particular, \u003cem\u003etrnK\u003c/em\u003e\u003csup\u003e\u003cem\u003eUUU\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003etrnT\u003c/em\u003e\u003csup\u003e\u003cem\u003eGGU\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003epsaB-psaA\u003c/em\u003e, \u003cem\u003endhJ\u003c/em\u003e-\u003cem\u003endhK\u003c/em\u003e, and \u003cem\u003erpl22\u003c/em\u003e-\u003cem\u003erps19\u003c/em\u003e-\u003cem\u003erpl2\u003c/em\u003e 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\u0026ndash;16 optimal codons was identified in \u003cem\u003eEucalyptus\u003c/em\u003e, 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 \u003cem\u003eEucalyptus\u003c/em\u003e plants. Phylogenetic reconstruction confirmed that the eucalypt hybrid formed a monophyletic clade with the pure species \u003cem\u003eE. urophylla\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe 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.\u003c/p\u003e","manuscriptTitle":"Comparative Analysis of Chloroplast Genome Structure and Phylogenetic Relationships Between a Typical Eucalypt Hybrid and Two Purebred Species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 09:20:28","doi":"10.21203/rs.3.rs-9082114/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bf4342cb-4dd9-42a8-8625-7dd592b1653d","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-14T20:39:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 09:20:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9082114","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9082114","identity":"rs-9082114","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}