The complete chloroplast genome sequences and phylogenetics of Cornus sanguinea L. and Cornus sericea L. (Cornaceae)

The complete chloroplast genome sequences and phylogenetics of Cornus sanguinea L. and Cornus sericea L. 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(Cornaceae) Eugenia Nikonorova, Alexandr Shevtsov, Nailya Tursunbay, Oxana Khapilina, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5965562/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 This study provides an in-depth analysis of the chloroplast genomes of two Cornus species, Cornus sanguinea L. and Cornus sericea L., which are significant both in ornamental horticulture and traditional medicine. These species were collected from the Botanical Garden of the VILAR, providing a unique geographic context for genetic examination. Our results indicated that the plastomes of both species have typical quadripartite structure of chloroplast DNA, with slight variations in the size of the Large Single Copy (LSC) and Small Single Copy (SSC) regions compared to other Cornus species. The complete chloroplast genome size of C. sericea and C. sanguinea was 158 244 and 158 663 bp, respectively. A total of 131 genes, including 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes were found. The study highlighted the role of simple sequence repeats (SSRs) in genomic differentiation, with a notable absence of tetra-, penta-, and hexa-nucleotide repeats in the studied genomes. This aspect of the genome could be vital for understanding species differentiation and evolution within the genus. Phylogenetic analyses placed C. sanguinea and C. sericea within a broader clade of Cornaceae and reflected their close relationship to other species in the Cornaceae family. Overall, our study provides new data about the structure and features of the C. sericea cp genome and adds the valuable information on cp genome C. sanguinea , that is necessary for further studies. chloroplast genome plastids Cornaceae Cornus dogwood Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The family Cornaceae includes 3 genera and over a hundred species, which, along with such families as Curtisiaceae, Grubbiaceae, Hydrangeaceae, Hydrostachyaceae, Loasaceae, and Nyssaceae, are the part of Cornales order (Thomas et al. 2021 ). The majority of species within the family belong to the genus Cornus . The dogwoods ( Cornus spp.) are not only prized for their ornamental beauty but play a significant role in nature, biology, and medicine (Kazimierski et al. 2019 ; Badoni et al. 2024 ). Tenuta et al. (2022) in the comprehensive review highlighted that the edible fruits of European and Asian Cornus species are a rich source of phytochemicals with nutritional and functional properties. For example, C. mas (cornelian cherry) fruits and leaves are used in traditional medicine for treatment of diabetes, obesity, atherosclerosis, skin diseases, gastrointestinal and rheumatic disorders (Dinda et al. 2016 ). It was shown that C. officinalis possess the high range of pharmacological properties, such as hepatic and renal protection, antitumor, neuroprotective, antidiabetic effect, anti-inflammatory, antioxidant and immunoregulatory activities, etc (Huang et al. 2018 ). The conserved nature of the chloroplast (cp) genome, along with its well-defined structure and gene content has provided valuable insights into the genetic diversity and evolutionary relationships within the Cornaceae family (Daniell et al. 2016 ; Fu et al. 2017 ; Guan et al. 2024 ). The first study of chloroplast DNA (cpDNA) variability by PCR-RFLP analysis of the C. sanguinea was performed by H. Liesebach and B. Götz ( 2008 ), who reported the presence of eight different haplotypes across different European populations without association with geographic occurrence and low level of variation (Liesebach and Götz 2008 ). Lv et al. ( 2019 ) reported the complete cp genome sequences of C. sunhangii , giving a foundation for further studies on these species (Lv et al. 2019 ). Li et al. (Li et al. 2020 ) also contributed to this field by characterizing the cp genome of C. bretschneideri , respectively, further expanding our understanding of the genetic features of these plants. However, despite the recognized importance of the Cornaceae, the phylogenetic relationships within this family have been controversial and complex (Xiang et al. 1996 ; Fu et al. 2017 ; Du et al. 2023 ; Guan et al. 2024 ). The classification of species within the Cornus genus has seen diverse interpretations, with the taxonomy undergoing multiple revisions as new evidence comes to light (Du et al. 2023 ). In other species it's been found that cp genomes can vary depending on the species and their geographic distribution, which is related to adaptation to local climatic conditions (Xiong et al. 2023 ). Keir et al. ( 2011 ) revealed the relatively low diversity in cpDNA haplotypes of C. nuttallii , collected from 20 native populations of southern California and northern Idaho, USA (Keir et al. 2011 ). In study of Guan et al. ( 2024 ) plastid data of species growing from China to the USA were studied (Guan et al. 2024 ) – however, they did not include any species native to Russia. In the VILAR Botanical Garden, two species of dogwood ( C. sanguinea and C. sericea ) are cultivated and included in the collection. These species are thoroughly studied, including their genetic resources. However, there is no data on the structure and characteristics of the cp genome of C. sericea (no verified sequence was available in the NCBI database at the time of writing), and the studies on the chloroplast genome of C. sanguinea are limited (only one representative - FCN1796 from China - has been identified). Therefore, the aim of this study was to characterize and compare the structure and gene organization of the plastid genomes of C. sanguinea and C. sericea from the medicinal plant collection of the VILAR Botanical Garden. Materials and Methods Plant material. Fresh mature leaves of Cornus sanguinea L. (voucher specimen VF 004.21) and Cornus sericea L. (voucher specimen VF 006.21) were collected in July at the Botanical Garden of the VILAR (55°33'51.8"N; 37°35'56.8"E), located in urban zone of Moscow with warm-summer humid continental climate (Fig. 1 ). Chloroplasts isolation. Chloroplasts were isolated using high ionic strength solutions based on a technique described by Bookjans et al. (Bookjans et al. 1984 ). Briefly, after incubation in the dark at 4°C, fresh leaves were homogenized in a Waring blender in 50 mM Tris, 25 mM EDTA, 10 mM mercaptoethanol, 0.1% BSA and 1.25 M NaCl (pH = 8.0). The homogenate was filtered and centrifuged for 5 minutes at 1500 g (Eppendorf, Germany), the pellet was resuspended in the homogenization buffer, and the centrifugation was repeated for 5 minutes at 1500 g . The resulting chloroplast pellet was resuspended in a 50 mM Tris, 10 mM EDTA (pH = 8.0) and used for DNA extraction. DNA extraction. CpDNA was extracted following the method described by Shi et al. (Shi et al. 2012 ). The analysis of the extracted DNA fragments was performed by DNA separation in a 0.8% agarose gel containing ethidium bromide. Results were documented using a Gel Doc system (Bio-Rad, USA) with Quantity One software (Bio-Rad, USA). DNA concentrations were measured fluorometrically with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, MA, USA). DNA library preparation was performed using the Illumina DNA Prep, (M) Tagmentation kit (Cat. No. 20060060, Illumina, USA), following the manufacturer's guidelines. Libraries were normalized and then pooled to receive a 14 pM concentration, including a 1% PhiX control. Whole-genome sequencing was performed on the MiSeq platform (Illumina, USA) with the MiSeq Reagent Kit v3, 600 Cycles (#MS-102-3003). Plastome annotation. Read quality was assessed via fastqc, and adapter trimming and filtering of low-quality reads were performed using BBduk within Geneious (Kearse et al. 2012 ). The GetOrganelle toolkit (Jin et al. 2020 ) was used for de novo plastome assembly. Specifically for the plastome of C. sanguinea , graph correction using Bandage (Wick et al. 2015 ) and reassembly were executed using the scripts join_spades_fastg_by_blast.py and get_organelle_from_assembly.py to receive an organelle-sufficient graph as described at GetOrganelle flowchart (Jin et al. 2020 ). Plastome annotations were carried out with GeSeq (Tillich et al. 2017 ), PGA (Qu et al. 2019 ), and CPGAVAS2 (Shi et al. 2019 ), using C. capitata (NC_084212.1) as the reference genome, with subsequent manual review and annotation corrections in Geneious. Simple sequence repeats (SSRs) were determined using the MISA software v2.1, 2020-08-25 (Beier et al. 2017 ), with set parameters for unit size and the minimum number of repeats: (1/10) (2/6) (3/5) (4/5) (5/5) (6/5) (Beier et al. 2017 ). Phylogenetic analysis . For the phylogenetic study, 60 plastomes from related species within the Cornaceae, Hydrangeaceae, Nyssaceae, Garryaceae, Curtisiaceae, Grubbiaceae families, and the Arabidopsis thaliana plastome used as an outgroup were downloaded from NSBI. The list of accession numbers and species used for phylogenetic analysis is available at Supplementary Table 1. Multiple sequence alignment was performed using MAFFT (Katoh 2002 ), implemented in Geneious platform, followed by trimming using TrimAl (Capella-Gutiérrez et al. 2009 ). The phylogenetic analysis was carried out using the neighbor-joining method with 500 bootstrap replicates and the Tamura-Nei model in Geneious (Kearse et al. 2012 ). Data visualization . Visualization of plastomes was done using the R programming language ((R Core Team 2018 ), version 4.2.1) and package "chloroplot" (Zheng et al. 2020 ). The visual representation of Inverted Repeats (IRs), Small Single Copy (SSC), Large Single Copy (LSC), and the genes located on them was performed using the online version of the IRscope tool (Amiryousefi et al. 2018 ). The IRScope webserver allows to processes ten plastomes at a time only, so we analyzed several representative Cornus species to compare their IR junctions. Phylogenetic tree was visualized using the R programming language ((R Core Team 2018 ), version 4.3.2) and package "ggtree". Results Characteristics of the C. sericea and C. sanguinea plastomes Initially, we assembled and characterized the cp genome sequence of C. sericea and C. sanguinea . As shown in Table 1 , the plastomes of the studied species have typical quadripartite structures and fell within the typical size parameters (130,000–160,000 bp), although they slightly exceeded the reference. The number of unique genes, rRNA, and tRNA matched the reference. The GC content showed minimal variation, whereas the lengths of LSC and SSC regions exceeded the reference by 130 to 619 bp; meanwhile, the size of IRs was slightly reduced. Notably, an additional gene and CDS were detected in the plastomes of C. sericea and C. sanguinea (see below). Table 1 The basic chloroplast genome characteristics of C. sericea and C. sanguinea Species C. sericea C. sanguinea C. capitata (reference) Genome size (bp) 158 244 158 663 157 199 GC content (%) 37.9 37.8 38.2 SSC Genome size (bp) 18 711 18 644 18 412 GC content (%) 32.0 31.9 32.4 LSC Genome size (bp) 87 459 87 925 86 306 GC content (%) 36.0 36.0 36.4 IRs Genome size (bp) 26 037 26 047 26 112 GC content (%) 43.1 43.0 43.1 CDS 86 (79) 86 (79) 85 (79) gene 131 (113) 131 (113) 130 (113) rRNA 8 (4) 8 (4) 8 (4) IRs 2 2 2 tRNA 37 (30) 37 (30) 37 (30) Note: the number of unique genes is shown in brackets The studied cp genomes had a typical structure and only slightly differenced from the reference. Therefore, on the next step we have performed an annotation of the resulted assemblies. Annotation of the C. sanguinea and C. sericea plastomes The preliminary result of the annotations using three different packages, GeSeq, PGA, and CPGAVAS2, and comparison with the existing reference plastome showed the multiple genes with inaccurate coordinates of gene start and end, differences in coordinates of introns and exons, errors in the annotation of some tRNAs and genes with short exons, etc., were identified. Typically, these inaccuracies involved genes with short exons, such as rps16, pafI (ycf3), rpoC1, and rps12 (a trans-splicing gene composed of 3 exons from two pre-mRNAs with 1 exon in LSC and pairs of the 2nd and 3rd exons in IRs). Errors were also found in tRNAs with introns or those that were unusually short (under 100 nucleotides), including trnT-CGU or trnG-UCC, trnM-CAU or trnfM-CAU, trnM-CAU or trnI-CAU, trnE-UUC or trnI-GAU tRNAs. Such types of misannotation have been described previously (Qu et al. 2023 ). The list of annotated genes is presented in Table 2 . Table 2 List of genes in annotated genomes of C. sanguinea and C. sericea Group C. sanguinea and C. sericea tRNA tRNA-Ala: trnA-UGC (2) tRNA-Arg: trnR-ACG (2), trnR-UCU tRNA-Asn: trnN-GUU (2) tRNA-Asp: trnD-GUC tRNA-Cys: trnC-GCA tRNA-Gln: trnQ-UUG tRNA-Glu: trnE-UUC tRNA-Gly: trnG-GCC, trnG-UCC* tRNA-His: trnH-GUG tRNA-Ile: trnI-CAU (2), trnI-GAU (2)* tRNA-Leu: trnL-CAA (2), trnL-UAA*, trnL-UAG tRNA-Lys: trnK-UUU* tRNA-Met: trnfM-CAU, trnM-CAU tRNA-Phe: trnF-GAA tRNA-Pro: trnP-UGG tRNA-Ser: trnS-GCU, trnS-GGA, trnS-UGA tRNA-Thr: trnT-GGU, trnT-UGU tRNA-Trp: trnW-CCA tRNA-Tyr: trnY-GUA tRNA-Val: trnV-GAC (2), trnV-UAC* rRNA rrn4.5 rRNA (2), rrn5 rRNA (2), rrn16 rRNA (2), rrn23 rRNA (2) NADH-dehydrogenase ndhA*, ndhB (2)*, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK Photosystem I psaA, psaB, psaC, psaI, psaJ, pafI (ycf3)**, pafII (ycf4), pbf1 Photosystem II psaA, psaB, psaC, psbD, psbH, psbI, psbJ, psbK, psbL, psbM, psbT, psbZ Large subunit of the ribosome (LSU) rpl14, rpl16*, rpl2 (2)*, rpl20, rpl22, rpl23 (2), rpl32, rpl33, rpl36 Small subunit of the ribosome (SSU) rps11, rps12 (2)**, rps14, rps15, rps16, rps18, rps19, rps2, rps3, rps4, rps7 (2), rps8 RNA-polymerase rpoA, rpoB, rpoC1*, rpoC2 Cytochromes ccsA, petA, petB*, petD*, petG, petL, petN, psbD, psbE ATP-synthase atpA, atpB, atpE, atpF*, atpH, atpI Other rbcL, cemA, clpP1**, infA, matK, accD, ycf1 (2), ycf2 (2) * or ** – one or two introns (2) – two gene copies After annotation the visualization of the plastomes was performed. Complete genomic maps for C. sanguinea, C. sericea , and the reference genome of C. capitata are provided in Fig. 2 . The duplicating genes located entirely or partially on the IRs encompass seven protein-encoding genes: ndhB, rpl2, rpl23, rps7, rps12, ycf2, and ycf1; seven tRNAs: trnI-GAU, trnA-UGC, trnL-CAA, trnI-CAU tRNA, trnR-ACG tRNA, trnV-GAC tRNA, trnN-GUU tRNA; along with all rRNAs – rrn4.5, rrn5, rrn16, rrn23. There were nine genes with a single intron – ndhA, ndhB, rpl16, rpl2, rps16, rpoC1, petB, petD, and atpF; and three genes featuring two introns – pafI (ycf3), rps12, and clpP1. Additionally, five tRNA genes include introns – trnG-UCC, trnI-GAU, trnL-UAA, trnK-UUU, trnV-UAC. IR Contraction and Expansion To evaluate the specific features of IRs, which, according to the literature data (Goulding et al. 1996 ; Zhu et al. 2016 ), may help in understanding the mechanisms of genome stability, and in identifying species-specific adaptations and phylogenetic relationships, we performed an analysis of the inverted repeats and their junctions with SSC and LSC in 8 representative Cornus species, revealing differences between the genomes studied (Fig. 3 ). The longest LSC was observed in C. sanguinea , whereas the shortest was in C. multivernosa . The length of SSC also differed between the studied species: the longest SSC was found in C. bretschneideri and the shortest in C. kousa . The rps19 gene was almost entirely located on the LSC, with its first 6 ( C. sericea ) and 46 ( C. sanguinea ) nucleotides on the IRB (LSC/IRb junction). The ycf1 gene was partially located on the SSC and IRa and was 5469 bp in both species. It starts 977 bp within IRa and extends 4,462 bp into the SSC in C. sericea , closely resembling the junction in C. bretschneideri and C. wilsoniana . When comparing IRs in some species shown in Fig. 3 , it is evident that this gene was partially on the SSC (4331–4498 bp) and on the IRa (977–1114 bp) in the all studied species. Copies of the ycf1 gene 1044 bp in length in C. sericea and 1059 bp in C. sanguinea were also found, located at the SSC/IRb junction. In the genome of C. capitata , which was used as a reference, the ycf1 gene was annotated only once, as well as in C. bretschneideri . Therefore, despite IRScope only generates visual data it has enabled a detailed evaluation of the structure of IRs and their junctions with SSC and LSC in the studied Cornus species. The search for SSRs SSRs or microsatellites are short tandemly repeated DNA motifs from 1 to 6 bp, and important sources of adaptive genetic variation (Kashi and King 2006 ). In the genomic analysis of C. sanguinea and C. sericea , a total of 53 SSRs were revealed for each species (Table 3 ). Notably, there was a complete lack of tetra-, penta-, and hexa-nucleotide repeats in the studied samples. When compare with reference C. capitata , the less frequency of SSRs was found. At the same time, in more distinct A. thaliana the other pattern of SSR was found, confirming the significance of SSRs search in species differentiation. Table 3 Classification and frequency of SSRs within the chloroplast genomes of C. sanguinea and C. sericea, C. capitata and A. thaliana Type Repeat C. sanguinea C. sericea C. capitata A. thaliana Mono- A 22 22 8 30 G - - - 1 T 29 29 10 38 Di- AT - - - 4 TA 1 1 1 2 Tri- TTA 1 1 - 1 AAT - - - 1 Phylogenetic study To elucidate the evolutionary relationships between species belonging to the Cornaceae, Hydrangeaceae, Nyssaceae, Garryaceae, Curtisiaceae, Grubbiaceae families, and Arabidopsis thaliana , which served as the outgroup, a phylogenetic tree was constructed based on their complete plastid genomes (Fig. 4 ). The sequenced plastomes of C. sericea and C. sanguinea appear within a cluster of Cornus species. This cluster was a part of a larger clade including Alangium species, suggesting that these genera share a common ancestor which is in line with the existing literature data (Du et al. 2023 ; Guan et al. 2024 ). The studied Cornus species formed four distinct clades, as it is described earlier: 1) the blue, white, or black-fruited dogwoods (BW); 2) the cornelian cherries (CC); 3) the big‐bracted dogwoods (BB); and 4) the dwarf dogwoods (DW) (Du et al. 2023 ). As it is seen from the results, the C. sanguinea sample was placed together with available in NCBI sample of C. sanguinea (MN380667.1) from China. The newly sequenced C. sericea plastome was placed together with C. sanguinea samples, C. bretschneideri, C. macrophylla, C. alba , and C. walteri , forming the sister branches. Both C. sericea and C. sanguinea plastomes were within BW clade. Pairwise alignment of C. sanguinea and C. sericea specimens showed an impressive 99.4% identity. However, this identity dropped to 87.8% when the reference sequence was added. Discussion In present study, the complete chloroplast genomes of C. sanguinea and C. sericea have been characterized, providing additional genetic information for these plants. The chloroplast genomes of Cornus species have been extensively studied due to their importance in understanding phylogenetic relationships and evolutionary adaptations within the genus. The chloroplast genome size for both C. sericea and C. sanguinea reported in our study shows slight deviations from the reference species C. capitata , with minor differences in the SSC and LSC regions. The cp genomes had typical size being 158 244 and 158 663 bp for C. sericea and C. sanguinea , respectively. For example, the cp genome size of C. elliptica reported by Lu et al. (2021) was 157,400 bp, while Li et al. ( 2020 ) described the 158,270 bp genome of C. bretschneideri . Lv et al. ( 2019 ) provided details on the 157,446 bp cp genome of C. sunhangii , and Yuan et al. ( 2021 ) reported the 158,451 bp genome of C. alba . A total of 131 genes were found, including 86 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. These results are align with some of previous studies (Lv et al. 2019 ; Guan et al. 2024 ). However, Li et al. ( 2020 ) characterized the complete chloroplast genome of C. bretschneideri , where the genome organization and gene content were comparable to the findings of our study with C. sericea and C. sanguinea , except for protein-coding genes, which number was a bit higher (132 vs 131) (Li et al. 2020 ). Additionally, at the study of Yuan et al. ( 2021 ) on C. alba the similar structural features, such as the size of LSC and SSC regions were found, whereas the number of protein-coding genes and tRNA was 132 and 38, respectively (Yuan et al. 2021 ). The variation in the length of IR, LSC, and SSC regions observed across different Cornus species in our study provides insight into the plastome structural dynamics and their evolutionary implications (Goulding et al. 1996 ; Kashi and King 2006 ; Zhu et al. 2016 ). The contraction and expansion at the IR boundaries, particularly affecting the rps19 and ycf1 genes, are phenomena widely reported in the literature and are thought to contribute to genomic plasticity and adaptation. In early study of Goulding et al. ( 1996 ), the authors studied the movement of the IR of chloroplast DNA in flowering plants and noted the frequent small expansion and contraction near the rps19 gene even closely related flowering species (Goulding et al. 1996 ). In cucumbers, comparative chloroplast genome analyses revealed significant variations in the LSC and SSC regions, especially in Indian ecotype, which may correlate with the plant's adaptation to temperature variations (Xia et al. 2023 ). This study provides insights into how structural changes in chloroplast genomes can influence plant responses to environmental stresses. In study Zhu et al. ( 2016 ), the authors showed that genes moved from the SC into the IR exhibited lower synonymous substitution rates comparable to other IR genes and vice versa – genes moved from the IR into the SC had higher rates of substitution like other SC genes (Zhu et al. 2016 ). The number of SSRs was higher than that in reference genome C. capitata , and there was a complete lack of tetra-, penta-, and hexa-nucleotide repeats in the studied samples. The results obtained differ from the study of Guan et al. ( 2024 ), where the only 25–31 SSRs were identified in 10 taxa of Cornus subg. Syncarpea ; at the same time, the mononucleotide repeats were more frequent in those cp genomes, which is consistent with the observed data (Guan et al. 2024 ). The phylogenetic placement of C. sericea and C. sanguinea within the Cornus clade and their close relationship to other species in the Cornaceae family supports the data of a shared evolutionary ancestry (Zhang et al. 2020 ). The results of the study are consistent with the available data indicating the presence of four distinct clades within Cornus genus (Du et al. 2023 ). In present study, both C. sericea and C. sanguinea plastomes were within BW clade. C. sanguinea cp genome was placed together with available in NCBI database sample of C. sanguinea (MN380667.1, isolate FCN1796) from China, indicating a high similarity between these cpDNA. According to DEC analysis performed by Du et al. (2022), the “BW clade likely had an ancestor in eastern Asia, which diversified and spread into North America and Europe”. However, at the same study the authors showed that the plastid phylogeny without nuclear data cannot provide a comprehensive analysis of the biogeography and evolution, resulting in species nesting discordance (Du et al. 2023 ). Also, the high genomic identity between these two species compared to the reference sequence highlights relatively low divergence within the Cornus genus and consistent with existing studies (Keir et al. 2011 ; Guan et al. 2024 ). Conclusion In summary, the cpDNA of two Cornus species, C. sericea and C. sanguinea , while conserved in overall structure, does display slight variations. The study has provided new insights into the structure and features of the C. sericea cp genome, clarified the available data about C. sanguinea cpDNA and their classification within the Cornus clade. The comparative plastome analysis of the species growing in the Botanical Garden of VILAR provides data for further studies on the possible impact of the biogeography on cpDNA variation and resolving the phylogenetic positions of these species within the Cornaceae family. Declarations Conflicts of Interest: The authors declare no conflicts of interest Funding: The study was carried out in accordance with the State task on the topic FGUU 2024-0001. Author Contribution EN designed the research, collected samples, analyzed the data, wrote the draft; AS and NT performed DNA isolation and sequencing; OK reviewed and commented on the manuscript; DB participated in the data analysis and edited the final version of the manuscript. Acknowledgements: The authors express their gratitude to the staff at the Botanical Garden of the VILAR who took care of the plants and made it possible to obtain samples for the research. Data Archiving Statement Sequence data that support the findings of this study have been deposited in GenBank (accession numbers PP811661 and PP818646) and will be released to the public database when they appear in print. 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American J of Botany 98:1327–1336. https://doi.org/10.3732/ajb.1000466 Li X, Ma Q, Zhou H, Yang Y, Li H, Wang J (2020) Characterization of the complete chloroplast genome of Cornus bretschneideri (cornaceae). Mitochondrial DNA Part B 5:543–544. https://doi.org/10.1080/23802359.2019.1710281 Liesebach H, Götz B (2008) Low Chloroplast DNA Diversity in Red Dogwood (Cornus sanguinea L.). Silvae Genetica 57:291–300. https://doi.org/10.1515/sg-2008-0044 Lv Z-Y, Huang X-H, Luo J, Zhang X, Deng T, Li Z-M (2019) The complete chloroplast genome sequence of Cornus sunhangii (Cornaceae). Mitochondrial DNA Part B 4:3242–3243. https://doi.org/10.1080/23802359.2019.1669090 Qu X-J, Moore MJ, Li D-Z, Yi T-S (2019) PGA: a software package for rapid, accurate, and flexible batch annotation of plastomes. Plant Methods 15:50. https://doi.org/10.1186/s13007-019-0435-7 Qu X-J, Zou D, Zhang R-Y, Stull GW, Yi T-S (2023) Progress, challenge and prospect of plant plastome annotation. Front Plant Sci 14:1166140. https://doi.org/10.3389/fpls.2023.1166140 R Core Team (2018) R: A language and environment for statistical computing Shi C, Hu N, Huang H, Gao J, Zhao Y-J, Gao L-Z (2012) An Improved Chloroplast DNA Extraction Procedure for Whole Plastid Genome Sequencing. PLoS ONE 7:e31468. https://doi.org/10.1371/journal.pone.0031468 Shi L, Chen H, Jiang M, Wang L, Wu X, Huang L, Liu C (2019) CPGAVAS2, an integrated plastome sequence annotator and analyzer. Nucleic Acids Research 47:W65–W73. https://doi.org/10.1093/nar/gkz345 Thomas SK, Liu X, Du Z, Dong Y, Cummings A, Pokorny L, Xiang Q (Jenny), Leebens‐Mack JH (2021) Comprehending Cornales: phylogenetic reconstruction of the order using the Angiosperms353 probe set. American J of Botany 108:1112–1121. https://doi.org/10.1002/ajb2.1696 Tillich M, Lehwark P, Pellizzer T, Ulbricht-Jones ES, Fischer A, Bock R, Greiner S (2017) GeSeq – versatile and accurate annotation of organelle genomes. Nucleic Acids Research 45:W6–W11. https://doi.org/10.1093/nar/gkx391 Wick RR, Schultz MB, Zobel J, Holt KE (2015) Bandage: interactive visualization of de novo genome assemblies. Bioinformatics 31:3350–3352. https://doi.org/10.1093/bioinformatics/btv383 Xia L, Wang H, Zhao X, Obel HO, Yu X, Lou Q, Chen J, Cheng C (2023) Chloroplast Pan-Genomes and Comparative Transcriptomics Reveal Genetic Variation and Temperature Adaptation in the Cucumber. IJMS 24:8943. https://doi.org/10.3390/ijms24108943 Xiang Q-Y, Brunsfeld SJ, Soltis DE, Soltis PS (1996) Phylogenetic Relationships in Cornus Based on Chloroplast DNA Restriction Sites: Implications for Biogeography and Character Evolution. Systematic Botany 21:515. https://doi.org/10.2307/2419612 Xiong Y, Xiong Y, Jia X, Zhao J, Yan L, Sha L, Liu L, Yu Q, Lei X, Bai S, Ma X (2023) Divergence in Elymus sibiricus is related to geography and climate oscillation: A new look from pan‐chloroplast genome data. J of Sytematics Evolution jse.13020. https://doi.org/10.1111/jse.13020 Yuan W, He S, Zhang S, Chang D, He Y (2021) The complete chloroplast genome sequence of Cornus Alba L. (Cornaceae). Mitochondrial DNA Part B 6:1997–1998. https://doi.org/10.1080/23802359.2021.1938727 Zhang C, Zhang T, Luebert F, Xiang Y, Huang C-H, Hu Y, Rees M, Frohlich MW, Qi J, Weigend M, Ma H (2020) Asterid Phylogenomics/Phylotranscriptomics Uncover Morphological Evolutionary Histories and Support Phylogenetic Placement for Numerous Whole-Genome Duplications. Molecular Biology and Evolution 37:3188–3210. https://doi.org/10.1093/molbev/msaa160 Zheng S, Poczai P, Hyvönen J, Tang J, Amiryousefi A (2020) Chloroplot: An Online Program for the Versatile Plotting of Organelle Genomes. Front Genet 11:576124. https://doi.org/10.3389/fgene.2020.576124 Zhu A, Guo W, Gupta S, Fan W, Mower JP (2016) Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates. New Phytologist 209:1747–1756. https://doi.org/10.1111/nph.13743 Additional Declarations No competing interests reported. Supplementary Files Supp.Table1.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-5965562","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":412551338,"identity":"ebd0811d-8d27-4922-bd6a-891aa175dd4f","order_by":0,"name":"Eugenia Nikonorova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYJCCA0AswwZmVgAxM3MDAQ3MQC0JDDwQLWdAAoyEtTCAtIDZjG1gEr8WeffzBw/8/HGYh0+6+eHjynm10fztQC0/Krbh1GJ4JpnhYE/CYR42mWPGhme3Hc+dcZixgbHnzG3cWhqSGQ7wgLRIJJhJNm47ltsA1MLM2IZHS/9jhoN/wFrSv/9snHMsdz4hLfISyQyHIbbkmDE2NtTkbiCkxUDiscFhmbR0kJZiyYZjB3I3ArUcxOcX+f7Exx/f2FjLyc9I3/ixoaYud975wwcf/KjAY8sBVP5hMHkAQx2yLQ2o/Dp8ikfBKBgFo2CEAgBOI1vBt+fncAAAAABJRU5ErkJggg==","orcid":"","institution":"All-Russian Research Institute of Medicinal and Aromatic Plants (VILAR)","correspondingAuthor":true,"prefix":"","firstName":"Eugenia","middleName":"","lastName":"Nikonorova","suffix":""},{"id":412551339,"identity":"90b25001-f7be-41f1-a046-d56de0da0d68","order_by":1,"name":"Alexandr Shevtsov","email":"","orcid":"","institution":"National Center for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Alexandr","middleName":"","lastName":"Shevtsov","suffix":""},{"id":412551340,"identity":"926ac510-8135-45f3-98e2-5f904e84b159","order_by":2,"name":"Nailya Tursunbay","email":"","orcid":"","institution":"National Center for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Nailya","middleName":"","lastName":"Tursunbay","suffix":""},{"id":412551341,"identity":"53459e2f-db18-4498-bae1-df8dc6b5d8a9","order_by":3,"name":"Oxana Khapilina","email":"","orcid":"","institution":"National Center for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Oxana","middleName":"","lastName":"Khapilina","suffix":""},{"id":412551342,"identity":"636fbd93-023c-42a4-ac83-ff86bc2c2e9c","order_by":4,"name":"Dmitry Baleev","email":"","orcid":"","institution":"All-Russian Research Institute of Medicinal and Aromatic Plants (VILAR)","correspondingAuthor":false,"prefix":"","firstName":"Dmitry","middleName":"","lastName":"Baleev","suffix":""}],"badges":[],"createdAt":"2025-02-05 12:08:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5965562/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5965562/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75933436,"identity":"842d9c58-0c47-465d-9fed-ecc9cf9d00ff","added_by":"auto","created_at":"2025-02-10 16:31:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":580406,"visible":true,"origin":"","legend":"\u003cp\u003ePhotos of respective \u003cem\u003eCornus sanguinea\u003c/em\u003e L. (A) and \u003cem\u003eCornus sericea \u003c/em\u003eL. (B) used in this study\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5965562/v1/2709dd282be07f533628c2f8.png"},{"id":75932231,"identity":"e0257226-8f68-4ade-9f41-93672d442def","added_by":"auto","created_at":"2025-02-10 16:15:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":319179,"visible":true,"origin":"","legend":"\u003cp\u003eMaps of the complete chloroplast genome of \u003cem\u003eC. sanguinea \u003c/em\u003eand\u003cem\u003e C. sericea. \u003c/em\u003eThe circles are commented from the inner to outer: \u003cem\u003e\u0026nbsp;\u003c/em\u003ethe length and location of the corresponding SSC, IRa and IRb, and LSC regions are shown in the inner circle; the GC content is represented as the proportion of the shaded parts where the zero-level based on the outer part of circle; the gene names and their codon usage bias are shown on the outermost layer, with pseudogenes marked with asterisks; genes are color-coded by their functional classification; genes inside the circle are transcribed clockwise, genes outside the circle anticlockwise (also represented with arrows).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5965562/v1/a2e9a9ba2941abe57dbf8cf9.png"},{"id":75932251,"identity":"613c9637-ab78-46bb-89c3-3aca658f479f","added_by":"auto","created_at":"2025-02-10 16:15:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":266984,"visible":true,"origin":"","legend":"\u003cp\u003eLSC/IR and IR/SSC of 8 representative \u003cem\u003eCornus\u003c/em\u003e species. Note: Two taxa of Cornus used in this study are depicted at the very bottom. JLB (IRb /LSC), JSB (IRb/SSC), JSA (SSC/IRa) and JLA (IRa/LSC) denote the junctions between each corresponding region. The genes located near the junctions are the realistically scaled with the bp distances. The arrows indicate the bp distance of the start or end coordinate of gene from the junction site.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5965562/v1/cd15580e779bbe9dda6a0b93.png"},{"id":75932235,"identity":"8a771290-283d-4afc-b2fc-c00a9d909f4b","added_by":"auto","created_at":"2025-02-10 16:15:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":96600,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree inferred by the neighbor-joining method with 500 bootstrap replicates and the Tamura-Nei model based on 60 representative species. Clades withing the \u003cem\u003eCornus \u003c/em\u003egenus are highlighted by different colors: blue color for BW clade, green – for CC clade, yellow – for BB clade, and red – for DW clade.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5965562/v1/db5c680ba8f3b6da37fdfc8f.png"},{"id":76148399,"identity":"feedff55-301e-4841-8963-5cc9125b38b0","added_by":"auto","created_at":"2025-02-12 21:16:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2083046,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5965562/v1/7a549f38-92ba-4844-874f-1fa634f619fb.pdf"},{"id":75932250,"identity":"b3f734ed-493b-4872-834d-7727ff01a4a3","added_by":"auto","created_at":"2025-02-10 16:15:46","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13996,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5965562/v1/e9eae432a412c7bedd20c0d9.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The complete chloroplast genome sequences and phylogenetics of Cornus sanguinea L. and Cornus sericea L. (Cornaceae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe family Cornaceae includes 3 genera and over a hundred species, which, along with such families as Curtisiaceae, Grubbiaceae, Hydrangeaceae, Hydrostachyaceae, Loasaceae, and Nyssaceae, are the part of \u003cem\u003eCornales\u003c/em\u003e order (Thomas et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The majority of species within the family belong to the genus \u003cem\u003eCornus\u003c/em\u003e. The dogwoods (\u003cem\u003eCornus\u003c/em\u003e spp.) are not only prized for their ornamental beauty but play a significant role in nature, biology, and medicine (Kazimierski et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Badoni et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Tenuta et al. (2022) in the comprehensive review highlighted that the edible fruits of European and Asian \u003cem\u003eCornus\u003c/em\u003e species are a rich source of phytochemicals with nutritional and functional properties. For example, \u003cem\u003eC. mas\u003c/em\u003e (cornelian cherry) fruits and leaves are used in traditional medicine for treatment of diabetes, obesity, atherosclerosis, skin diseases, gastrointestinal and rheumatic disorders (Dinda et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It was shown that \u003cem\u003eC. officinalis\u003c/em\u003e possess the high range of pharmacological properties, such as hepatic and renal protection, antitumor, neuroprotective, antidiabetic effect, anti-inflammatory, antioxidant and immunoregulatory activities, etc (Huang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe conserved nature of the chloroplast (cp) genome, along with its well-defined structure and gene content has provided valuable insights into the genetic diversity and evolutionary relationships within the Cornaceae family (Daniell et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Guan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The first study of chloroplast DNA (cpDNA) variability by PCR-RFLP analysis of the \u003cem\u003eC. sanguinea\u003c/em\u003e was performed by H. Liesebach and B. G\u0026ouml;tz (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), who reported the presence of eight different haplotypes across different European populations without association with geographic occurrence and low level of variation (Liesebach and G\u0026ouml;tz \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Lv et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported the complete cp genome sequences of \u003cem\u003eC. sunhangii\u003c/em\u003e, giving a foundation for further studies on these species (Lv et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Li et al. (Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) also contributed to this field by characterizing the cp genome of \u003cem\u003eC. bretschneideri\u003c/em\u003e, respectively, further expanding our understanding of the genetic features of these plants. However, despite the recognized importance of the Cornaceae, the phylogenetic relationships within this family have been controversial and complex (Xiang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Du et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Guan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The classification of species within the \u003cem\u003eCornus\u003c/em\u003e genus has seen diverse interpretations, with the taxonomy undergoing multiple revisions as new evidence comes to light (Du et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In other species it's been found that cp genomes can vary depending on the species and their geographic distribution, which is related to adaptation to local climatic conditions (Xiong et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Keir et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) revealed the relatively low diversity in cpDNA haplotypes of \u003cem\u003eC. nuttallii\u003c/em\u003e, collected from 20 native populations of southern California and northern Idaho, USA (Keir et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In study of Guan et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) plastid data of species growing from China to the USA were studied (Guan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) \u0026ndash; however, they did not include any species native to Russia.\u003c/p\u003e \u003cp\u003eIn the VILAR Botanical Garden, two species of dogwood (\u003cem\u003eC. sanguinea\u003c/em\u003e and \u003cem\u003eC. sericea\u003c/em\u003e) are cultivated and included in the collection. These species are thoroughly studied, including their genetic resources. However, there is no data on the structure and characteristics of the cp genome of \u003cem\u003eC. sericea\u003c/em\u003e (no verified sequence was available in the NCBI database at the time of writing), and the studies on the chloroplast genome of \u003cem\u003eC. sanguinea\u003c/em\u003e are limited (only one representative - FCN1796 from China - has been identified). Therefore, the aim of this study was to characterize and compare the structure and gene organization of the plastid genomes of \u003cem\u003eC. sanguinea\u003c/em\u003e and \u003cem\u003eC. sericea\u003c/em\u003e from the medicinal plant collection of the VILAR Botanical Garden.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cem\u003ePlant material.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFresh mature leaves of \u003cem\u003eCornus sanguinea\u003c/em\u003e L. (voucher specimen VF 004.21) and \u003cem\u003eCornus sericea\u003c/em\u003e L. (voucher specimen VF 006.21) were collected in July at the Botanical Garden of the VILAR (55\u0026deg;33'51.8\"N; 37\u0026deg;35'56.8\"E), located in urban zone of Moscow with warm-summer humid continental climate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eChloroplasts isolation.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eChloroplasts were isolated using high ionic strength solutions based on a technique described by Bookjans et al. (Bookjans et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Briefly, after incubation in the dark at 4\u0026deg;C, fresh leaves were homogenized in a Waring blender in 50 mM Tris, 25 mM EDTA, 10 mM mercaptoethanol, 0.1% BSA and 1.25 M NaCl (pH\u0026thinsp;=\u0026thinsp;8.0). The homogenate was filtered and centrifuged for 5 minutes at 1500 \u003cem\u003eg\u003c/em\u003e (Eppendorf, Germany), the pellet was resuspended in the homogenization buffer, and the centrifugation was repeated for 5 minutes at 1500 \u003cem\u003eg\u003c/em\u003e. The resulting chloroplast pellet was resuspended in a 50 mM Tris, 10 mM EDTA (pH\u0026thinsp;=\u0026thinsp;8.0) and used for DNA extraction.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDNA extraction.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCpDNA was extracted following the method described by Shi et al. (Shi et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The analysis of the extracted DNA fragments was performed by DNA separation in a 0.8% agarose gel containing ethidium bromide. Results were documented using a Gel Doc system (Bio-Rad, USA) with Quantity One software (Bio-Rad, USA). DNA concentrations were measured fluorometrically with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, MA, USA). DNA library preparation was performed using the Illumina DNA Prep, (M) Tagmentation kit (Cat. No. 20060060, Illumina, USA), following the manufacturer's guidelines. Libraries were normalized and then pooled to receive a 14 pM concentration, including a 1% PhiX control. Whole-genome sequencing was performed on the MiSeq platform (Illumina, USA) with the MiSeq Reagent Kit v3, 600 Cycles (#MS-102-3003).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePlastome annotation.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eRead quality was assessed via fastqc, and adapter trimming and filtering of low-quality reads were performed using BBduk within Geneious (Kearse et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The GetOrganelle toolkit (Jin et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) was used for \u003cem\u003ede novo\u003c/em\u003e plastome assembly. Specifically for the plastome of \u003cem\u003eC. sanguinea\u003c/em\u003e, graph correction using Bandage (Wick et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and reassembly were executed using the scripts join_spades_fastg_by_blast.py and get_organelle_from_assembly.py to receive an organelle-sufficient graph as described at GetOrganelle flowchart (Jin et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Plastome annotations were carried out with GeSeq (Tillich et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), PGA (Qu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and CPGAVAS2 (Shi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), using \u003cem\u003eC. capitata\u003c/em\u003e (NC_084212.1) as the reference genome, with subsequent manual review and annotation corrections in Geneious. Simple sequence repeats (SSRs) were determined using the MISA software v2.1, 2020-08-25 (Beier et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), with set parameters for unit size and the minimum number of repeats: (1/10) (2/6) (3/5) (4/5) (5/5) (6/5) (Beier et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePhylogenetic analysis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFor the phylogenetic study, 60 plastomes from related species within the Cornaceae, Hydrangeaceae, Nyssaceae, Garryaceae, Curtisiaceae, Grubbiaceae families, and the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e plastome used as an outgroup were downloaded from NSBI. The list of accession numbers and species used for phylogenetic analysis is available at Supplementary Table\u0026nbsp;1. Multiple sequence alignment was performed using MAFFT (Katoh \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), implemented in Geneious platform, followed by trimming using TrimAl (Capella-Guti\u0026eacute;rrez et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The phylogenetic analysis was carried out using the neighbor-joining method with 500 bootstrap replicates and the Tamura-Nei model in Geneious (Kearse et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eData visualization\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eVisualization of plastomes was done using the R programming language ((R Core Team \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), version 4.2.1) and package \"chloroplot\" (Zheng et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The visual representation of Inverted Repeats (IRs), Small Single Copy (SSC), Large Single Copy (LSC), and the genes located on them was performed using the online version of the IRscope tool (Amiryousefi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The IRScope webserver allows to processes ten plastomes at a time only, so we analyzed several representative \u003cem\u003eCornus\u003c/em\u003e species to compare their IR junctions. Phylogenetic tree was visualized using the R programming language ((R Core Team \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), version 4.3.2) and package \"ggtree\".\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCharacteristics of the C. sericea and C. sanguinea plastomes\u003c/h2\u003e \u003cp\u003eInitially, we assembled and characterized the cp genome sequence of \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the plastomes of the studied species have typical quadripartite structures and fell within the typical size parameters (130,000\u0026ndash;160,000 bp), although they slightly exceeded the reference. The number of unique genes, rRNA, and tRNA matched the reference. The GC content showed minimal variation, whereas the lengths of LSC and SSC regions exceeded the reference by 130 to 619 bp; meanwhile, the size of IRs was slightly reduced. Notably, an additional gene and CDS were detected in the plastomes of \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e (see below).\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 basic chloroplast genome characteristics of \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC. sericea\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. sanguinea\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eC. capitata\u003c/em\u003e (reference)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eGenome size (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e158 244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e158 663\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e157 199\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eGC content (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenome size (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18 644\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 412\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGC content (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenome size (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87 459\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87 925\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e86 306\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGC content (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIRs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenome size (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26 047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26 112\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGC content (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eCDS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86 (79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86 (79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85 (79)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003egene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e131 (113)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e131 (113)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e130 (113)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003erRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eIRs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003etRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37 (30)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eNote: the number of unique genes is shown in brackets\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe studied cp genomes had a typical structure and only slightly differenced from the reference. Therefore, on the next step we have performed an annotation of the resulted assemblies.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnnotation of the C. sanguinea and C. sericea plastomes\u003c/h3\u003e\n\u003cp\u003eThe preliminary result of the annotations using three different packages, GeSeq, PGA, and CPGAVAS2, and comparison with the existing reference plastome showed the multiple genes with inaccurate coordinates of gene start and end, differences in coordinates of introns and exons, errors in the annotation of some tRNAs and genes with short exons, etc., were identified. Typically, these inaccuracies involved genes with short exons, such as rps16, pafI (ycf3), rpoC1, and rps12 (a trans-splicing gene composed of 3 exons from two pre-mRNAs with 1 exon in LSC and pairs of the 2nd and 3rd exons in IRs). Errors were also found in tRNAs with introns or those that were unusually short (under 100 nucleotides), including trnT-CGU or trnG-UCC, trnM-CAU or trnfM-CAU, trnM-CAU or trnI-CAU, trnE-UUC or trnI-GAU tRNAs. Such types of misannotation have been described previously (Qu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The list of annotated genes is presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\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\u003eList of genes in annotated genomes of \u003cem\u003eC. sanguinea\u003c/em\u003e and \u003cem\u003eC. sericea\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC. sanguinea\u003c/em\u003e and \u003cem\u003eC. sericea\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\u003etRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etRNA-Ala: trnA-UGC (2)\u003c/p\u003e \u003cp\u003etRNA-Arg: trnR-ACG (2), trnR-UCU\u003c/p\u003e \u003cp\u003etRNA-Asn: trnN-GUU (2)\u003c/p\u003e \u003cp\u003etRNA-Asp: trnD-GUC\u003c/p\u003e \u003cp\u003etRNA-Cys: trnC-GCA\u003c/p\u003e \u003cp\u003etRNA-Gln: trnQ-UUG\u003c/p\u003e \u003cp\u003etRNA-Glu: trnE-UUC\u003c/p\u003e \u003cp\u003etRNA-Gly: trnG-GCC, trnG-UCC*\u003c/p\u003e \u003cp\u003etRNA-His: trnH-GUG\u003c/p\u003e \u003cp\u003etRNA-Ile: trnI-CAU (2), trnI-GAU (2)*\u003c/p\u003e \u003cp\u003etRNA-Leu: trnL-CAA (2), trnL-UAA*, trnL-UAG\u003c/p\u003e \u003cp\u003etRNA-Lys: trnK-UUU*\u003c/p\u003e \u003cp\u003etRNA-Met: trnfM-CAU, trnM-CAU\u003c/p\u003e \u003cp\u003etRNA-Phe: trnF-GAA\u003c/p\u003e \u003cp\u003etRNA-Pro: trnP-UGG\u003c/p\u003e \u003cp\u003etRNA-Ser: trnS-GCU, trnS-GGA, trnS-UGA\u003c/p\u003e \u003cp\u003etRNA-Thr: trnT-GGU, trnT-UGU\u003c/p\u003e \u003cp\u003etRNA-Trp: trnW-CCA\u003c/p\u003e \u003cp\u003etRNA-Tyr: trnY-GUA\u003c/p\u003e \u003cp\u003etRNA-Val: trnV-GAC (2), trnV-UAC*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003erRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003errn4.5 rRNA (2), rrn5 rRNA (2), rrn16 rRNA (2), rrn23 rRNA (2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNADH-dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003endhA*, ndhB (2)*, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhotosystem I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epsaA, psaB, psaC, psaI, psaJ, pafI (ycf3)**, pafII (ycf4),\u003c/p\u003e \u003cp\u003epbf1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhotosystem II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epsaA, psaB, psaC, psbD, psbH, psbI, psbJ, psbK, psbL, psbM, psbT, psbZ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarge subunit of the ribosome (LSU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erpl14, rpl16*, rpl2 (2)*, rpl20, rpl22, rpl23 (2), rpl32, rpl33, rpl36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmall subunit of the ribosome (SSU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erps11, rps12 (2)**, rps14, rps15, rps16, rps18, rps19, rps2, rps3, rps4, rps7 (2), rps8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRNA-polymerase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erpoA, rpoB, rpoC1*, rpoC2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCytochromes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eccsA, petA, petB*, petD*, petG, petL, petN, psbD, psbE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATP-synthase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eatpA, atpB, atpE, atpF*, atpH, atpI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erbcL, cemA, clpP1**, infA, matK, accD, ycf1 (2), ycf2 (2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e* or ** \u0026ndash; one or two introns\u003c/p\u003e \u003cp\u003e(2) \u0026ndash; two gene copies\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter annotation the visualization of the plastomes was performed. Complete genomic maps for \u003cem\u003eC. sanguinea, C. sericea\u003c/em\u003e, and the reference genome of \u003cem\u003eC. capitata\u003c/em\u003e are provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The duplicating genes located entirely or partially on the IRs encompass seven protein-encoding genes: ndhB, rpl2, rpl23, rps7, rps12, ycf2, and ycf1; seven tRNAs: trnI-GAU, trnA-UGC, trnL-CAA, trnI-CAU tRNA, trnR-ACG tRNA, trnV-GAC tRNA, trnN-GUU tRNA; along with all rRNAs \u0026ndash; rrn4.5, rrn5, rrn16, rrn23. There were nine genes with a single intron \u0026ndash; ndhA, ndhB, rpl16, rpl2, rps16, rpoC1, petB, petD, and atpF; and three genes featuring two introns \u0026ndash; pafI (ycf3), rps12, and clpP1. Additionally, five tRNA genes include introns \u0026ndash; trnG-UCC, trnI-GAU, trnL-UAA, trnK-UUU, trnV-UAC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIR Contraction and Expansion\u003c/h3\u003e\n\u003cp\u003eTo evaluate the specific features of IRs, which, according to the literature data (Goulding et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), may help in understanding the mechanisms of genome stability, and in identifying species-specific adaptations and phylogenetic relationships, we performed an analysis of the inverted repeats and their junctions with SSC and LSC in 8 representative \u003cem\u003eCornus\u003c/em\u003e species, revealing differences between the genomes studied (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The longest LSC was observed in \u003cem\u003eC. sanguinea\u003c/em\u003e, whereas the shortest was in \u003cem\u003eC. multivernosa\u003c/em\u003e. The length of SSC also differed between the studied species: the longest SSC was found in \u003cem\u003eC. bretschneideri\u003c/em\u003e and the shortest in \u003cem\u003eC. kousa\u003c/em\u003e. The rps19 gene was almost entirely located on the LSC, with its first 6 (\u003cem\u003eC. sericea\u003c/em\u003e) and 46 (\u003cem\u003eC. sanguinea\u003c/em\u003e) nucleotides on the IRB (LSC/IRb junction). The ycf1 gene was partially located on the SSC and IRa and was 5469 bp in both species. It starts 977 bp within IRa and extends 4,462 bp into the SSC in \u003cem\u003eC. sericea\u003c/em\u003e, closely resembling the junction in \u003cem\u003eC. bretschneideri\u003c/em\u003e and \u003cem\u003eC. wilsoniana\u003c/em\u003e. When comparing IRs in some species shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, it is evident that this gene was partially on the SSC (4331\u0026ndash;4498 bp) and on the IRa (977\u0026ndash;1114 bp) in the all studied species. Copies of the ycf1 gene 1044 bp in length in \u003cem\u003eC. sericea\u003c/em\u003e and 1059 bp in \u003cem\u003eC. sanguinea\u003c/em\u003e were also found, located at the SSC/IRb junction. In the genome of \u003cem\u003eC. capitata\u003c/em\u003e, which was used as a reference, the ycf1 gene was annotated only once, as well as in \u003cem\u003eC. bretschneideri\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTherefore, despite IRScope only generates visual data it has enabled a detailed evaluation of the structure of IRs and their junctions with SSC and LSC in the studied \u003cem\u003eCornus\u003c/em\u003e species.\u003c/p\u003e\n\u003ch3\u003eThe search for SSRs\u003c/h3\u003e\n\u003cp\u003eSSRs or microsatellites are short tandemly repeated DNA motifs from 1 to 6 bp, and important sources of adaptive genetic variation (Kashi and King \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In the genomic analysis of \u003cem\u003eC. sanguinea\u003c/em\u003e and \u003cem\u003eC. sericea\u003c/em\u003e, a total of 53 SSRs were revealed for each species (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, there was a complete lack of tetra-, penta-, and hexa-nucleotide repeats in the studied samples. When compare with reference \u003cem\u003eC. capitata\u003c/em\u003e, the less frequency of SSRs was found. At the same time, in more distinct \u003cem\u003eA. thaliana\u003c/em\u003e the other pattern of SSR was found, confirming the significance of SSRs search in species differentiation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClassification and frequency of SSRs within the chloroplast genomes of \u003cem\u003eC. sanguinea\u003c/em\u003e and \u003cem\u003eC. sericea, C. capitata\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRepeat\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC. sanguinea\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. sericea\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eC. capitata\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eA. thaliana\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\u003eMono-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDi-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTri-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic study\u003c/h2\u003e \u003cp\u003eTo elucidate the evolutionary relationships between species belonging to the Cornaceae, Hydrangeaceae, Nyssaceae, Garryaceae, Curtisiaceae, Grubbiaceae families, and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, which served as the outgroup, a phylogenetic tree was constructed based on their complete plastid genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The sequenced plastomes of \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e appear within a cluster of \u003cem\u003eCornus\u003c/em\u003e species. This cluster was a part of a larger clade including \u003cem\u003eAlangium\u003c/em\u003e species, suggesting that these genera share a common ancestor which is in line with the existing literature data (Du et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Guan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The studied \u003cem\u003eCornus\u003c/em\u003e species formed four distinct clades, as it is described earlier: 1) the blue, white, or black-fruited dogwoods (BW); 2) the cornelian cherries (CC); 3) the big‐bracted dogwoods (BB); and 4) the dwarf dogwoods (DW) (Du et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As it is seen from the results, the \u003cem\u003eC. sanguinea\u003c/em\u003e sample was placed together with available in NCBI sample of \u003cem\u003eC. sanguinea\u003c/em\u003e (MN380667.1) from China. The newly sequenced \u003cem\u003eC. sericea\u003c/em\u003e plastome was placed together with \u003cem\u003eC. sanguinea\u003c/em\u003e samples, \u003cem\u003eC. bretschneideri, C. macrophylla, C. alba\u003c/em\u003e, and \u003cem\u003eC. walteri\u003c/em\u003e, forming the sister branches. Both \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e plastomes were within BW clade.\u003c/p\u003e \u003cp\u003ePairwise alignment of \u003cem\u003eC. sanguinea\u003c/em\u003e and \u003cem\u003eC. sericea\u003c/em\u003e specimens showed an impressive 99.4% identity. However, this identity dropped to 87.8% when the reference sequence was added.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn present study, the complete chloroplast genomes of \u003cem\u003eC. sanguinea\u003c/em\u003e and \u003cem\u003eC. sericea\u003c/em\u003e have been characterized, providing additional genetic information for these plants. The chloroplast genomes of \u003cem\u003eCornus\u003c/em\u003e species have been extensively studied due to their importance in understanding phylogenetic relationships and evolutionary adaptations within the genus. The chloroplast genome size for both \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e reported in our study shows slight deviations from the reference species \u003cem\u003eC. capitata\u003c/em\u003e, with minor differences in the SSC and LSC regions. The cp genomes had typical size being 158 244 and 158 663 bp for \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e, respectively. For example, the cp genome size of \u003cem\u003eC. elliptica\u003c/em\u003e reported by Lu et al. (2021) was 157,400 bp, while Li et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) described the 158,270 bp genome of \u003cem\u003eC. bretschneideri\u003c/em\u003e. Lv et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) provided details on the 157,446 bp cp genome of \u003cem\u003eC. sunhangii\u003c/em\u003e, and Yuan et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported the 158,451 bp genome of \u003cem\u003eC. alba\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eA total of 131 genes were found, including 86 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. These results are align with some of previous studies (Lv et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Guan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, Li et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) characterized the complete chloroplast genome of \u003cem\u003eC. bretschneideri\u003c/em\u003e, where the genome organization and gene content were comparable to the findings of our study with \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e, except for protein-coding genes, which number was a bit higher (132 vs 131) (Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, at the study of Yuan et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) on \u003cem\u003eC. alba\u003c/em\u003e the similar structural features, such as the size of LSC and SSC regions were found, whereas the number of protein-coding genes and tRNA was 132 and 38, respectively (Yuan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe variation in the length of IR, LSC, and SSC regions observed across different \u003cem\u003eCornus\u003c/em\u003e species in our study provides insight into the plastome structural dynamics and their evolutionary implications (Goulding et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Kashi and King \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The contraction and expansion at the IR boundaries, particularly affecting the rps19 and ycf1 genes, are phenomena widely reported in the literature and are thought to contribute to genomic plasticity and adaptation. In early study of Goulding et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), the authors studied the movement of the IR of chloroplast DNA in flowering plants and noted the frequent small expansion and contraction near the rps19 gene even closely related flowering species (Goulding et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). In cucumbers, comparative chloroplast genome analyses revealed significant variations in the LSC and SSC regions, especially in Indian ecotype, which may correlate with the plant's adaptation to temperature variations (Xia et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This study provides insights into how structural changes in chloroplast genomes can influence plant responses to environmental stresses. In study Zhu et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the authors showed that genes moved from the SC into the IR exhibited lower synonymous substitution rates comparable to other IR genes and vice versa \u0026ndash; genes moved from the IR into the SC had higher rates of substitution like other SC genes (Zhu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe number of SSRs was higher than that in reference genome \u003cem\u003eC. capitata\u003c/em\u003e, and there was a complete lack of tetra-, penta-, and hexa-nucleotide repeats in the studied samples. The results obtained differ from the study of Guan et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), where the only 25\u0026ndash;31 SSRs were identified in 10 taxa of \u003cem\u003eCornus\u003c/em\u003e subg. \u003cem\u003eSyncarpea\u003c/em\u003e; at the same time, the mononucleotide repeats were more frequent in those cp genomes, which is consistent with the observed data (Guan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe phylogenetic placement of \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e within the \u003cem\u003eCornus\u003c/em\u003e clade and their close relationship to other species in the Cornaceae family supports the data of a shared evolutionary ancestry (Zhang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The results of the study are consistent with the available data indicating the presence of four distinct clades within \u003cem\u003eCornus\u003c/em\u003e genus (Du et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In present study, both \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e plastomes were within BW clade. \u003cem\u003eC. sanguinea\u003c/em\u003e cp genome was placed together with available in NCBI database sample of \u003cem\u003eC. sanguinea\u003c/em\u003e (MN380667.1, isolate FCN1796) from China, indicating a high similarity between these cpDNA. According to DEC analysis performed by Du et al. (2022), the \u0026ldquo;BW clade likely had an ancestor in eastern Asia, which diversified and spread into North America and Europe\u0026rdquo;. However, at the same study the authors showed that the plastid phylogeny without nuclear data cannot provide a comprehensive analysis of the biogeography and evolution, resulting in species nesting discordance (Du et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Also, the high genomic identity between these two species compared to the reference sequence highlights relatively low divergence within the \u003cem\u003eCornus\u003c/em\u003e genus and consistent with existing studies (Keir et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Guan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, the cpDNA of two \u003cem\u003eCornus\u003c/em\u003e species, \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e, while conserved in overall structure, does display slight variations. The study has provided new insights into the structure and features of the \u003cem\u003eC. sericea\u003c/em\u003e cp genome, clarified the available data about \u003cem\u003eC. sanguinea\u003c/em\u003e cpDNA and their classification within the \u003cem\u003eCornus\u003c/em\u003e clade. The comparative plastome analysis of the species growing in the Botanical Garden of VILAR provides data for further studies on the possible impact of the biogeography on cpDNA variation and resolving the phylogenetic positions of these species within the Cornaceae family.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThe study was carried out in accordance with the State task on the topic FGUU 2024-0001.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eEN designed the research, collected samples, analyzed the data, wrote the draft; AS and NT performed DNA isolation and sequencing; OK reviewed and commented on the manuscript; DB participated in the data analysis and edited the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThe authors express their gratitude to the staff at the Botanical Garden of the VILAR who took care of the plants and made it possible to obtain samples for the research.\u003c/p\u003e \u003cp\u003eData Archiving Statement Sequence data that support the findings of this study have been deposited in GenBank (accession numbers PP811661 and PP818646) and will be released to the public database when they appear in print.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eStatement Sequence data that support the findings of this study have been deposited in GenBank (accession numbers PP811661 and PP818646) and will be released to the public database when they appear in print.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmiryousefi A, Hyv\u0026ouml;nen J, Poczai P (2018) IRscope: an online program to visualize the junction sites of chloroplast genomes. 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Mitochondrial DNA Part B 6:1997\u0026ndash;1998. https://doi.org/10.1080/23802359.2021.1938727\u003c/li\u003e\n\u003cli\u003eZhang C, Zhang T, Luebert F, Xiang Y, Huang C-H, Hu Y, Rees M, Frohlich MW, Qi J, Weigend M, Ma H (2020) Asterid Phylogenomics/Phylotranscriptomics Uncover Morphological Evolutionary Histories and Support Phylogenetic Placement for Numerous Whole-Genome Duplications. Molecular Biology and Evolution 37:3188\u0026ndash;3210. https://doi.org/10.1093/molbev/msaa160\u003c/li\u003e\n\u003cli\u003eZheng S, Poczai P, Hyv\u0026ouml;nen J, Tang J, Amiryousefi A (2020) Chloroplot: An Online Program for the Versatile Plotting of Organelle Genomes. Front Genet 11:576124. https://doi.org/10.3389/fgene.2020.576124\u003c/li\u003e\n\u003cli\u003eZhu A, Guo W, Gupta S, Fan W, Mower JP (2016) Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates. New Phytologist 209:1747\u0026ndash;1756. https://doi.org/10.1111/nph.13743\u003c/li\u003e\n\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":"chloroplast genome, plastids, Cornaceae, Cornus, dogwood","lastPublishedDoi":"10.21203/rs.3.rs-5965562/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5965562/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study provides an in-depth analysis of the chloroplast genomes of two \u003cem\u003eCornus\u003c/em\u003e species, \u003cem\u003eCornus sanguinea\u003c/em\u003e L. and \u003cem\u003eCornus sericea\u003c/em\u003e L., which are significant both in ornamental horticulture and traditional medicine. These species were collected from the Botanical Garden of the VILAR, providing a unique geographic context for genetic examination. Our results indicated that the plastomes of both species have typical quadripartite structure of chloroplast DNA, with slight variations in the size of the Large Single Copy (LSC) and Small Single Copy (SSC) regions compared to other \u003cem\u003eCornus\u003c/em\u003e species. The complete chloroplast genome size of \u003cem\u003eC. sericea\u003c/em\u003e and \u003cem\u003eC. sanguinea\u003c/em\u003e was 158 244 and 158 663 bp, respectively. A total of 131 genes, including 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes were found. The study highlighted the role of simple sequence repeats (SSRs) in genomic differentiation, with a notable absence of tetra-, penta-, and hexa-nucleotide repeats in the studied genomes. This aspect of the genome could be vital for understanding species differentiation and evolution within the genus. Phylogenetic analyses placed \u003cem\u003eC. sanguinea\u003c/em\u003e and \u003cem\u003eC. sericea\u003c/em\u003e within a broader clade of Cornaceae and reflected their close relationship to other species in the Cornaceae family. Overall, our study provides new data about the structure and features of the \u003cem\u003eC. sericea\u003c/em\u003e cp genome and adds the valuable information on cp genome \u003cem\u003eC. sanguinea\u003c/em\u003e, that is necessary for further studies.\u003c/p\u003e","manuscriptTitle":"The complete chloroplast genome sequences and phylogenetics of Cornus sanguinea L. and Cornus sericea L. (Cornaceae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-10 16:15:40","doi":"10.21203/rs.3.rs-5965562/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":"cbaabcde-da3d-46de-bf00-604fb5845ab1","owner":[],"postedDate":"February 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-12T21:08:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-10 16:15:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5965562","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5965562","identity":"rs-5965562","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}