The complete chloroplast genome sequence of the medicinal plant Ardisia crispa (Myrsinaceae)

The complete chloroplast genome sequence of the medicinal plant Ardisia crispa (Myrsinaceae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The complete chloroplast genome sequence of the medicinal plant Ardisia crispa (Myrsinaceae) Juan Ye, Qin Luo, Yun-hu Lang, Ning Ding, Ying-quan Jian, Zhi-kun Wu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4013297/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Ardisia crispa , a member of the Myrsinaceae family, possesses significant horticultural and medicinal properties as an ethnomedicine. The study aimed to analyze the chloroplast genome of A. crispa and compare it with other Ardisia species, revealing a length of 156,785 bp with a quadripartite structure and 131 genes, including 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Furthermore, 59 simple sequence repeat (SSR) sites were identified in the genome. Examination of codon usage within the chloroplast genome indicated a greater inclination towards A/U nucleotides over G/C nucleotides, with leucine displaying the highest frequency among amino acids. The chloroplast genomes of the nine Ardisia species demonstrate conserved gene content and quantity, presenting more consistent boundaries and decreased variability. In the phylogenetic tree, A. crispa is clustered with A. crispa var dielsii , suggesting a close relationship with A. mamillata and A. pedalis . This study involved the construction and analysis of the chloroplast genome structure of A. crispa , as well as phylogenetic analysis using extensive chloroplast genome sequence data from Ardisia plants. This research is crucial for understanding the genetic basis of A. crispa and the adaptive evolution within the Ardisia genus. Biological sciences/Biological techniques/Genomic analysis/Comparative genomics Biological sciences/Biotechnology/Genomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Ardisia crispa (Thunb.) A. DC., a member of the Myrsinaceae family, is predominantly found in the provinces south of the Yangtze River Basin in China. The root of this plant is utilized in traditional medicine and is a key component of the renowned Hmong medicinal specialty, Radix Ardisia, in Guizhou 1,3 . Modern research has identified A. crispa as containing triterpene glycosides, flavonoids, isocoumarins, and other bioactive compounds, known for their therapeutic properties such as relieving heat, soothing the throat, and activating tendons. This plant is commonly used to treat conditions like sore throat, tonsillitis, nephritis, and edema, earning it the nickname ''aryngeal medicine'' 2 . Two subspecies of A. crispa have been identified based on morphological differences, specifically A. crispa var dielsii (characterized by shorter stature and narrow lanceolate leaves) and A. crispa var amplifolia (recognized for its stout stature and broad leaves) 4 . However, the lack of a scientific foundation for this classification hinders a comprehensive understanding of the plant's genetic background, conservation of germplasm resources, and phylogenetic evolution. Furthermore, the structural features of the plastid genome, functional classification, codon preference analysis, and differences in the chloroplast genome between the two variants have not been thoroughly explored. This knowledge gap not only limits our understanding of A. crispa and its variants but also complicates the accurate identification of mixed pseudo-products involving A. crispa in medicinal herb preparations. Chloroplasts are semi-autonomous organelles found in plants, characterized by having the second largest genome in plant cells 5 . The chloroplast genome typically consists of a large single copy (LSC) region, a small single copy (SSC) region, and two inverted repeats (IR) 6 . Due to its uniparental inheritance, moderate mutation rate, and ease of sequencing, the chloroplast genome is often regarded as a more efficient resource compared to nuclear and mitochondrial genomes. Consequently, it is frequently utilized for investigating the origins and evolution of plants, elucidating phylogenetic relationships among different taxonomic classes, and for species identification purposes 7 . In recent years, advancements in high-throughput sequencing technologies have facilitated the successful assembly, annotation, and analysis of a substantial number of subspecies chloroplast genomes. The application of chloroplast genome analysis has yielded promising results in the investigation of identification, genetic relationships, and phylogenetics of various species, including Sabia , Phoebe , Vaccinium , Hibiscus rosa - sinensis , Dalbergia hainanensis , Litsea , and Zingiber 8–14 . Therefore, this research aimed to utilize high-throughput sequencing technology to sequence, assemble, and annotate the complete chloroplast genome of A. crispa . Additionally, comparative analysis of structural features and phylogenetic relationships among A. crispa and its varieties A. crispa var dielsii and A. crispa var amplifolia , as well as other Ardisia species, was conducted using bioinformatics tools. The findings of this study contribute to the understanding of species identification, phylogeny, and conservation efforts related to A. crispa and other medicinal herbs. Results Chloroplast genome assembly and annotation. The chloroplast genome of A. crispa exhibits a structural composition typical of most angiosperms, characterized by a cyclic double-stranded molecule with a quadratic configuration (Fig. 1 ). The genome spans a total length of 156,785 bp, with a GC content of 37.0%. Specifically, the large single copy (LSC) region measures 86,342 bp, the small single copy (SSC) region measures 18,417 bp, and the inverted repeat (IR) region measures 26,014 bp. The GC contents of the LSC, IR, and SSC regions are 35.0%, 43.0%, and 30.1%, respectively. A total of 131 genes were annotated, including 86 protein-coding genes, 37 tRNA genes, 8 rRNA genes, 15 genes with 2 copies ( rrn4.5 、 rrn5 、 rrn16 、 rrn23 、 trnA-UGC 、 trnI-CAU 、 trnI-GAU 、 trnL-CAA 、 trnN-GUU 、 trnR-ACG 、 trnV-GAC 、 rps12 、 rps7 、 rpl2 、 ndhB ), and 21 genes had 1 intron ( trnA-UGC (×2)、 trnG-UCC、trnI-GAU (×2)、 trnK-UUU 、 trnL-UAA、trnV-UAC、rps12 (×2)、 rps16、rpl16、rpl2 (×2)、 rpoC1、petB、petD、atpF、ndhA、ndhB (×2) ), and 2 genes with 2 introns ( ycf3 , clpP ) ( Table 1 .). Table 1 Gene composition of A. crispa chloroplast genome. Genes marked with the sign are the genewith a single ( * ) or double ( ** ) introns and duplicated genes (×2). Gene function Gene type Gene names Number Self-replication Ribosomal RNAs rrn4.5 (×2)、 rrn5 (×2)、 rrn16 (×2)、 rrn23 (×2) 8 Transfer RNAs trnA-UGC* (×2)、 trnC-GCA、trnD-GUC、trnE-UUC、trnF-GAA、trnG-GCC、trnG-UCC*、trnH-GUG、trnI-CAU (×2)、 trnI-GAU* (×2)、 trnK-UUU*、trnL-CAA (×2)、 trnL-UAA*、trnL-UAG、trnM-CAU、trnN-GUU (×2)、 trnP-UGG、trnQ-UUG、trnR-ACG (×2)、 trnR-UCU、trnS-GCU、trnS-GGA、trnS-UGA、trnT-GGU、trnT-UGU、trnV-GAC (×2)、 trnV-UAC*、trnW-CCA、trnY-GUA、trnfM-CAU 37 Proteins of small ribosomal subunit rps11、rps12** (×2)、 rps14、rps15、rps16*、rps18、rps19、rps2、rps3、rps4、rps7 (×2)、 rps8 14 Proteins of large ribosomal subunit rpl14、rpl16*、rpl2* (×2) 、rpl20、rpl22、rpl32、rpl33、rpl36 9 Subunits of RNA polymerase rpoA、rpoB、rpoC1*、rpoC2 4 Photosynthesis Subunits of photosystem I psaA、psaB、psaC、psaI、psaJ 5 Subunits of photosystem II psbA、psbB、psbC、psbD、psbE、psbF、psbH、psbI、psbJ、psbK、psbL、psbM、psbN、psbT、psbZ 15 Subunits of cytochrome b/f complex petA、petB*、petD*、petG、petL、petN 6 Subunits of ATP synthase atpA、atpB、atpE、atpF*、atpH、atpI 6 Protease clpP** 1 Large subunit of rubisco rbcL 1 NADH dehydrogenase subunit ndhA*、ndhB* (×2) 、ndhC、ndhD、ndhE、ndhF、ndhG、ndhH、ndhI、ndhJ、ndhK 12 Other genes Maturase matK 1 Envelope membrane protein cemA 1 Acetyl-CoA carboxylase accD 1 c-type cytochrome synthesis gene ccsA 1 Translation initiation factor InfA 1 Genes of unknown function Conserved hypothetical chloroplast ORF ycf1、ycf15 (×2)、 ycf2 (×2)、 ycf3**、ycf4、ycf68 8 Repeat sequences analysis. The chloroplast genome of A. crispa contained 59 SSRs, consisting of 44 single-nucleotide repeats, 5 dinucleotide repeats, 8 tetranucleotide repeats, and 2 pentanucleotide repeats, with no trinucleotide or hexanucleotide repeats identified. The predominant types of SSRs were A/T repeats ( 41 ) (Fig. 2 A). Additionally, a total of 49 long repetitive sequences were identified, comprising 22 forward repeats, 26 palindromic repeats, and 1 inverted repeat. No complementary repetitive sequences were observed, with the lengths of forward and palindromic repetitive sequences primarily falling within the range of 30 to 49 bp (Fig. 2 B). A total of 38 tandem repeats were identified, ranging in length from 10 to 30 base pairs. Codon analysis. The analysis of chloroplast codon statistics in A. crispa revealed that the 86 protein-coding genes encompassed 61 distinct codon species, totaling 52,261 codons that encode 20 different amino acids, including 3 termination codons (Fig. 3 ). Among the amino acid codons, leucine (Leu), serine (Ser), and arginine (Arg) were each encoded by six codons, with frequencies of 5,218 (9.98%), 4,779 (9.14%), and 3,216 (6.15%), respectively. Leucine was found to be the most commonly utilized codon, followed by serine, while tryptophan (Trp) was the least utilized with only 688 codons (1.32%). Among all the codons observed in the chloroplast genome of A. crispa , the codon AAA exhibited the highest frequency of 2,182 occurrences with a relative synonymous codon usage (RSCU) value of 1.35, while the codon GCG had the lowest frequency of 221 occurrences with an RSCU of 1.22. A total of 35 codons in the genome had an RSCU value greater than or equal to 1, with 8 ending in G or C and 27 ending in A or U. Additionally, the A. crispa chloroplast genome displayed an effective number of codons (ENc) value of 55.71, a codon adaptation index (CAI) value of 0.652, and GC, GC1, GC2, and GC3 contents of 37.04%, 36.89%, 36.61%, and 37.62%, respectively. IR contraction and expansion in the chloroplast genome. The study compared the boundaries between the inverted repeat (IR) and large single copy (LSC) regions in the chloroplast genomes of 9 species of Ardisia . Results indicated the presence of 4 distinct boundaries in the chloroplast genome of Ardisia , with variations in the genes located at these boundaries and their respective lengths (Fig. 4 ). Specifically, the A. crispa and the remaining 8 species exhibited the presence of rpl22 gene on the left side of the genome-wide LSC/IRb boundary (JLB), rpl2 gene on the right side, and rps19 gene spanning the JLB. Notably, an expansion of the rps19 gene into the LSC region was observed in A. crispa and A. crispa var. dielsii , with a 240 bp expansion, while A. crispa var. amplifolia , A. crenata , A. crenata var. Bicolor , A. japonica , A. polysticta showed a 232 bp expansion, and a 69 bp expansion in A. gigantifolia and A. bullata . The analysis of the IRb/SSC (JSB) boundary expansion revealed that, with the exception of A. japonica , whose JSB boundary was positioned to the left of the ndhF gene, the ndhF gene of the remaining 8 species encompassed the JSB boundary. However, the degree of expansion exhibited slight variation, with A. crispa , A. crispa var. dielsii , A. gigantifolia , and A. bullat , expanding by 5 bp into the IRb region, while the remaining 4 species expanded by 3 bp. The analysis of the SSC/IRa (JSA) and LSC/IRa (JLA) boundaries in nine Ardisia species revealed variations in the expansion of ycf1 and trnH genes, respectively. Specifically, the ycf1 gene exhibited expansion ranging from 4,600 bp to 4,614 bp towards the SSC region, while the trnH gene spanned the JLA boundary. Additionally, the rps1 gene of A. crenata and A. polysticta were found to be located at the JLA boundary. Comparative chloroplast genomic and nucleotide diversity analyses. In this study, the chloroplast genome sequences of 9 species of Ardisia were compared and analyzed to evaluate the extent of differences. A. crispa (OP626693) was used as the reference genome, and the mVISTA online tool was employed for the analysis (Fig. 5 ). The nucleotide polymorphism analysis revealed a mean nucleotide diversity (Pi) value of 0.00459 among the 9 species. Six highly variable regions ( trnT-psbD , ndhB-trnL , rpl32-trnL , trnL-ccsA , trnL-ndhB ) were identified when Pi > 0.02, with one region in IRa, IRb, and LSC, and two regions in SSC showing significant variability (Fig. 6 ). Phylogenetic analyses. In order to ascertain the phylogenetic placement of A. crispa , the Bayesian inference (BI) phylogenetic tree was generated using 29 chloroplast genome sequences from 22 Ardisia species (Fig. 7 ). The analysis revealed that A. crispa and A. crispa var. dielsii formed a sister relationship on a single branch, showing closer affinity to A. mamillata and A. pedalis . Conversely, A. crispa var. amplifolia clustered with A. crenata var. bicolor on a separate branch, indicating a closer relationship to A. crenata . Furthermore, the findings of this study provide support for the taxonomic separation of the Primulaceae from the Myrsinaceae. Discussion In this study, the chloroplast genome of A. crispa was successfully sequenced, assembled, and annotated. Consistent with the characteristics of most angiosperms, the chloroplast genome of Paris mairei exhibits a typical tetrameric structure, with the GC content in the sequences of each region following the pattern of IRs > LSC > SSC. This distribution may be attributed to the presence of high GC content rRNA genes in the IR region, which aligns with findings from previous studies on Paris mairei , A. crenata , A. crenata var. bicolor , A. crispa var. dielsii , and A. crispa var. amplifolia 15–17 . Repetitive sequences are prevalent in chloroplast genomes, with their type, number, and distribution varying among species or populations. Interspersed repeats within the chloroplast genome of A. crispa have been identified as valuable tools in genetic variation, structure analysis, and species identification 18–21 . Among the three types detected (forward, reverse, and palindromic), forward and palindromic repeats were found to be the most prevalent, with repeat sequence lengths predominantly falling within the range of 30–49 base pairs. These findings align with previous studies on chloroplast genome repeats in various plant species, including A, B, and C, among others 17,22 . In this study, 59 SSRs were identified, with the predominant occurrence of single-nucleotide repeats composed of A or T. This observation suggests a high prevalence of A or T nucleotides in the base composition of the A. crispa chloroplast genome. The SSRs identified in the A. crispa chloroplast genome may serve as valuable resources for the development of molecular markers and species identification within the Ardisia . Codon preference is the unequal use of synonymous codons encoding the same amino acids by species. Codon usage bias is an important feature of genome evolution and is important for the study of molecular evolution and gene ectopic expression 23 . The Relative Synonymous Codon Usage (RSCU) metric quantifies the ratio between the observed frequency of a codon and its expected frequency based on theoretical calculations. A value of RSCU = 1 indicates equal usage frequency among synonymous codons, suggesting no preference in codon usage. Values of RSCU > 1 suggest a strong preference in codon usage, while values of RSCU < 1 indicate a weak preference. Mutations in the third position of codons are subject to less selective pressure compared to mutations in the first and second positions, and are often associated with changes in amino acid species. The codon's 3rd base composition and content is one of the most important indicators of genomic preference, and higher plants tend to use codons ending in A/U 24 . In the analysis of codon preference in the chloroplast genome of A. crispa , 35 codons with RSCU ≥ 1 were identified, of which 27 codons terminated in A/U. This observation suggests that synonymous codons in the chloroplast genome of A. crispa exhibit a preference for ending in A/U, consistent with findings in other genomes such as Phyllanthaceae 25 , Notopterygium 26 , Cinnamomum camphora 27 , among others. Furthermore, the ENc value of the A. crispa chloroplast genome was calculated to be 55.71, indicating a mild preference. The GC and GC3 contents of the A. crispa chloroplast genome were both below 50%, suggesting a bias towards the use of A and T bases, a pattern that is similar to the results of Dendrobium devonianum 28 and Glycyrrhiza eurycarpa 29 . The chloroplast genome's inverted repeat (IR) region is a prevalent feature in the genomes of many higher plants, and its dynamic contraction and expansion are widely recognized as a key evolutionary process contributing to variations in chloroplast genome size 30 . Expansion of the IR region can facilitate the incorporation of genes located at the genome's periphery. Furthermore, the presence of reverse repeat sequences within the IR region can result in the formation of intact genes or partial gene fragments on the opposite side of the region. This study examined the chloroplast genome boundaries of nine Ardisia species, revealing significant differences between the JLB and JLA boundaries, with the JLB and JSA boundaries showing more conservation. The absence of the ycf1 gene in A. crispa , A. crispa var. dielsii , and A. crispa var. amplifolia , as well as the presence of a pseudogene ycf1 at the JSB border, has been documented 31 . Previous research suggests that variations in selective pressure on the ycf1 gene contribute to differences in evolutionary rates 32–33 . The presence of the ndhF gene was limited to the SSC region in A. japonia , while the rps1 gene was found in the IRa region in A. polysticta , A. gigantifolia and A. crenata . The expansion of the rps19 gene into the IRb region was observed in A. bullata and A. gigantifolia . The phenomenon of boundary expansion and contraction was evident in the analysis of chloroplast gene boundaries in other plants within the same genus. Similar results were found in the chloroplast genome boundary analysis of species such as Rubia cordifolia 34 , Polygala sibirica 35 , and Triticum 36 , indicating that changes in chloroplast genome boundaries do not follow a consistent pattern. Nucleotide diversity can be calculated to quantify differences in cp genomes at the sequence level 37 . These regions may undergo accelerated nucleotide substitution at the species level, suggesting their potential for use as molecular markers in plant identification and phylogenetic analysis 38 . The results of nucleotide polymorphism analysis in this study showed that the non-coding regions of the cp genome sequences of the 9 species of Ardisia were highly variable. There were obvious differences in the spacer regions of the trnT - psbD , rpl32 - trnL , trnL - ccsA , trnL - ndh B, and ndhB - trnL genes, and these regions of variability can provide the basis for the development of molecular markers, species identification, and DNA barcode screening of Ardisia . The chloroplast genome has demonstrated efficacy in elucidating phylogenetic relationships among plant taxa 39–40 . In this investigation, a Bayesian inference phylogenetic tree was constructed utilizing 29 chloroplast whole genomes of Lysimachia christinae (Primulaceae) as an outgroup. The analysis revealed a close relationship between A. crispa and A. crispa var. dielsii , with both taxa forming a distinct clade. However, A. crispa var. amplifolia did not cluster with these two taxa, indicating significant intraspecific variation within Hypericum, potentially influenced by geographical factors. The clustering of A. crispa var. amplifolia with A. crenata and A. crenata var. bicolor at 100% support aligns with the ITS and ITS2 sequence identifications documented in previous literature 41–42 . Consistent with earlier research, our findings suggest a closer relationship between A. crenata and A. crenata var. bicolor , as well as A. polysticta 17,43 . Our study utilized a phylogenetic analysis of chloroplasts to distinguish Myrsinaceae and Primulaceae into separate branches with strong support, advocating for the continued classification of Myrsinaceae as a distinct taxonomic group. Conclusion This study involved sequencing the chloroplast genome of A. crispa , conducting a comprehensive analysis of its sequence, structure, and characteristics, identifying differential sequences in the chloroplast genome of A. crispa and its two variants, and investigating the phylogenetic relationships of Ardisia . The findings not only enhanced the knowledge of the chloroplast genome of A. crispa , but also served as a valuable resource for taxonomic identification and phylogenetic analysis of the genus Ardisia . This has significant implications for the conservation of Ardisia germplasm resources and the identification of valuable genetic resources. Material and methods Sample collection. The fresh leaves of sample (Fig. 8 ) were collected from the Ceheng county, Guizhou Province, China, (coordinates: E105°47′29.73″, N24°59′59.51″; altitude: 933 m). It was identified as Ardisia crispa (Thunb.) A. DC.) by Associate Professor Yan Fulin of Guizhou University of Traditional Chinese Medicine. The voucher specimen (with collection numbers of YFL_2021040307) has been deposited in the Herbarium of Guizhou University of Traditional Chinese Medicine (GZYGH), Guizhou, China. The collection of plant materials complies with the wild plant protection regulations of the People′s Republic of China, and we obtained the permission of local authorities on forestry and the grassland bureau in Guizhou province in China.</p DNA extraction and chloroplast genome sequencing. Total DNA was extracted from the fresh leaves of A. crispa by the modified CTAB method 42 . Sequencing was carried out on the Illumina HiSeq XTen to generate approximately 3 GB 150 bp reads at Beijing Genomics Institute (BGI, Wuhan, China). Genome assembly and annotation. The filtered reads were assembled into a complete chloroplast genome by the program GetOrganelle v1.5 45 . In this pipeline, the complete chloroplast genome reads were extracted from total genomic reads and were subsequently assembled using SPAdes version 3.10 46 . The genes were annotated using PGA 47 and Geneious 11.0.3 48 with the published complete chloroplast genome of A. crenata (GenBank accession number: NC_059021) as the reference. Transfer RNAs (tRNAs) were confirmed by their specific structure predicted by tRNAscan-SE 2.0 49 . The OGDRAW ( https://chlorobox.mpimp-golm.mpg.de/OGDraw.html ) 50 was used to draw a detailed physical map of the A. crispa chloroplast genome. Chloroplast genome structural analysis. The online software REPuter ( https://bibiserv.cebitec.unibielefeld.de/reputer ) was employed to analyze forward (F), palindromic (P), reverse (R), and complement (C) repeats, with the following settings: Minimum repeat size of 3 bp and hamming Distance of 30 bp 51 . We used the default parameters in the online Tandem Repeats Finder ( http://tandem.bu.edu/trf/trf.html ) 52 to search for tandem repeats in DNA sequences. The online software MISA 53 was applied to predict SSRs with parameter thresholds set at 1, 2, 3, 4, 5, and 6, and nucleotide parameters of 10, 5, 4, 3, 3, 3, and 3, and the distance between two SSRs was not less than 100 bp. Relative synonymous codon usage (RSCU) analysis of the chloroplast genome of A. crispa was performed using condon W ( https://galaxy.pasteur.fr/?form=codonw ) online software. By using CUSP online software ( http://imed.med.ucm.es/EMBOSS/ ) 54 , we calculated the effective codon count (ENc), codon adaptation index (CAI), and counted the total codon GC content (GCall), the GC contents of positions 1, 2, and 3 (GC1, GC2, and GC3), and the GC content of position 3 of the synonymous codons (GC3s). Genome comparison. The genes of the boundaries in the chloroplast genomes of the Ardisia species were compared and visually represented using IRscope 55 ( https://irscope.shinyapps.io/irapp/ ) to reveal contraction and expansion of the IR regions. The comparative analysis of the whole sequence identity of the chloroplast genomes was performed using mVISTA 56 with the chloroplast genome of A.crispa (OP626693) as the reference sequence. Phylogenetic analysis. A phylogenetic analysis was conducted based on chloroplast genomes from 29 species, including those of the one A. crispa sequenced and assembled in this study and another 28 downloaded from GenBank. Twenty-nine complete plastid sequences were aligned using MAFFT v7.017 57 , and a BI phylogenetic tree was constructed using MrBayes 3.2.7 58 with Lysimachia christinae (Primulaceae) as the outgroup. Declarations Data availability The complete chloroplast genomes and annotations are available at the NCBI database ( Ardisia crispa : OP762693). Competing interests The authors declare no competing interests. Author Contribution F.L.Yan designed the research. Z.K.Wu, Y.Q.J., S.H.Wei, F.L.Yan and L.D. collected the samples, J.Ye, F.L.Yan, Q.Luo, and Y.H.Lang conceived the experiments, J.Ye, F.L.Yan, Q.Luo, and Y.H.Lang did computational analysis and deposited sequences. J.Ye, F.L.Yan, and Y.H.Lang wrote the manuscript. All authors have read and approved the manuscript. Acknowledgements This study was supported by the National Key Research and Development Program Project (2018YFC1708101), Guizhou Provincial Science and Technology Program Project (No. Qiankezhongyindi [2022] 4016), Guizhou Modern Industrial Technology System Construction of Chinese Herbal Medicines (No. GZCYTX2019-2024), and State Administration of Traditional Chinese Medicine Seedling Breeding of Chinese Herbal Medicines Seeds Required for National Essential Drugs (Guizhou) Base Construction Project (2014–2017). Additional information Correspondence and requests for materials should be addressed to J.Ye or F.L.Yan. References Guizhou Medical Products Administration. 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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-4013297","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":281602751,"identity":"72604409-accf-4af1-a68e-242de05ed179","order_by":0,"name":"Juan Ye","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Ye","suffix":""},{"id":281602752,"identity":"5752ff0f-b1f9-4481-8d74-63589fd19ef6","order_by":1,"name":"Qin Luo","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Luo","suffix":""},{"id":281602753,"identity":"18a4b410-41ea-409f-8ede-6e328d630635","order_by":2,"name":"Yun-hu Lang","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yun-hu","middleName":"","lastName":"Lang","suffix":""},{"id":281602754,"identity":"ad97be61-5ad5-45b8-999d-dd4a8fc9d6df","order_by":3,"name":"Ning Ding","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Ding","suffix":""},{"id":281602755,"identity":"d61cd38e-d469-4b83-901a-e53333965213","order_by":4,"name":"Ying-quan Jian","email":"","orcid":"","institution":"Guizhou Hanfang Pharmaceutical Co., LTD","correspondingAuthor":false,"prefix":"","firstName":"Ying-quan","middleName":"","lastName":"Jian","suffix":""},{"id":281602757,"identity":"88377bba-5337-4ca8-8360-d6cf6f122a6f","order_by":5,"name":"Zhi-kun Wu","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhi-kun","middleName":"","lastName":"Wu","suffix":""},{"id":281602758,"identity":"7276a566-9438-456a-98e4-337a5ef3db1e","order_by":6,"name":"Sheng-hua Wei","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sheng-hua","middleName":"","lastName":"Wei","suffix":""},{"id":281602760,"identity":"9740b740-d4fd-42f6-a15e-6fd95c77a276","order_by":7,"name":"Fu-lin Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYJCCDwwFEgz2B44ffADhJxDUwTiDwUCCgeHgmWQDUrQAqcMHzCSI0mJw/OzBZh4DizzGtgNplV/+HGbgZ88xYPi5A4+WM3mJQC0Sxcw8B4/dluE5zCDZ88aAsfcMHi0HcswfA7UktkkcSLstIXGYweBGjgEzYxseLeffGIJsSeyRf2BWLGFwmMGeoJYbORAtMxgOmDF+SADaIkFAi+SNN4aNc4BaNjCcSZZmOJDOI3HmWcHBXjxa+M7nGDa8qagDajl+8OOPP9Zy/O3JGx/8xKNF4QASh5mHoZkHxDiAVS0UyDcgcRh/MNThUzwKRsEoGAUjFAAAc9BXFECFCFoAAAAASUVORK5CYII=","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Fu-lin","middleName":"","lastName":"Yan","suffix":""}],"badges":[],"createdAt":"2024-03-04 17:08:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4013297/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4013297/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-66563-3","type":"published","date":"2024-08-16T15:57:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53253328,"identity":"4988d425-a69e-4f37-90f8-921b162e78f3","added_by":"auto","created_at":"2024-03-22 13:13:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1844411,"visible":true,"origin":"","legend":"\u003cp\u003eChloroplast genome map of \u003cem\u003eA. crispa. \u003c/em\u003eGenes shown inside the circle are transcribed clockwise, whereas genes outside are transcribed counterclockwise. The light gray inner circle shows the AT content, the dark gray corresponds to the GC content.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4013297/v1/2675f802922b28000f417e0e.jpg"},{"id":53252577,"identity":"218a7172-2b3e-4d77-bfc4-c79f83299b03","added_by":"auto","created_at":"2024-03-22 13:05:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126553,"visible":true,"origin":"","legend":"\u003cp\u003eRepeat type and number of \u003cem\u003eA. crispa \u003c/em\u003echloroplast genome (A: SSR type B: long repeat type and frequency of use)\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4013297/v1/8d528a3319035cbe4f731a7b.jpg"},{"id":53253330,"identity":"e06afbc3-69ab-4505-9e76-9f00e00c99ee","added_by":"auto","created_at":"2024-03-22 13:13:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101862,"visible":true,"origin":"","legend":"\u003cp\u003eRelative synonymous codon usage (RSCU) for protein-coding genes in \u003cem\u003eA. crispa\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4013297/v1/ffa83de4dfd68ed446c0a157.png"},{"id":53252573,"identity":"21d4a88f-6188-4f75-8003-603c439d4b72","added_by":"auto","created_at":"2024-03-22 13:05:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1187092,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of IR/SC boundary of chloroplast genomes of nine \u003cem\u003eArdisia\u003c/em\u003e species\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4013297/v1/3b8ba78e979489ea029b37dd.jpg"},{"id":53252574,"identity":"0f35c42c-140e-4d41-b128-be9f69fda71f","added_by":"auto","created_at":"2024-03-22 13:05:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3271236,"visible":true,"origin":"","legend":"\u003cp\u003eGlobal alignment analysis of the nine \u003cem\u003eArdisia\u003c/em\u003e chloroplast genomes\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4013297/v1/b482380f018afe637d807055.jpg"},{"id":53252583,"identity":"025745fe-f455-427e-af65-2c3eccbf6821","added_by":"auto","created_at":"2024-03-22 13:05:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":230190,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of nucleotide diversity (Pi) values among the nine \u003cem\u003eArdisia\u003c/em\u003e species chloroplast genome sequences.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4013297/v1/709f4e6c5871559f2cc7a8e1.png"},{"id":53252576,"identity":"48ead394-9ec9-4146-9ee3-a9071e6f2b10","added_by":"auto","created_at":"2024-03-22 13:05:15","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":772954,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis based on\u003cem\u003e \u003c/em\u003echloroplast genome sequences by BI tree\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4013297/v1/1c24b928325a40abf11b512e.jpg"},{"id":53252590,"identity":"f3cb4da8-ac26-4323-a018-61ef6db8f09d","added_by":"auto","created_at":"2024-03-22 13:05:17","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":470224,"visible":true,"origin":"","legend":"\u003cp\u003eThe plant of \u003cem\u003eA. crispa\u003c/em\u003e and its growing environment (A: Growing environment B: Living plants C: Flowers)\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4013297/v1/8d3ede46c9415f80967bb924.jpg"},{"id":63071429,"identity":"36df8a90-2639-483c-baa3-ab9452f05078","added_by":"auto","created_at":"2024-08-22 20:07:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8788448,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4013297/v1/affe4204-4bcb-4674-b7a5-d02bc396e548.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The complete chloroplast genome sequence of the medicinal plant Ardisia crispa (Myrsinaceae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eArdisia crispa\u003c/em\u003e (Thunb.) A. DC., a member of the Myrsinaceae family, is predominantly found in the provinces south of the Yangtze River Basin in China. The root of this plant is utilized in traditional medicine and is a key component of the renowned Hmong medicinal specialty, Radix Ardisia, in Guizhou\u003csup\u003e1,3\u003c/sup\u003e. Modern research has identified \u003cem\u003eA. crispa\u003c/em\u003e as containing triterpene glycosides, flavonoids, isocoumarins, and other bioactive compounds, known for their therapeutic properties such as relieving heat, soothing the throat, and activating tendons. This plant is commonly used to treat conditions like sore throat, tonsillitis, nephritis, and edema, earning it the nickname ''aryngeal medicine''\u003csup\u003e2\u003c/sup\u003e. Two subspecies of \u003cem\u003eA. crispa\u003c/em\u003e have been identified based on morphological differences, specifically \u003cem\u003eA. crispa\u003c/em\u003e var \u003cem\u003edielsii\u003c/em\u003e (characterized by shorter stature and narrow lanceolate leaves) and \u003cem\u003eA. crispa\u003c/em\u003e var \u003cem\u003eamplifolia\u003c/em\u003e (recognized for its stout stature and broad leaves)\u003csup\u003e4\u003c/sup\u003e. However, the lack of a scientific foundation for this classification hinders a comprehensive understanding of the plant's genetic background, conservation of germplasm resources, and phylogenetic evolution. Furthermore, the structural features of the plastid genome, functional classification, codon preference analysis, and differences in the chloroplast genome between the two variants have not been thoroughly explored. This knowledge gap not only limits our understanding of \u003cem\u003eA. crispa\u003c/em\u003e and its variants but also complicates the accurate identification of mixed pseudo-products involving \u003cem\u003eA. crispa\u003c/em\u003e in medicinal herb preparations. Chloroplasts are semi-autonomous organelles found in plants, characterized by having the second largest genome in plant cells\u003csup\u003e5\u003c/sup\u003e. The chloroplast genome typically consists of a large single copy (LSC) region, a small single copy (SSC) region, and two inverted repeats (IR)\u003csup\u003e6\u003c/sup\u003e. Due to its uniparental inheritance, moderate mutation rate, and ease of sequencing, the chloroplast genome is often regarded as a more efficient resource compared to nuclear and mitochondrial genomes. Consequently, it is frequently utilized for investigating the origins and evolution of plants, elucidating phylogenetic relationships among different taxonomic classes, and for species identification purposes\u003csup\u003e7\u003c/sup\u003e. In recent years, advancements in high-throughput sequencing technologies have facilitated the successful assembly, annotation, and analysis of a substantial number of subspecies chloroplast genomes. The application of chloroplast genome analysis has yielded promising results in the investigation of identification, genetic relationships, and phylogenetics of various species, including \u003cem\u003eSabia\u003c/em\u003e, \u003cem\u003ePhoebe\u003c/em\u003e, \u003cem\u003eVaccinium\u003c/em\u003e, \u003cem\u003eHibiscus rosa\u003c/em\u003e-\u003cem\u003esinensis\u003c/em\u003e, \u003cem\u003eDalbergia hainanensis\u003c/em\u003e, \u003cem\u003eLitsea\u003c/em\u003e, and \u003cem\u003eZingiber\u003c/em\u003e\u003csup\u003e8\u0026ndash;14\u003c/sup\u003e. Therefore, this research aimed to utilize high-throughput sequencing technology to sequence, assemble, and annotate the complete chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e. Additionally, comparative analysis of structural features and phylogenetic relationships among \u003cem\u003eA. crispa\u003c/em\u003e and its varieties \u003cem\u003eA. crispa\u003c/em\u003e var \u003cem\u003edielsii\u003c/em\u003e and \u003cem\u003eA. crispa\u003c/em\u003e var \u003cem\u003eamplifolia\u003c/em\u003e, as well as other \u003cem\u003eArdisia\u003c/em\u003e species, was conducted using bioinformatics tools. The findings of this study contribute to the understanding of species identification, phylogeny, and conservation efforts related to \u003cem\u003eA. crispa\u003c/em\u003e and other medicinal herbs.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eChloroplast genome assembly and annotation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e exhibits a structural composition typical of most angiosperms, characterized by a cyclic double-stranded molecule with a quadratic configuration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The genome spans a total length of 156,785 bp, with a GC content of 37.0%. Specifically, the large single copy (LSC) region measures 86,342 bp, the small single copy (SSC) region measures 18,417 bp, and the inverted repeat (IR) region measures 26,014 bp. The GC contents of the LSC, IR, and SSC regions are 35.0%, 43.0%, and 30.1%, respectively. A total of 131 genes were annotated, including 86 protein-coding genes, 37 tRNA genes, 8 rRNA genes, 15 genes with 2 copies ( \u003cem\u003errn4.5\u003c/em\u003e、\u003cem\u003errn5\u003c/em\u003e、\u003cem\u003errn16\u003c/em\u003e、\u003cem\u003errn23\u003c/em\u003e、\u003cem\u003etrnA-UGC\u003c/em\u003e、\u003cem\u003etrnI-CAU\u003c/em\u003e、\u003cem\u003etrnI-GAU\u003c/em\u003e、\u003cem\u003etrnL-CAA\u003c/em\u003e、\u003cem\u003etrnN-GUU\u003c/em\u003e、\u003cem\u003etrnR-ACG\u003c/em\u003e、\u003cem\u003etrnV-GAC\u003c/em\u003e、\u003cem\u003erps12\u003c/em\u003e、\u003cem\u003erps7\u003c/em\u003e、\u003cem\u003erpl2\u003c/em\u003e、\u003cem\u003endhB\u003c/em\u003e ), and 21 genes had 1 intron ( \u003cem\u003etrnA-UGC\u003c/em\u003e (\u0026times;2)、\u003cem\u003etrnG-UCC、trnI-GAU\u003c/em\u003e (\u0026times;2)、\u003cem\u003etrnK-UUU\u003c/em\u003e、\u003cem\u003etrnL-UAA、trnV-UAC、rps12\u003c/em\u003e (\u0026times;2)、\u003cem\u003erps16、rpl16、rpl2\u003c/em\u003e (\u0026times;2)、\u003cem\u003erpoC1、petB、petD、atpF、ndhA、ndhB\u003c/em\u003e (\u0026times;2) ), and 2 genes with 2 introns ( \u003cem\u003eycf3\u003c/em\u003e, \u003cem\u003eclpP\u003c/em\u003e ) ( Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGene composition of \u003cem\u003eA. crispa\u003c/em\u003e chloroplast genome. Genes marked with the sign are the genewith a single (\u003cem\u003e*\u003c/em\u003e) or double (\u003cem\u003e**\u003c/em\u003e) introns and duplicated genes (\u0026times;2).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene function\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGene names\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eSelf-replication\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRibosomal RNAs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003errn4.5\u003c/em\u003e(\u0026times;2)、\u003cem\u003errn5\u003c/em\u003e(\u0026times;2)、\u003cem\u003errn16\u003c/em\u003e(\u0026times;2)、\u003cem\u003errn23\u003c/em\u003e(\u0026times;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTransfer RNAs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003etrnA-UGC*\u003c/em\u003e(\u0026times;2)、\u003cem\u003etrnC-GCA、trnD-GUC、trnE-UUC、trnF-GAA、trnG-GCC、trnG-UCC*、trnH-GUG、trnI-CAU\u003c/em\u003e(\u0026times;2)、\u003cem\u003etrnI-GAU*\u003c/em\u003e(\u0026times;2)、\u003cem\u003etrnK-UUU*、trnL-CAA\u003c/em\u003e(\u0026times;2)、\u003cem\u003etrnL-UAA*、trnL-UAG、trnM-CAU、trnN-GUU\u003c/em\u003e(\u0026times;2)、\u003cem\u003etrnP-UGG、trnQ-UUG、trnR-ACG\u003c/em\u003e(\u0026times;2)、\u003cem\u003etrnR-UCU、trnS-GCU、trnS-GGA、trnS-UGA、trnT-GGU、trnT-UGU、trnV-GAC\u003c/em\u003e(\u0026times;2)、\u003cem\u003etrnV-UAC*、trnW-CCA、trnY-GUA、trnfM-CAU\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteins of small ribosomal subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erps11、rps12**\u003c/em\u003e(\u0026times;2)、\u003cem\u003erps14、rps15、rps16*、rps18、rps19、rps2、rps3、rps4、rps7\u003c/em\u003e(\u0026times;2)、\u003cem\u003erps8\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteins of large ribosomal subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpl14、rpl16*、rpl2*\u003c/em\u003e(\u0026times;2)\u003cem\u003e、rpl20、rpl22、rpl32、rpl33、rpl36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of RNA polymerase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpoA、rpoB、rpoC1*、rpoC2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003ePhotosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of photosystem I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsaA、psaB、psaC、psaI、psaJ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of photosystem II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsbA、psbB、psbC、psbD、psbE、psbF、psbH、psbI、psbJ、psbK、psbL、psbM、psbN、psbT、psbZ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of cytochrome b/f complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epetA、petB*、petD*、petG、petL、petN\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of ATP synthase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eatpA、atpB、atpE、atpF*、atpH、atpI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eclpP**\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarge subunit of rubisco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erbcL\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNADH dehydrogenase subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003endhA*、ndhB*\u003c/em\u003e(\u0026times;2)\u003cem\u003e、ndhC、ndhD、ndhE、ndhF、ndhG、ndhH、ndhI、ndhJ、ndhK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eOther genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaturase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ematK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnvelope membrane protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecemA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcetyl-CoA carboxylase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eaccD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec-type cytochrome synthesis gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eccsA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTranslation initiation factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eInfA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes of unknown function\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConserved hypothetical chloroplast ORF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eycf1、ycf15\u003c/em\u003e(\u0026times;2)、\u003cem\u003eycf2\u003c/em\u003e(\u0026times;2)、\u003cem\u003eycf3**、ycf4、ycf68\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\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\u003e \u003cb\u003eRepeat sequences analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e contained 59 SSRs, consisting of 44 single-nucleotide repeats, 5 dinucleotide repeats, 8 tetranucleotide repeats, and 2 pentanucleotide repeats, with no trinucleotide or hexanucleotide repeats identified. The predominant types of SSRs were A/T repeats (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additionally, a total of 49 long repetitive sequences were identified, comprising 22 forward repeats, 26 palindromic repeats, and 1 inverted repeat. No complementary repetitive sequences were observed, with the lengths of forward and palindromic repetitive sequences primarily falling within the range of 30 to 49 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). A total of 38 tandem repeats were identified, ranging in length from 10 to 30 base pairs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCodon analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe analysis of chloroplast codon statistics in \u003cem\u003eA. crispa\u003c/em\u003e revealed that the 86 protein-coding genes encompassed 61 distinct codon species, totaling 52,261 codons that encode 20 different amino acids, including 3 termination codons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among the amino acid codons, leucine (Leu), serine (Ser), and arginine (Arg) were each encoded by six codons, with frequencies of 5,218 (9.98%), 4,779 (9.14%), and 3,216 (6.15%), respectively. Leucine was found to be the most commonly utilized codon, followed by serine, while tryptophan (Trp) was the least utilized with only 688 codons (1.32%). Among all the codons observed in the chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e, the codon AAA exhibited the highest frequency of 2,182 occurrences with a relative synonymous codon usage (RSCU) value of 1.35, while the codon GCG had the lowest frequency of 221 occurrences with an RSCU of 1.22. A total of 35 codons in the genome had an RSCU value greater than or equal to 1, with 8 ending in G or C and 27 ending in A or U. Additionally, the \u003cem\u003eA. crispa\u003c/em\u003e chloroplast genome displayed an effective number of codons (ENc) value of 55.71, a codon adaptation index (CAI) value of 0.652, and GC, GC1, GC2, and GC3 contents of 37.04%, 36.89%, 36.61%, and 37.62%, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIR contraction and expansion in the chloroplast genome.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe study compared the boundaries between the inverted repeat (IR) and large single copy (LSC) regions in the chloroplast genomes of 9 species of \u003cem\u003eArdisia\u003c/em\u003e. Results indicated the presence of 4 distinct boundaries in the chloroplast genome of \u003cem\u003eArdisia\u003c/em\u003e, with variations in the genes located at these boundaries and their respective lengths (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Specifically, the \u003cem\u003eA. crispa\u003c/em\u003e and the remaining 8 species exhibited the presence of \u003cem\u003erpl22\u003c/em\u003e gene on the left side of the genome-wide LSC/IRb boundary (JLB), \u003cem\u003erpl2\u003c/em\u003e gene on the right side, and \u003cem\u003erps19\u003c/em\u003e gene spanning the JLB. Notably, an expansion of the \u003cem\u003erps19\u003c/em\u003e gene into the LSC region was observed in \u003cem\u003eA. crispa\u003c/em\u003e and \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003edielsii\u003c/em\u003e, with a 240 bp expansion, while \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003eamplifolia\u003c/em\u003e, \u003cem\u003eA. crenata\u003c/em\u003e, \u003cem\u003eA. crenata\u003c/em\u003e var. \u003cem\u003eBicolor\u003c/em\u003e, \u003cem\u003eA. japonica\u003c/em\u003e, \u003cem\u003eA. polysticta\u003c/em\u003e showed a 232 bp expansion, and a 69 bp expansion in \u003cem\u003eA. gigantifolia\u003c/em\u003e and \u003cem\u003eA. bullata\u003c/em\u003e. The analysis of the IRb/SSC (JSB) boundary expansion revealed that, with the exception of \u003cem\u003eA. japonica\u003c/em\u003e, whose JSB boundary was positioned to the left of the \u003cem\u003endhF\u003c/em\u003e gene, the \u003cem\u003endhF\u003c/em\u003e gene of the remaining 8 species encompassed the JSB boundary. However, the degree of expansion exhibited slight variation, with \u003cem\u003eA. crispa\u003c/em\u003e, \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003edielsii\u003c/em\u003e, \u003cem\u003eA. gigantifolia\u003c/em\u003e, and \u003cem\u003eA. bullat\u003c/em\u003e, expanding by 5 bp into the IRb region, while the remaining 4 species expanded by 3 bp. The analysis of the SSC/IRa (JSA) and LSC/IRa (JLA) boundaries in nine \u003cem\u003eArdisia\u003c/em\u003e species revealed variations in the expansion of \u003cem\u003eycf1\u003c/em\u003e and \u003cem\u003etrnH\u003c/em\u003e genes, respectively. Specifically, the \u003cem\u003eycf1\u003c/em\u003e gene exhibited expansion ranging from 4,600 bp to 4,614 bp towards the SSC region, while the \u003cem\u003etrnH\u003c/em\u003e gene spanned the JLA boundary. Additionally, the \u003cem\u003erps1\u003c/em\u003e gene of \u003cem\u003eA. crenata\u003c/em\u003e and \u003cem\u003eA. polysticta\u003c/em\u003e were found to be located at the JLA boundary.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eComparative chloroplast genomic and nucleotide diversity analyses.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this study, the chloroplast genome sequences of 9 species of \u003cem\u003eArdisia\u003c/em\u003e were compared and analyzed to evaluate the extent of differences. \u003cem\u003eA. crispa\u003c/em\u003e (OP626693) was used as the reference genome, and the mVISTA online tool was employed for the analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The nucleotide polymorphism analysis revealed a mean nucleotide diversity (Pi) value of 0.00459 among the 9 species. Six highly variable regions (\u003cem\u003etrnT-psbD\u003c/em\u003e, \u003cem\u003endhB-trnL\u003c/em\u003e, \u003cem\u003erpl32-trnL\u003c/em\u003e, \u003cem\u003etrnL-ccsA\u003c/em\u003e, \u003cem\u003etrnL-ndhB\u003c/em\u003e) were identified when Pi\u0026thinsp;\u0026gt;\u0026thinsp;0.02, with one region in IRa, IRb, and LSC, and two regions in SSC showing significant variability (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic analyses.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to ascertain the phylogenetic placement of \u003cem\u003eA. crispa\u003c/em\u003e, the Bayesian inference (BI) phylogenetic tree was generated using 29 chloroplast genome sequences from 22 \u003cem\u003eArdisia\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The analysis revealed that \u003cem\u003eA. crispa\u003c/em\u003e and \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003edielsii\u003c/em\u003e formed a sister relationship on a single branch, showing closer affinity to \u003cem\u003eA. mamillata\u003c/em\u003e and \u003cem\u003eA. pedalis\u003c/em\u003e. Conversely, \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003eamplifolia\u003c/em\u003e clustered with \u003cem\u003eA. crenata\u003c/em\u003e var. \u003cem\u003ebicolor\u003c/em\u003e on a separate branch, indicating a closer relationship to \u003cem\u003eA. crenata\u003c/em\u003e. Furthermore, the findings of this study provide support for the taxonomic separation of the Primulaceae from the Myrsinaceae.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, the chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e was successfully sequenced, assembled, and annotated. Consistent with the characteristics of most angiosperms, the chloroplast genome of Paris mairei exhibits a typical tetrameric structure, with the GC content in the sequences of each region following the pattern of IRs\u0026thinsp;\u0026gt;\u0026thinsp;LSC\u0026thinsp;\u0026gt;\u0026thinsp;SSC. This distribution may be attributed to the presence of high GC content rRNA genes in the IR region, which aligns with findings from previous studies on \u003cem\u003eParis mairei\u003c/em\u003e, \u003cem\u003eA. crenata\u003c/em\u003e, \u003cem\u003eA. crenata\u003c/em\u003e var. \u003cem\u003ebicolor\u003c/em\u003e, \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003edielsii\u003c/em\u003e, and \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003eamplifolia\u003c/em\u003e\u003csup\u003e15\u0026ndash;17\u003c/sup\u003e. Repetitive sequences are prevalent in chloroplast genomes, with their type, number, and distribution varying among species or populations. Interspersed repeats within the chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e have been identified as valuable tools in genetic variation, structure analysis, and species identification\u003csup\u003e18\u0026ndash;21\u003c/sup\u003e. Among the three types detected (forward, reverse, and palindromic), forward and palindromic repeats were found to be the most prevalent, with repeat sequence lengths predominantly falling within the range of 30\u0026ndash;49 base pairs. These findings align with previous studies on chloroplast genome repeats in various plant species, including A, B, and C, among others\u003csup\u003e17,22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, 59 SSRs were identified, with the predominant occurrence of single-nucleotide repeats composed of A or T. This observation suggests a high prevalence of A or T nucleotides in the base composition of the \u003cem\u003eA. crispa\u003c/em\u003e chloroplast genome. The SSRs identified in the \u003cem\u003eA. crispa\u003c/em\u003e chloroplast genome may serve as valuable resources for the development of molecular markers and species identification within the \u003cem\u003eArdisia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eCodon preference is the unequal use of synonymous codons encoding the same amino acids by species. Codon usage bias is an important feature of genome evolution and is important for the study of molecular evolution and gene ectopic expression\u003csup\u003e23\u003c/sup\u003e. The Relative Synonymous Codon Usage (RSCU) metric quantifies the ratio between the observed frequency of a codon and its expected frequency based on theoretical calculations. A value of RSCU\u0026thinsp;=\u0026thinsp;1 indicates equal usage frequency among synonymous codons, suggesting no preference in codon usage. Values of RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1 suggest a strong preference in codon usage, while values of RSCU\u0026thinsp;\u0026lt;\u0026thinsp;1 indicate a weak preference. Mutations in the third position of codons are subject to less selective pressure compared to mutations in the first and second positions, and are often associated with changes in amino acid species. The codon's 3rd base composition and content is one of the most important indicators of genomic preference, and higher plants tend to use codons ending in A/U\u003csup\u003e24\u003c/sup\u003e. In the analysis of codon preference in the chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e, 35 codons with RSCU\u0026thinsp;\u0026ge;\u0026thinsp;1 were identified, of which 27 codons terminated in A/U. This observation suggests that synonymous codons in the chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e exhibit a preference for ending in A/U, consistent with findings in other genomes such as Phyllanthaceae\u003csup\u003e25\u003c/sup\u003e, \u003cem\u003eNotopterygium\u003c/em\u003e\u003csup\u003e26\u003c/sup\u003e, \u003cem\u003eCinnamomum camphora\u003c/em\u003e\u003csup\u003e27\u003c/sup\u003e, among others. Furthermore, the ENc value of the \u003cem\u003eA. crispa\u003c/em\u003e chloroplast genome was calculated to be 55.71, indicating a mild preference. The GC and GC3 contents of the \u003cem\u003eA. crispa\u003c/em\u003e chloroplast genome were both below 50%, suggesting a bias towards the use of A and T bases, a pattern that is similar to the results of \u003cem\u003eDendrobium devonianum\u003c/em\u003e\u003csup\u003e28\u003c/sup\u003e and \u003cem\u003eGlycyrrhiza eurycarpa\u003c/em\u003e\u003csup\u003e29\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe chloroplast genome's inverted repeat (IR) region is a prevalent feature in the genomes of many higher plants, and its dynamic contraction and expansion are widely recognized as a key evolutionary process contributing to variations in chloroplast genome size\u003csup\u003e30\u003c/sup\u003e. Expansion of the IR region can facilitate the incorporation of genes located at the genome's periphery. Furthermore, the presence of reverse repeat sequences within the IR region can result in the formation of intact genes or partial gene fragments on the opposite side of the region.\u003c/p\u003e \u003cp\u003eThis study examined the chloroplast genome boundaries of nine \u003cem\u003eArdisia\u003c/em\u003e species, revealing significant differences between the JLB and JLA boundaries, with the JLB and JSA boundaries showing more conservation. The absence of the \u003cem\u003eycf1\u003c/em\u003e gene in \u003cem\u003eA. crispa\u003c/em\u003e, \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003edielsii\u003c/em\u003e, and \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003eamplifolia\u003c/em\u003e, as well as the presence of a pseudogene ycf1 at the JSB border, has been documented\u003csup\u003e31\u003c/sup\u003e. Previous research suggests that variations in selective pressure on the ycf1 gene contribute to differences in evolutionary rates\u003csup\u003e32\u0026ndash;33\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe presence of the \u003cem\u003endhF\u003c/em\u003e gene was limited to the SSC region in \u003cem\u003eA. japonia\u003c/em\u003e, while the \u003cem\u003erps1\u003c/em\u003e gene was found in the IRa region in \u003cem\u003eA. polysticta\u003c/em\u003e, \u003cem\u003eA. gigantifolia\u003c/em\u003e and \u003cem\u003eA. crenata\u003c/em\u003e. The expansion of the \u003cem\u003erps19\u003c/em\u003e gene into the IRb region was observed in \u003cem\u003eA. bullata\u003c/em\u003e and \u003cem\u003eA. gigantifolia\u003c/em\u003e. The phenomenon of boundary expansion and contraction was evident in the analysis of chloroplast gene boundaries in other plants within the same genus. Similar results were found in the chloroplast genome boundary analysis of species such as \u003cem\u003eRubia cordifolia\u003c/em\u003e\u003csup\u003e34\u003c/sup\u003e, \u003cem\u003ePolygala sibirica\u003c/em\u003e\u003csup\u003e35\u003c/sup\u003e, and \u003cem\u003eTriticum\u003c/em\u003e\u003csup\u003e36\u003c/sup\u003e, indicating that changes in chloroplast genome boundaries do not follow a consistent pattern.\u003c/p\u003e \u003cp\u003eNucleotide diversity can be calculated to quantify differences in cp genomes at the sequence level\u003csup\u003e37\u003c/sup\u003e. These regions may undergo accelerated nucleotide substitution at the species level, suggesting their potential for use as molecular markers in plant identification and phylogenetic analysis\u003csup\u003e38\u003c/sup\u003e. The results of nucleotide polymorphism analysis in this study showed that the non-coding regions of the cp genome sequences of the 9 species of \u003cem\u003eArdisia\u003c/em\u003e were highly variable. There were obvious differences in the spacer regions of the \u003cem\u003etrnT\u003c/em\u003e-\u003cem\u003epsbD\u003c/em\u003e, \u003cem\u003erpl32\u003c/em\u003e-\u003cem\u003etrnL\u003c/em\u003e, \u003cem\u003etrnL\u003c/em\u003e-\u003cem\u003eccsA\u003c/em\u003e, \u003cem\u003etrnL\u003c/em\u003e-\u003cem\u003endh\u003c/em\u003eB, and \u003cem\u003endhB\u003c/em\u003e-\u003cem\u003etrnL\u003c/em\u003e genes, and these regions of variability can provide the basis for the development of molecular markers, species identification, and DNA barcode screening of \u003cem\u003eArdisia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe chloroplast genome has demonstrated efficacy in elucidating phylogenetic relationships among plant taxa\u003csup\u003e39\u0026ndash;40\u003c/sup\u003e. In this investigation, a Bayesian inference phylogenetic tree was constructed utilizing 29 chloroplast whole genomes of \u003cem\u003eLysimachia christinae\u003c/em\u003e (Primulaceae) as an outgroup. The analysis revealed a close relationship between \u003cem\u003eA. crispa\u003c/em\u003e and \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003edielsii\u003c/em\u003e, with both taxa forming a distinct clade. However, \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003eamplifolia\u003c/em\u003e did not cluster with these two taxa, indicating significant intraspecific variation within Hypericum, potentially influenced by geographical factors. The clustering of \u003cem\u003eA. crispa\u003c/em\u003e var. \u003cem\u003eamplifolia\u003c/em\u003e with \u003cem\u003eA. crenata\u003c/em\u003e and \u003cem\u003eA. crenata\u003c/em\u003e var. \u003cem\u003ebicolor\u003c/em\u003e at 100% support aligns with the ITS and ITS2 sequence identifications documented in previous literature\u003csup\u003e41\u0026ndash;42\u003c/sup\u003e. Consistent with earlier research, our findings suggest a closer relationship between \u003cem\u003eA. crenata\u003c/em\u003e and \u003cem\u003eA. crenata\u003c/em\u003e var. \u003cem\u003ebicolor\u003c/em\u003e, as well as \u003cem\u003eA. polysticta\u003c/em\u003e\u003csup\u003e17,43\u003c/sup\u003e. Our study utilized a phylogenetic analysis of chloroplasts to distinguish Myrsinaceae and Primulaceae into separate branches with strong support, advocating for the continued classification of Myrsinaceae as a distinct taxonomic group.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study involved sequencing the chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e, conducting a comprehensive analysis of its sequence, structure, and characteristics, identifying differential sequences in the chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e and its two variants, and investigating the phylogenetic relationships of \u003cem\u003eArdisia\u003c/em\u003e. The findings not only enhanced the knowledge of the chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e, but also served as a valuable resource for taxonomic identification and phylogenetic analysis of the genus \u003cem\u003eArdisia\u003c/em\u003e. This has significant implications for the conservation of \u003cem\u003eArdisia\u003c/em\u003e germplasm resources and the identification of valuable genetic resources.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e \u003cb\u003eSample collection.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe fresh leaves of sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) were collected from the Ceheng county, Guizhou Province, China, (coordinates: E105\u0026deg;47\u0026prime;29.73\u0026Prime;, N24\u0026deg;59\u0026prime;59.51\u0026Prime;; altitude: 933 m). It was identified as \u003cem\u003eArdisia crispa\u003c/em\u003e (Thunb.) A. DC.) by Associate Professor Yan Fulin of Guizhou University of Traditional Chinese Medicine. The voucher specimen (with collection numbers of YFL_2021040307) has been deposited in the Herbarium of Guizhou University of Traditional Chinese Medicine (GZYGH), Guizhou, China. The collection of plant materials complies with the wild plant protection regulations of the People\u0026prime;s Republic of China, and we obtained the permission of local authorities on forestry and the grassland bureau in Guizhou province in China.\u003c/p \u003cp\u003e \u003cb\u003eDNA extraction and chloroplast genome sequencing.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTotal DNA was extracted from the fresh leaves of \u003cem\u003eA. crispa\u003c/em\u003e by the modified CTAB method\u003csup\u003e42\u003c/sup\u003e. Sequencing was carried out on the Illumina HiSeq XTen to generate approximately 3 GB 150 bp reads at Beijing Genomics Institute (BGI, Wuhan, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenome assembly and annotation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe filtered reads were assembled into a complete chloroplast genome by the program GetOrganelle v1.5\u003csup\u003e45\u003c/sup\u003e. In this pipeline, the complete chloroplast genome reads were extracted from total genomic reads and were subsequently assembled using SPAdes version 3.10\u003csup\u003e46\u003c/sup\u003e. The genes were annotated using PGA\u003csup\u003e47\u003c/sup\u003e and Geneious 11.0.3\u003csup\u003e48\u003c/sup\u003e with the published complete chloroplast genome of \u003cem\u003eA. crenata\u003c/em\u003e (GenBank accession number: NC_059021) as the reference. Transfer RNAs (tRNAs) were confirmed by their specific structure predicted by tRNAscan-SE 2.0\u003csup\u003e49\u003c/sup\u003e. The OGDRAW (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chlorobox.mpimp-golm.mpg.de/OGDraw.html\u003c/span\u003e\u003cspan address=\"https://chlorobox.mpimp-golm.mpg.de/OGDraw.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e50\u003c/sup\u003e was used to draw a detailed physical map of the \u003cem\u003eA. crispa\u003c/em\u003e chloroplast genome.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChloroplast genome structural analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe online software REPuter (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bibiserv.cebitec.unibielefeld.de/reputer\u003c/span\u003e\u003cspan address=\"https://bibiserv.cebitec.unibielefeld.de/reputer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to analyze forward (F), palindromic (P), reverse (R), and complement (C) repeats, with the following settings: Minimum repeat size of 3 bp and hamming Distance of 30 bp\u003csup\u003e51\u003c/sup\u003e. We used the default parameters in the online Tandem Repeats Finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tandem.bu.edu/trf/trf.html\u003c/span\u003e\u003cspan address=\"http://tandem.bu.edu/trf/trf.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e52\u003c/sup\u003e to search for tandem repeats in DNA sequences. The online software MISA\u003csup\u003e53\u003c/sup\u003e was applied to predict SSRs with parameter thresholds set at 1, 2, 3, 4, 5, and 6, and nucleotide parameters of 10, 5, 4, 3, 3, 3, and 3, and the distance between two SSRs was not less than 100 bp. Relative synonymous codon usage (RSCU) analysis of the chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e was performed using condon W (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://galaxy.pasteur.fr/?form=codonw\u003c/span\u003e\u003cspan address=\"https://galaxy.pasteur.fr/?form=codonw\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) online software. By using CUSP online software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://imed.med.ucm.es/EMBOSS/\u003c/span\u003e\u003cspan address=\"http://imed.med.ucm.es/EMBOSS/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e54\u003c/sup\u003e, we calculated the effective codon count (ENc), codon adaptation index (CAI), and counted the total codon GC content (GCall), the GC contents of positions 1, 2, and 3 (GC1, GC2, and GC3), and the GC content of position 3 of the synonymous codons (GC3s).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenome comparison.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe genes of the boundaries in the chloroplast genomes of the \u003cem\u003eArdisia\u003c/em\u003e species were compared and visually represented using IRscope\u003csup\u003e55\u003c/sup\u003e(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://irscope.shinyapps.io/irapp/\u003c/span\u003e\u003cspan address=\"https://irscope.shinyapps.io/irapp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to reveal contraction and expansion of the IR regions. The comparative analysis of the whole sequence identity of the chloroplast genomes was performed using mVISTA\u003csup\u003e56\u003c/sup\u003e with the chloroplast genome of \u003cem\u003eA.crispa\u003c/em\u003e (OP626693) as the reference sequence.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA phylogenetic analysis was conducted based on chloroplast genomes from 29 species, including those of the one \u003cem\u003eA. crispa\u003c/em\u003e sequenced and assembled in this study and another 28 downloaded from GenBank. Twenty-nine complete plastid sequences were aligned using MAFFT v7.017\u003csup\u003e57\u003c/sup\u003e, and a BI phylogenetic tree was constructed using MrBayes 3.2.7\u003csup\u003e58\u003c/sup\u003e with \u003cem\u003eLysimachia christinae\u003c/em\u003e (Primulaceae) as the outgroup.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe complete chloroplast genomes and annotations are available at the NCBI database (\u003cem\u003eArdisia crispa\u003c/em\u003e: OP762693).\u003c/p\u003e \u003c/div\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eF.L.Yan designed the research. Z.K.Wu, Y.Q.J., S.H.Wei, F.L.Yan and L.D. collected the samples, J.Ye, F.L.Yan, Q.Luo, and Y.H.Lang conceived the experiments, J.Ye, F.L.Yan, Q.Luo, and Y.H.Lang did computational analysis and deposited sequences. J.Ye, F.L.Yan, and Y.H.Lang wrote the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis study was supported by the National Key Research and Development Program Project (2018YFC1708101), Guizhou Provincial Science and Technology Program Project (No. Qiankezhongyindi [2022] 4016), Guizhou Modern Industrial Technology System Construction of Chinese Herbal Medicines (No. GZCYTX2019-2024), and State Administration of Traditional Chinese Medicine Seedling Breeding of Chinese Herbal Medicines Seeds Required for National Essential Drugs (Guizhou) Base Construction Project (2014\u0026ndash;2017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to J.Ye or F.L.Yan.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGuizhou Medical Products Administration. 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MRBAYES: Bayesian inference of phylogenetic trees. \u003cem\u003eBioinformatics\u003c/em\u003e. \u003cstrong\u003e17\u003c/strong\u003e, 754\u0026ndash;755. https://doi.org/10.1093/bioinformatics/17.8.754 (2001).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4013297/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4013297/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eArdisia crispa\u003c/em\u003e, a member of the Myrsinaceae family, possesses significant horticultural and medicinal properties as an ethnomedicine. The study aimed to analyze the chloroplast genome of \u003cem\u003eA. crispa\u003c/em\u003e and compare it with other \u003cem\u003eArdisia\u003c/em\u003e species, revealing a length of 156,785 bp with a quadripartite structure and 131 genes, including 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Furthermore, 59 simple sequence repeat (SSR) sites were identified in the genome. Examination of codon usage within the chloroplast genome indicated a greater inclination towards A/U nucleotides over G/C nucleotides, with leucine displaying the highest frequency among amino acids. The chloroplast genomes of the nine \u003cem\u003eArdisia\u003c/em\u003e species demonstrate conserved gene content and quantity, presenting more consistent boundaries and decreased variability. In the phylogenetic tree, \u003cem\u003eA. crispa\u003c/em\u003e is clustered with \u003cem\u003eA. crispa\u003c/em\u003e var \u003cem\u003edielsii\u003c/em\u003e, suggesting a close relationship with \u003cem\u003eA. mamillata\u003c/em\u003e and \u003cem\u003eA. pedalis\u003c/em\u003e. This study involved the construction and analysis of the chloroplast genome structure of \u003cem\u003eA. crispa\u003c/em\u003e, as well as phylogenetic analysis using extensive chloroplast genome sequence data from \u003cem\u003eArdisia\u003c/em\u003e plants. This research is crucial for understanding the genetic basis of \u003cem\u003eA. crispa\u003c/em\u003e and the adaptive evolution within the \u003cem\u003eArdisia\u003c/em\u003e genus.\u003c/p\u003e","manuscriptTitle":"The complete chloroplast genome sequence of the medicinal plant Ardisia crispa (Myrsinaceae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-22 13:05:08","doi":"10.21203/rs.3.rs-4013297/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-30T09:35:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-17T07:33:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-17T06:58:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145548118633312903017554747972769601802","date":"2024-05-17T01:54:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273285771114852474817443835635541513795","date":"2024-05-15T11:22:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"835da7d4-b077-476e-a9fb-e7f0bd4f613e","date":"2024-04-25T11:49:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-24T10:00:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-24T09:49:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-20T03:03:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-20T02:53:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-03-04T13:25:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a6d01341-7762-4a16-a680-fc572c112688","owner":[],"postedDate":"March 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29651819,"name":"Biological sciences/Biological techniques/Genomic analysis/Comparative genomics"},{"id":29651820,"name":"Biological sciences/Biotechnology/Genomics"}],"tags":[],"updatedAt":"2024-08-22T19:38:06+00:00","versionOfRecord":{"articleIdentity":"rs-4013297","link":"https://doi.org/10.1038/s41598-024-66563-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-08-16 15:57:10","publishedOnDateReadable":"August 16th, 2024"},"versionCreatedAt":"2024-03-22 13:05:08","video":"","vorDoi":"10.1038/s41598-024-66563-3","vorDoiUrl":"https://doi.org/10.1038/s41598-024-66563-3","workflowStages":[]},"version":"v1","identity":"rs-4013297","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4013297","identity":"rs-4013297","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}