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The chloroplast (cp) genome is a widely used tool for exploring plant evolutionary relationships, yet its effectiveness in fully resolving these relationships remains uncertain. Integrating cp genome data with nuclear DNA information offers a more comprehensive view but often requires separate datasets. In response, we employed the same raw read sequencing data to construct cp genome-based trees and nuclear DNA phylogenetic trees using Read2Tree, a cost-efficient method for extracting conserved nuclear gene sequences from raw read data, focusing on the Aurantioideae subfamily, which includes Citrus and its relatives. The resulting nuclear DNA trees were consistent with existing nuclear evolutionary relationships derived from high-throughput sequencing, but diverged from cp genome-based trees. To elucidate the underlying complex evolutionary processes causing these discordances, we implemented an integrative workflow that utilized multiple alignments of each gene generated by Read2Tree, in conjunction with other phylogenomic methods. Our analysis revealed that incomplete lineage sorting predominantly drives these discordances, while introgression and ancient introgression also contribute to topological discrepancies within certain clades. This study underscores the cost-effectiveness of using the same raw sequencing data for both cp and nuclear DNA analyses in understanding plant evolutionary relationships., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Hymenasplenium is one of the two genera in the large fern family Aspleniaceae. A previous study explored the molecular phylogeny of this genus using several chloroplast DNA fragments and identified three major clades, one of which is the monophyletic Old World clade with southwestern China as its diversity center. To date, there were only a few studies conducted on chloroplast genomes in Hymenasplenium or Aspleniaceae, limiting the understanding of the plastome features and its role in evolution of this group. Here, we studied the complete chloroplast genomes of 12 Hymenasplenium species covering all four subclades of the Old World clade distributed in China. The length of the Hymenasplenium plastomes ranged from 151,617 to 151,930 bp, and contained 129 genes in total, comprising 87 protein-coding, 34 tRNA, and eight rRNA genes. The GC content ranged from 41.8% to 42.1%. Comparative analyses of the Hymenasplenium chloroplast genomes displayed conserved genomic structure and identical gene arrangement. A total of 1,375 simple sequence repeats and 1,639 large repeats were detected. In addition, we detailed hypervariable regions that can be helpful for further phylogenetic research and species delimitation in Hymenasplenium . Furthermore, we supported phylogenetic relationships among major groups as well as possible cryptic speciation found in previous research in the genus. Our study provides new insights into evolutionary history and basic resources for phylogenetic and taxonomic studies of the genus Hymenasplenium ., Competing Interests: The authors declare that they have no competing interests., (© 2024 Chang et al.)

Salix, an economically and ecologically multifunctional tree species widely distributed in China, encompasses five ornamental species sequenced in this study, which are highly beneficial for plant phytoremediation due to their ability to absorb heavy metals. This research utilized high-throughput sequencing to acquire chloroplast genome sequences of Salix, analyzing their gene composition and structural characteristics, identifying potential molecular markers, and laying a foundation for Salix identification and resource classification. Chloroplast DNA was extracted from the leaves of Salix argyracea, Salix dasyclados, Salix eriocephala, Salix integra 'Hakuro Nishiki', and Salix suchowensis using an optimized CTAB method. Sequencing was conducted on the Illumina NovaSeq PE150 platform, and bioinformatics tools were employed to compare the structural features and variations within the chloroplast genomes of the Salix. Analysis revealed high similarity among the chloroplast genome sequences of the five Salix species, with a subsequent examination identifying 276, 269, 270, 273, and 273 SSR loci, respectively, along with unique simple repeat sequences in each variety. Comparison of chloroplast genomes across 22 Salix highlighted variations in regions such as matK-trnQ, ndhC-trnV, psbE-petL, rpl36-rps8, and ndhB-rps7, which may serve as valuable molecular markers for willow resource classification studies. In this study, chloroplast genome sequencing and structural analysis of Salix not only enhances the genetic resources of Salix but also forms a critical basis for the development of molecular markers and the exploration of interspecific phylogeny in the genus., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Chloroplasts, distinctive subcellular organelles found exclusively in plant species, contain three membranes: the outer, inner, and thylakoid membranes. They also have three soluble compartments: the intermembrane space, stroma, and thylakoid lumen. Accordingly, delicate sorting mechanisms are required to ensure proper protein targeting to these sub-chloroplast compartments. Except for most outer membrane proteins, chloroplast interior proteins possess N-terminal cleavable transit peptides as primary import signals. After the cleavage of transit peptides, which occurs during or after import into chloroplasts, the inner and thylakoid membrane proteins, as well as stromal and thylakoid luminal proteins, are further sorted based on additional targeting signals. In this review, we aim to recapitulate the mechanisms by which proteins are targeted to chloroplasts and subsequently sorted into sub-chloroplast compartments, with a focus on the design principles of sorting signals present in chloroplast proteins., Competing Interests: Declarations. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Eomecon chionantha Hance, an endemic species in China, has a long medical history in Chinese ethnic minority medicine and is known for its anti-inflammatory and analgesic effects. However, studies of E. chionantha are lacking. In this study, we investigated the characteristics of the E. chionantha chloroplast genome and determined the taxonomic position of E. chionantha in Papaveraceae via phylogenetic analysis. In addition, we determined molecular markers to identify E. chionantha at the molecular level by comparing the chloroplast genomes of E. chionantha and its closely related species. The complete chloroplast genomic information indicated that E. chionantha chloroplast DNA (178,808 bp) contains 99 protein-coding genes, 8 rRNAs, and 37 tRNAs. Meanwhile, we were able to identify a total of 54 simple sequence repeats through our analysis. Our findings from the phylogenetic analysis suggest that E. chionantha shares a close relationship with four distinct species, namely Macleaya microcarpa, Coreanomecon hylomeconoides, Hylomecon japonica, and Chelidonium majus. Additionally, using the Kimura two-parameter model, we successfully identified five hypervariable regions (ycf4-cemA, ycf3-trnS-GGA, trnC-GCA-petN, rpl32-trnL-UAG, and psbI-trnS-UGA). To the best of our knowledge, this is the first report of the complete chloroplast genome of E. chionantha, providing a scientific reference for further understanding of E. chionantha from the perspective of the chloroplast genome and establishing a solid foundation for the future identification, taxonomic determination and evolutionary analysis of this species., (© 2024. The Author(s) under exclusive licence to The Botanical Society of Japan.)

The taxonomic concepts and phylogenetic relations among genera of the family Mniaceae have given rise to much controversy in recent years, including Mnium, Plagiomnium, and Pohlia. Chloroplast genome study of these genera will be helpful to reflect the fact of this relationship. In this study, we sequenced three species in the Plagiomnium genus using an Illumina HiSeq 4000 platform. The complete chloroplast genomes of P. rostratum, P. succulentum and P. vesicatum were 125,196 bp, 124,689 bp, and 124,663 bp in length, which all contained a quadripartite structure including two copies of the invert repeats (IR, 10,120 bp, 9,818 bp, and 9,665 bp), one large single copy region (LSC, 86,395 bp, 86,299 bp, and 86,532 bp), and one single copy region (SSC, 18,561 bp, 18,754 bp, and 18,801 bp). The overall GC contents were 29.8%, 30.5%, and 30.5% respectively. The simple sequence repeats (SSRs) were detected in conjunction with Plagiomnium acutum, with variable sites genes observed: rpoC2, ycf1, and ycf2. Combined with the other three sequences published in Mniaceae, analyses of codon usage, repeats sequences, GC contents, and gene features revealed similarities among the seven species in Mniaceae. The trend of nucleotide diversity (Pi) in the seven complete chloroplast genomes showed Pi > 0.056: trnI-rpl23, petG-petL-psbE, trnK-chlB, trnG-trnR-atpA, rpoB-trnC-ycf66, ndhB, trnN-ndhF, and rps15-ycf1. We confirmed the phylogenetic relationships that Plagiomnium genus is a sister group with Mnium, while the Pohlia genus is not a monophyletic group. Phylogenetic analyses corroborated the monophyly of Mniaceae and supported the transfer of the Pohlia genus into Mniaceae., Competing Interests: Declarations. Ethical approval: Not applicable. Conflict of interest: The authors declare that they have no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Rubia alata Wall (R. alata) and Rubia ovatifolia Z. Ying Zhang (R. ovatifolia) are unique medicinal plants native to China. Sequencing their chloroplast genomes is important for understanding species differentiation and establishing phylogenetic relationships., Methods and Results: The chloroplast genomes of R. alata and R. ovatifolia were sequenced using the Illumina HiSeq platform. The chloroplast genome of R. alata is 154,973 base pairs (bp) in length, containing a large single-copy region (LSC) of 84,801 bp, a small single-copy region (SSC) of 17,138 bp, and a pair of inverted repeats (IRs) of the same length. The length of the chloroplast genome, LSC, SSC, and IR regions of R. ovatifolia is 26,517 bp, 84,716 bp, 17,116 bp and 26,517 bp, respectively. Codon usag e analysis revealed that R. alata had the highest frequency of Aspartic acid (Asp) (1650 occurrences) in protein-coding sequences (CDS), while R. ovatifolia showed the highest frequency of Tyrosine (Try) (1479 occurrences). Comparative analysis of chloroplast genomes across seven species from the genus Rubia identified the most divergent coding regions, including rps16, psbI-trns-CGA, and petN, while plastid rRNAs were the most conserved. Phylogenetic analysis showed R. alata clustering with R. cordifolia (66.3% support), and R. ovatifolia clustering with Rubia podantha (100% support)., Conclusions: These findings enhance our understanding of the chloroplast genome structure in Rubia species and provide molecular information for the future development and utilization of R. alata and R. ovatifolia resources., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethical approval and consent to participate The authors confirm that all methods comply with local and national regulations. Consent for publication This manuscript has been posted as a preprint on Research Square with https://doi.org/10.21203/rs.3.rs-3896731/v1, which is available from: https://www.Researchsquare.com/article/rs-3896731/v1., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Cinnamomum daphnoides (Siebold & Zucc 1846) is a rare and endangered island species with a unique Sino-Japanese distribution pattern. However, inormation regarding the species' chloroplast (cp) genome, structural features, and the phylogenetic relationship is still lacking. We utilized high-throughput sequencing technology to assemble and annotate the first cp genome of C. daphnoides (GenBank OR654104), followed by genomic characterization and phylogenetic analysis to fill the gaps in this species' cp genome. Our analysis showed that the cp genome has a quadripartite structure spanning 152,765 bp with a GC content of 39.15%. The genome encodes 126 genes, which include 36 tRNA genes, 8 rRNA genes, and 82 mRNA genes. Specifically, 44 genes are related to photosynthesis, 59 are associated with self-replication, six are other genes, and four have unknown functionality. The Codon usage bias in the genome exhibits a preference for A/U bases. We identified 29 interspaced repeat sequences that belonging to three types of repeat sequences. A total of 217 cpSSR loci were detected with single nucleotide repeats (59.91%) being the most frequent loci, mainly composed of A/T repeats. Our selection pressure analysis revealed that the ycf2 gene experienced strong positive selection (Ka/Ks = 1.81, P > 0.844). Further, we identified three highly variable fragments (psbM, psbT, and ycf1) that can be utilized as specific DNA barcoding markers for species definition and population genetic studies. We conducted boundary analysis, which showed that the structure and gene sequence of the two species were highly conserved. Finally, our phylogenetic analysis supports that C. daphnoides is close to C. cassia in the Cinnamomum genes, indicating that the two species share a common ancestry. Overall, providing genomic information on C. daphnoides will be beneficial for the conservation and utilization of endangered plant genetic resources. It will also serve as a reference for the identification of species and the phylogenetic analysis of Cinnamomum. This information will be useful in future research., Competing Interests: Declarations Conflict of interest No potential conflict of interest was reported by the authors. Ethical Approval This article does not contain any studies with animals performed by any of the authors. The plant materials used in this study were collected from the arboretum of Zhejiang Academy of Forestry, and the collection process was in accordance with the regulations of the academy and relevant local laws and regulations., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Potato (Solanum tuberosum) is considered worldwide as one of the most important non-cereal food crops. As a result of its adaptability and worldwide production area, potato displays a vast phenotypical variability as well as genomic diversity. Chloroplast genomes have long been a core issue in plant molecular evolution and phylogenetic studies, and have an important role in revealing photosynthetic mechanisms, metabolic regulations and the adaptive evolution of plants. We sequenced the complete chloroplast genome of the Hungarian cultivar White Lady, which is 155 549 base pairs (bp) in length and is characterised by the typical quadripartite structure composed of a large- and small single-copy region (85 991 bp and 18 374 bp, respectively) interspersed by two identical inverted repeats (25 592 bp). The genome consists of 127 genes of which 82 are protein-coding, eight are ribosomal RNAs and 37 are transfer RNAs. The overall gene content and distribution of the genes on the White Lady chloroplast was the same as found in other potato chloroplasts. The alignment of S. tuberosum chloroplast genome sequences resulted in a highly resolved tree, with 10 out of the 13 nodes recovered having bootstrap values over 90%. By comparing the White Lady chloroplast genome with available S. tuberosum sequences we found that gene content and synteny are highly conserved. The new chloroplast sequence can support further studies of genetic diversity, resource conservation, evolution and applied agricultural research. The new sequence can support further potato genetic diversity and evolutionary studies, resource conservation, and also applied agricultural research., Competing Interests: Declarations. Conflict of interest: The authors have no competing interests to declare that are relevant to the content of this article. Ethical approval: No ethical approval was required for the study., (© 2024. The Author(s).)

Key Message: This study describes an optimized plastid genetic engineering platform to produce full marker-free transplastomic plants with transgene integrated at homoplasmy in one step in tissue culture. Plastid engineering is attractive for both biotechnology and crop improvement due to natural bio-confinement from maternal inheritance, the absence of transgene positional effects and silencing, the ability to express transgenes in operons, and unparalleled production of heterologous proteins. While plastid engineering has had numerous successes in the production of high-value compounds, no transplastomic plants have been approved for use in agriculture. In order for transplastomic plants to be used in agriculture, the removal of antibiotic selection genes is required. In this work, we developed an optimized strategy to generate homoplasmic marker-free lines of potato (Solanum tuberosum) in a single transformation event. To achieve marker-free transplastomic lines, vectors were redesigned to enable integration of the transgene cassette into the plastid genome, while maintaining the selection cassette on the vector backbone. After an initial round of tissue culture with selection, the selective pressure was removed, leading to the elimination of the vector backbone, while retaining the integrated transgene cassette at homoplasmy. Marker-free transplastomic lines produced using this strategy had a normal phenotype, and transgene integration was stable across generations. The new vectors developed in this work for the generation of marker-free transplastomics will represent a valuable alternative platform for routine plastid genetic engineering in higher plants. It is also anticipated that this approach will contribute to speed the path to commercialization of these novel transplastomic plant varieties., Competing Interests: Declarations. Conflict of interest: CNS is editor-in-chief of Plant Cell Reports. He had no editorial role in peer review or the decision for this manuscript., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

To maintain CO 2 fixation in the Calvin-Benson-Bassham cycle, multistep regulation of the chloroplast ATP synthase (CF 1 F o ) is crucial to balance the ATP output of photosynthesis with protection of the apparatus. A well-studied mechanism is thiol modulation; a light/dark regulation through reversible cleavage of a disulfide in the CF 1 F o γ-subunit. The disulfide hampers ATP synthesis and hydrolysis reactions in dark-adapted CF 1 F o from land plants by increasing the required transmembrane electrochemical proton gradient ([Formula: see text]). Here, we show in Chlamydomonas reinhardtii that algal CF 1 F o is differently regulated in vivo. A specific hairpin structure in the γ-subunit redox domain disconnects activity regulation from disulfide formation in the dark. Electrochromic shift measurements suggested that the hairpin kept wild-type CF 1 F o active, whereas the enzyme was switched off in algal mutant cells expressing a plant-like hairpin structure. The hairpin segment swap resulted in an elevated [Formula: see text] threshold to activate plant-like CF 1 F o , increased by ~1.4 photosystem (PS) I charge separations. The resulting dark-equilibrated [Formula: see text] dropped in the mutants by ~2.7 PSI charge separation equivalents. Photobioreactor experiments showed no phenotypes in autotrophic aerated mutant cultures. In contrast, chlorophyll fluorescence measurements under heterotrophic dark conditions point to an altered dark metabolism in cells with the plant-like CF 1 F o as the result of bioenergetic deviations from wild-type. Our results suggest that the lifestyle of C. reinhardtii requires a specific CF 1 F o dark regulation that partakes in metabolic coupling between the chloroplast and acetate-fueled mitochondria., Competing Interests: Competing interests statement:The authors declare no competing interest.

Photosynthesis in fluctuating light requires coordinated adjustments of diffusion conductance and biochemical capacity, but the role of chloroplast ATP synthase activity (g H + ) in dynamic photosynthesis is not well understood. In this study, we measured gas exchange, chlorophyll fluorescence and electrochromic shift signals in fluctuating light for leaves of tomato (Solanum lycopersicum) and maize (Zea mays). During the transition from sun to shade, simultaneous increases in g H + , effective quantum yield of PSII, and net CO 2 assimilation rate (A N ) occurred in tomato but uncoupled in maize, indicating that g H   +  limited A N during the sun-to-shade transition in tomato but not in maize. During the shade-to-sun transition, g H   +  increased simultaneously with stomatal conductance, mesophyll conductance and Rubisco carboxylation capacity in tomato, suggesting that g H + is an overlooked factor affecting light induction of A N in tomato. By comparison, g H in fluctuating light differs between species, and chloroplast ATP synthase may be a potential target for improving dynamic photosynthesis in crops such as tomato.+  maintained at high levels in maize and its A N was mainly restricted by stomatal conductance. Our results reveal that the kinetics of g H + in fluctuating light differs between species, and chloroplast ATP synthase may be a potential target for improving dynamic photosynthesis in crops such as tomato., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Berberidis Radix (Sankezhen), a typical multi-origin Chinese medicinal material, originates from the dried roots of plants of the Berberis genus and is used to treat various ailments. These species have similar morphologies, potentially leading to misidentifications that can impact medicine efficacy. Therefore, developing suitable molecular markers to identify medicinal species is imperative. Furthermore, discrepancies exist in the taxonomy of the Berberis genus. In the present study, we de novo assembled the chloroplast genomes of six Berberis species (Berberis woomungensis C. Y. Wu, Berberis pruinosa Franch., Berberis thunbergii DC., Berberis chinensis Poir., Berberis wilsoniae Hemsl., and Berberis sp.) that commonly constitute Berberidis Radix and compared them with previously reported genomes. Our comparative analysis revealed similarities in genome structure, relative synonymous codon usage, amino acid frequency, repeats, and substitutions. Higher synonymous substitutions, indicative of predominant purifying selection on protein-coding genes, were observed compared to non-synonymous substitutions. However, positive selection was identified in six genes across 29 Berberis species-accD, matK, ndhD, rbcL, ycf1, and ycf2-highlighting their potential roles in adaptive responses to specific environmental conditions within the genus. Inverted repeats expansion and contraction affected the rate of mutations and were associated with the phylogenetic classification of Berberis. Our phylogenetic analysis supported the division of the Berberis complex into four genera, which corroborates previous studies involving extensive sampling. We identified the ndhD-ccsA region as the most polymorphic region and applied this region to Chinese patent medicines containing Berberidis Radix through metabarcoding. The metabarcoding analysis confirmed that five Berberis species commonly constitute Berberidis Radix in Chinese patent medicines. In conclusion, this study provides insight into the molecular evolution of the chloroplast genome and the phylogeny of the Berberis genus. In addition, metabarcoding provides insight into the species composition of Berberidis Radix in Chinese patent medicines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The chloroplast genome has considerable potential to enhance crop productivity, but it remains underutilized in breeding because it is difficult to modify. This study elucidates the potential of recently developed chloroplast-targeted C-to-T base editors in facilitating the use of the chloroplast genome for crop breeding. The herbicide metribuzin interferes with photosynthesis by binding to the D1 protein of photosystem II, encoded by the chloroplast genome. Naturally occurring D1 mutants with V219I or A251V substitutions are known to have resistance to some herbicides including metribuzin. Here, using the base editors, we introduced these substitutions and showed that the A251V single mutation and the V219 & A251V double mutations conferred significant metribuzin resistance to Arabidopsis thaliana. The V219I & A251V double mutants exhibited increased metribuzin resistance and grew better than the A251V single mutants. Furthermore, the double mutants grew as well as wild-type plants in the absence of metribuzin. The single and double mutants, which are a challenge to obtain through traditional mutagenesis and crossbreeding methods, can be relatively easily generated using C-to-T base editors. In view of the conservation of V219 and A251 across numerous species, C-to-T base editing can potentially confer metribuzin resistance to a wide range of crops. Compared to nuclear genes, chloroplast genes are also less likely to spread into wild populations. Our findings suggest that chloroplast-targeting C-to-T base editors will find many roles in future crop breeding efforts., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Lasianthus species are widely used in traditional Chinese folk medicine with high medicinal value. However, source materials and herbarium specimens are often misidentified due to morphological characteristics and commonly used DNA barcode fragments are not sufficient for accurately identifying Lasianthus species. To improve the molecular methods for distinguishing among Lasianthus species, we report the complete chloroplast (CP) genomes of Lasianthus attenuatus, Lasianthus henryi, Lasianthus hookeri, Lasianthus sikkimensis, obtained via high-throughput Illumina sequencing., Results: These showed CP genomes size of 160164-160246 bp and a typical quadripartite structure, including a large single-copy region (86675-86848 bp), a small single-copy region (17177-17326 bp), and a pair of inverted repeats (28089-28135 bp). As a whole, the gene order, GC content and IR/SC boundary structure were remarkably similar among of the four Lasianthus CP genomes, the partial gene length and IR, LSC and SSC regions length are still different. The average GC content of the CP genomes was 36.71-36.75%, and a total of 129 genes were detected, including 83 different protein-coding genes, 8 different rRNA genes and 38 different tRNA genes. Furthermore, we compared our 4 complete CP genomes data with publicly available CP genome data from six other Lasianthus species, and we initially screened eleven highly variable region fragments were initially screened. We then evaluated the identification efficiency of eleven highly variable region fragments and 5 regular barcode fragments. Ultimately, we found that the optimal combination fragment' ITS2 + psaI-ycf4' could authenticated the Lasianthus species well. Additionally, the results of genome comparison of Rubiaceae species showed that the coding region is more conservative than the non-coding region, and the ycf1 gene shows the most significant variation. Finally, 49 species of CP genome sequences belonging to 16 genera of the Rubiaceae family were used to construct phylogenetic trees., Conclusions: Our research is the first to analyze the chloroplast genomes of four species of Lasianthus in detail and we ultimately determined that the combination fragment' ITS2 + psaI-ycf4' is the optimal barcode combination for identifying the genus of Lasianthus. Meanwhile, we gathered the available CP genome sequences from the Rubiaceae and used them to construct the most comprehensive phylogenetic tree for the Rubiaceae family. These investigations provide an important reference point for further studies in the species identification, genetic diversity, and phylogenetic analyses of Rubiaceae species., (© 2024. The Author(s).)

The Malvaceae family, comprising 9 subfamilies and 4,225 species, includes economically significant taxa, such as Ceiba pentandra, Gossypium ekmanianum, Gossypium stephensii, Kokia drynarioides, Talipariti hamabo, and Durio zibethinus. Chloroplast (cp) genome research is crucial for elucidating the evolutionary divergence and species identification within this family. In this study, we assembled and annotated cp genomes of six Malvaceae species, conducting comprehensive comparative genomic and phylogenomic analyses. The assembled genomes range from 160,495 to 163,970 bp in size, with 125-129 genes annotated. Notable differences were observed in the IR (inverted repeat) regions, and SSR analysis revealed that Durio zibethinus has the highest number of specific variation sites. Among the six species, Talipariti hamabo uniquely exhibits more palindromic repeats than forward repeats. Seven highly mutated regions were identified, offering potential markers for species identification. Phylogenetic reconstruction using the maximum likelihood method revealed two primary clades within Malvaceae: Byttneriina and Malvadendrina. Within Malvadendrina, the subfamily Helicteroideae represents the earliest divergence, followed by Sterculioideae. This study provides a robust phylogenetic framework and valuable insights into the classification and evolutionary history of Malvaceae species., Competing Interests: Declarations. Ethical approval: The study was conducted the plant material that complies with relevant institutional, national, and international guidelines and legislation. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background : Xi Junecry ( Pinellia ternata ), a perennial herb of the Araceae family, is indigenous to Xinxian County, Henan Province, China, and is regarded as a premium variety among similar medicinal materials. However, the lack of comprehensive genetic information on Xi Junecry germplasm resources has constrained the cultivation and identification of high-quality varieties. Methods : In this study, six chloroplast genomes of Xi Junecry were assembled and annotated using high-throughput sequencing. Subsequently, comparative analyses were conducted, and a phylogenetic tree was constructed. Results : The six Xi Junecry chloroplast genome lengths ranged from 157,456 to 158,406 bp, and the GC content was between 36.0% and 36.2%. A total of 265 single nucleotide polymorphism sites were identified across the six genomes, with a whole-genome nucleotide diversity (Pi) value of 0.00084. Among the four genomic regions, the small single-copy region exhibited the highest Pi, followed by the large single-copy region, while the inverted repeat region showed the lowest. Nucleotide polymorphism in coding regions was significantly lower than in non-coding regions. Nine hypervariable regions were identified, as follows: ndhE-ndhG , trnN-GUU-ndhF, trnS-GCU-trnG-UCC , atpB-rbcL , psaI , accD-ycf4 , psbE-petL , psaC-ndhE , and psbI-trnG-UCC . Positive selection sites were detected in the accD and rbcL genes. Phylogenetic analysis clustered the six Xi Junecry samples into a distinct clade, separating them from other regional Pinellia samples. Conclusions : These findings elucidate the genetic variation levels in Xi Junecry and provide high-variability loci for population history inference, genetic diversity assessment, species domestication studies, and new cultivar development.

Background: The genus Impatiens, which includes both annual and perennial herbs, holds considerable ornamental, economic, and medicinal value. However, it posed significant challenges for taxonomic and systematic reconstruction. This was largely attributed to its high intraspecific diversity and low interspecific variation in morphological characteristics. In this study, we sequenced samples from 12 Impatiens species native to China and assessed their phylogenetic resolution using the complete chloroplast genome, in conjunction with published samples of Impatiens. In addition, a comparative analysis of chloroplast genomes were conducted to explore the evolution of the chloroplast genome in Impatiens., Results: The chloroplast genomes of 12 Impatiens species exhibited high similarity to previously published samples in terms of genome size, gene content, and sequence. The chloroplast genome of Impatiens exhibited a typical four-part structure, with lengths ranging from 146,987 bp(I. morsei)- 152,872 bp(I. jinpingensis). Our results identified 10 mutant hotspot regions (rps16, rps16-trnG, trnS-trnR, and rpoB-trnC) that could serve as effective molecular markers for phylogenetic analyses and species identification within the Impatiens. Phylogenetic analyses supported the classification of Impatiens as a monophyletic taxon. The identified affinities supported the taxonomic classification of the subgenus Clavicarpa within the Impatiens, with subgenus Clavicarpa being the first taxon to diverge. In phylogenetic tree,the Impatiens was divided into eight distinct clades. The results of ancestral trait reconstruction suggested that the ancestral traits of Impatiens included a perennial life cycle, four sepals and three pollen grooves. However, the ancestral morphology regarding fruit shape, flower colour, and spacing length remained ambiguous., Conclude: Our study largely supported the family-level taxonomic treatment of Impatiens species in China and demonstrated the utility of whole chloroplast genome sequences for phylogenetic resolution. Comparative analysis of the chloroplast genomes of Impatiens facilitated the development of molecular markers.The results of ancestral trait reconstruction showed that the ancestor type of habit was perennial, the number of sepals was 4, and morphology and number of aperture was 3 colpus. The traits of capsule shape, flower colour, and spur length underwent a complex evolutionary process. Our results provided data support for further studies and some important new insights into the evolution of the Impatiens., Competing Interests: Declarations. Ethics approval and consent to participate: The collecting of all samples in this study followed the Regulations on the Protection of Wild Plants of China, the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. All methods were carried out in accordance with relevant guidelines and regulations. Consent for publications: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Pueraria is an edible and medicinal raw material, which is of great value to the pharmaceutical and food industries. Nonetheless, due to morphological diversity and complex domestication history, the classification of Pueraria plants is ambiguous. As the varieties on the market are mixed, the species are difficult to distinguish, and their morphological characteristics are similar to the physical and chemical properties. It is difficult to accurately identify them by traditional identification methods. Chloroplast (cp) genomes are widely used in species identification and phylogenetic studies to achieve accurate identification of medicinal plants, and can also provide more reference information for phylogenetic studies. Based on interspecific and intraspecific sampling, the cp genomes of eight species or varieties of Pueraria plants were examined in this study., Results: The study unveiled that the cp genome size varied from 151,555 to 153,668 base pairs (bp), with the total GC content ranging from 35.4 to 37.0%. Moreover, it was discerned that the cp genome contained between 128 and 135 genes. Comparative analysis indicated that the highest number of Simple Sequence Repeats (SSRs) was identified in P. montana and P. alopecuroides, with a preponderance of these SSRs being rich in Adenine (A) and Thymine (T) nucleotides. Complete comparison and sliding window analysis of the cp genome established that the non-coding region exhibited greater sequence differences than the coding region, and that the large single copy (LSC) region demonstrated higher nucleotide polymorphism levels. Fourteen highly variable loci such as rpoB,ycf1,rbcL,trnF-GAA-trnL,psbC-psbD, and ycf4-cemA were detected as potential molecular markers for Pueraria species identification. Moreover, the phylogenetic tree demonstrated that other Pueraria species had the most distant relationship with Haymondia wallichii and Toxicopueraria peduncularis, thereby offering fresh perspectives into the species classification of Pueraria. The molecular clock analysis results indicate that the divergence time of Pueraria may occur at ∼6.46 Ma. It is speculated that the cold climate may be the cause of Pueraria species diversity and promote the radiation of the genus., Conclusion: This research provides theoretical backing and serves as a reference point for the identification and taxonomical classification of Pueraria species. The findings will prove beneficial in future studies on the preservation of medicinal resources, phylogenetic relationships, and genetic engineering of Pueraria plants., Competing Interests: Declarations. Ethics approval and consent to participate: This study including the collection of plant samples complies with relevant institutional, national, and international guidelines and legislation. All the necessary permissions have been granted for this research. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Numerous species of Ardisia are widely used for their medicinal and ornamental values in China. However, accurately identifying Ardisia species at the molecular level remains a challenge due to the morphological similarities among different species, the complexity of interspecific variation, and the limited availability of genetic markers. In this study, we reported 20 chloroplast genomes of Ardisia species from China and combined them with 8 previously published chloroplast genomes to conduct a comprehensive analysis for phylogenetic relationships and adaptive evolution., Results: For the 28 Ardisia species analyzed in this study, the size of the chloroplast genomes ranged from 155,088 bp to 156,999 bp, and all exhibited a typical tetrad structure with conserved gene content and number. Each genome contained 85-88 protein-coding genes, 36-37 tRNA genes, and 8 rRNA genes. Comparative analysis showed that the genomic structures and gene order were relatively conserved with slight variations in the inverted repeat regions (IRs). Simple sequence repeats (SSRs) were predominantly single nucleotide repeats, while repeat sequences were mainly composed of palindromic and forward repeats. Twelve highly variable regions were identified as potential DNA barcodes for species identification and phylogenetic analysis of Ardisia. The phylogenetic tree supported the division of the subgenus Bladhia s.l. into two subgenera: Bladhia s.str. and Odontophylla (Yang) Huang. Further investigation revealed that two protein-coding genes (rbcL and rpoC2) were under positive selection and might be associated with the adaptation of Ardisia species to shaded environments., Conclusion: Our study analyzed the chloroplast genomes of 20 Ardisia species from China to explore their phylogenetic relationships and adaptive evolution. By combining these results with data from eight previously published chloroplast genomes, the essential characteristics of Ardisia chloroplast genomes were clarified. The research establishes a theoretical basis for the classification, identification, and comprehension of the adaptive evolution of Ardisia species., Competing Interests: Declarations. Ethics approval and consent to participate: The collection of all samples completely complies with national and local legislation permission. Plant samples used in the study were not included in the list of national key protected plants and were not collected from the national park or nature reserve when we collected them. According to national and local legislation, no specifc permission was required for collecting these plants when we collected them. Voucher specimens were prepared and deposited at the Herbarium of Guangxi Institute of Botany, Chinese Academy of Sciences (IBK). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Guizhou Province is one of the most important karst regions of southwest China, with 22 Primulina species. These species are highly habitat-specialized and dependent on the soils of the karst region, and many inter-species classifications are unclear. Therefore, studying the chloroplast genomes and estimating the divergence times of there species can not only provide a better understanding of interspecific relationships but also help to know the species speciation and divergence in the karst environment. So, we sequenced and assembled the chloroplast genomes of nine Primulina species (including six endemic species of Guizhou) and conducted chloroplast genome comparison analysis and phylogenetic study. The chloroplast genome structures of the nine Primulina species were quadripartite with total lengths of 152,869-153,364 base pairs (bp) and GC content of 37.55-37.64%. There are 132 functional genes annotated, respectively. A total of 375 simple sequence repeats and 375 interspersed nuclear elements were identified. The 30 highly preferred codons identified were used at similar frequencies in different species, respectively. A phylogenetic tree constructed on the basis of the 38 chloroplast genomes showed that Primulina species form a monophyletic group. Eleven mutational hotspot regions that could serve as potential molecular markers were identified, of which two regions near the 3' and 5' ends of the ycf1 gene were of appropriate size and can serve as molecular markers for phylogenetic studies of Primulina. The results of molecular clock analyses indicate that the three major branches of Primulina begin to diverge in the Miocene, and the number of species proliferated in the Pliocene and Pleistocene. Most of the species of Primulina in Guizhou Province were formed in the Pleistocene and rapidly diverged within a short period of time. This research study enriches the genetic resource information of Primulina and deepens the understanding of the phylogenetic relationships of the genus., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: The plants collected in the study are not listed on the List of National Key Protected Wild Plants in China or the IUCN red list of threatened soecies, and were collected following international, national and local legislation, with no special permission required for sample collection., (© 2024. The Author(s).)

Background/objectives: Sium serra is distributed in Korea, China, and Japan. It was first identified as the genus Pimpinella and then reclassified as Sium by Kitagawa. Some Sium species are used as herbal medicine and are often confused with the similar form Ligusticum sinense . In this study, we analyzed the cp genome of S. serra and conducted comparative analyses with the cp genomes of related taxa., Methods: We extracted gDNA from fresh leaves and sequenced it using Illumina HiSeq2500. For the chloroplast genome assembly, de novo assembly was performed using Velvet v1.2.07. For the annotation, GeSeq and NCBI BLASTN were used. Afterwards, related taxa were analyzed using programs such as DnaSP and MISA., Results: S. serra was excluded from the study on the chloroplast (cp) genome in Sium because it was classified as Pimpinella in China. Therefore, this study aimed to analyze the cp genome of S. serra for the first time and its location within the genus Sium . The complete cp genome of S. serra was 154,755 bp in length, including a pair of inverted repeats, each 26,255 bp, a large single-copy region of 84,581 bp, and a small single-copy region of 17,664 bp. The cp genome comprised 79 protein-coding, 30 tRNA, and 4 rRNA genes. Furthermore, six regions of high nucleotide diversity were identified in the genus Sium . In the genus Sium , 1630 repeats that can serve as markers were also identified. Eight protein-coding genes with high K A /K S values were under positive selection in the Sium . Our phylogenetic analyses suggest that S. serra was positioned with high bootstrap support within the Sium of the tribe Oenantheae , specifically in the southern Palearctic subclade., Conclusions: In this study, the S. serra chloroplast genome was sequenced and assembled. The genus Sium formed a monophyletic group; however, as not all the Sium species were included in this study, further research is necessary. This study can serve as foundational data not only for Sium but also for the tribe Oenantheae.

Desmodesmus (Sphaeropleales, Chlorophyceae) is a genus of freshwater green algae widely distributed in aquatic ecosystems and known for its potential in biotechnology and bioremediation. We present the complete chloroplast genome sequences of two Desmodesmus species, D. hystrix and D. perforatus. Comparative analysis revealed significant differences in their quadripartite structures, particularly in the lengths of the small single copy regions. We observed variations in G + C content across genomic regions, with inverted repeat regions showing a higher G + C content. Microsatellite analysis revealed that A/T mononucleotides were the most abundant type, while dispersed repeat analysis suggested a potential link to expansion of the single-copy region in D. perforatus. Gene annotation, codon usage analysis, and comparison of genome rearrangement highlighted both similarities and differences between the species. Nucleotide diversity analysis revealed higher variability in intergenic regions than in coding regions, with IR regions displaying relative conservation. Comparative analyses of genomes and IR border arrangements demonstrated that the Desmodesmus species exhibit substantial differences in genome structure and gene arrangement among Scenedesmaceae. Phylogenetic analysis, based on 51 single-copy genes from 34 species of Sphaeropleales provided additional information about phylogenetic relationships within Sphaeropleales. These findings contribute to our understanding of comparative plastid genomics in green algae., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Background: The genus Amorphophallus (Araceae) contains approximately 250 species, most of which have high ecological and economic significance. The chloroplast genome data and the comprehensive analysis of the chloroplast genome structure of Amorphophallus is limited. In this study, four chloroplast genomes of Amorphophallus were sequenced and assembled. For the first time, comparative analyses of chloroplast genomes were conducted on the 13 Amorphophallus species in conjunction with nine published sequences., Results: The Amorphophallus chloroplast genomes exhibited typical quadripartite structures with lengths ranging from 164,417 to 177,076 bp. These structures consisted of a large single copy (LSC, 90,705 - 98,561 bp), a small single copy (SSC, 14,172 - 21,575 bp), and a pair of inverted repeats (IRs, 26,225 - 35,204 bp). The genomes contain 108 - 113 unique genes, including 76 - 79 protein-coding genes, 28 - 29 tRNA genes, and 4 rRNA genes. The molecular structure, gene order, content, codon usage, long repeats, and simple sequence repeats (SSRs) within Amorphophallus were generally conserved. However, several variations in intron loss and gene expansion on the IR-SSC boundary regions were found among these 13 genomes. Four mutational hotspot regions, including trnM-atpE, atpB, atpB-rbcL and ycf1 were identified. They could identify and phylogeny future species in the genus Amorphophallus. Positive selection was found for rpl36, ccsA, rpl16, rps4, rps8, rps11, rps12, rps14, clpP, rps3, ycf1, rpl20, rps2, rps18, rps19, atpA, atpF, rpl14, rpoA, rpoC1, rpoC2 and rps15 based on the analyses of Ka/Ks ratios. Phylogenetic inferences based on the complete chloroplast genomes revealed a sister relationship between Amorphophallus and Caladieae. All Amorphophallus species formed a monophyletic evolutionary clade and were divided into three groups, including CA-II, SEA, and CA-I. Amorphophallus albus, A. krausei, A. kachinensis and A. konjac were clustered into the CA-II clade, A. paeoniifolius and A. titanum were clustered into the SEA clade, A. muelleri 'zhuyajin1', Amorphophallus sp, A. coaetaneus, A. tonkinensis and A. yunnanensis were clustered into CA- I clade., Conclusions: The genome structure and gene content of Amorphophallus chloroplast genomes are consistent across various species. In this study, the structural variation and comparative genome of chloroplast genomes of Amorphophallus were comprehensively analyzed for the first time. The results provide important genetic information for species classification, identification, molecular breeding, and evolutionary exploration of the genus Amorphophallus., Competing Interests: Declarations. Ethics approval and consent to participate: The materials involved in the article does not an endangered or protected species; therefore, permission is not required to collect this species. Research on these species, including the collection of plant materials has been carried out in accordance with guidelines provided by Kunming University. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Genomic analysis is crucial for better understanding the evolutionary history of species and for their conservation. Shizhenia pinguicula is a rare medicinal plant endemic to China. However, the complete chloroplast genome of this species has not been reported to date. Insufficient genomic research on S. pinguicula has hindered effective conservation efforts for this valuable plant., Methods: In this study, we sequenced and assembled the first complete chloroplast genome of S. pinguicula using Illumina sequencing technology. We conducted a comparative analysis of its chloroplast genome with related species and reconstructed phylogenetic relationships., Results: The chloroplast genome of S. pinguicula exhibited a typical quadripartite structure with a length of 158,658 bp. A total of 123 genes, 118 simple sequence repeats, and 51 dispersed repetitive sequences were identified. The inverted repeat boundaries were significantly expanded, along with the pseudogenization and loss of multiple NDH genes. Codon usage bias is primarily influenced by natural selection and other factors, with the ycf3 gene under positive selection. Additionally, 10 hypervariable regions were detected for species identification and evolutionary studies. Phylogenetic analysis indicated that Ponerorchis gracilis and Hemipilia yajiangensis form a clade, with S. pinguicula as their sister species, located at the basal position of the Ponerorchis - Hemipilia alliance., Conclusions: The chloroplast genome structure and gene content of S. pinguicula exhibit certain degrees of variation compared to other species within the Orchidinae subtribe. This genome should be useful for further investigations into the biology of Shizhenia and the development of biodiversity conservation strategies.

Background: Zingiber Mill., a morphologically diverse herbaceous perennial genus of Zingiberaceae, is distributed mainly in tropical to warm-temperate Asia. In China, species of Zingiber have crucial medicinal, edible, and horticultural values; however, their phylogenetic relationships remain unclear., Methods: To address this issue, the complete plastomes of the 29 Zingiber accessions were assembled and characterized. Comparative plastome analysis and phylogenetic analysis were conducted to develop genomic resources and elucidate the intraspecific phylogeny of Zingiber ., Results: The newly reported plastomes ranged from 161,495 to 163,880 bp in length with highly conserved structure. Results of comparative analysis suggested that IR expansions/contractions and changes of repeats were the main reasons that influenced the genome size of the Zingiber plastome. A large number of SSRs and six highly variable regions ( rpl20 , clpP , ycf1 , petA - psbJ , rbcL - accD , and rpl32 - trnL ) have been identified, which could serve as potential DNA markers for future population genetics or phylogeographic studies on this genus. The well-resolved plastome phylogeny suggested that Zingiber could be divided into three clades, corresponding to sect. Pleuranthesis (sect. Zingiber + sect. Dymczewiczia ) and sect. Cryptanthium ., Conclusions: Overall, this study provided a robust phylogeny of Zingiber plants in China, and the newly reported plastome data and plastome-derived markers will be of great significance for the accurate identification, protection, and agricultural management of Zingiber resources in the future.

Background: Cephalanthera nanchuanica is a terrestrial orchid species and has been red listed as a second-grade protected plant due to its limited distributions in China. Initially classified within a monotypic genus Tangtsinia, this species was later reassigned to Cephalanthera based on morphological and molecular data. However, previous phylogenetic analyses of Cephalanthera using several segment sequences exhibited a low discriminatory power in delineating its relationships., Results: In this study, we characterized and comparatively analyzed the complete chloroplast (cp) genome of C. nanchuanica with those of six previously reported Cephalanthera species. Our findings revealed that the cp genome of C. nanchuanica had the typical quadripartite structure, with a size of 161,365 bp and a GC content of 37.27%. A total of 113 unique genes were annotated, among which nearly half of protein-encoding genes (RSCU > 1) showed a preference in codon usage. No structural rearrangements were observed among the cp genomes of Cephalanthera species, except for C. humilis, which displayed structural alterations due to gene loss, relocation, and inverted repeat (IR) expansion/contraction. The cp genomes of Cephalanthera species were highly conserved, with only a small number of SSRs detected, most of which preferred A/T bases. Comparative analysis of cp genomes indicated that IR and coding regions were less divergent than single copy and non-coding regions and eight mutational hotspots were identified. Phylogenetic analysis suggested that the tribe Neottieae was a monophyletic group, divided into five clades. Palmorchis was the earliest-diverging lineage, followed by Cephalanthera. Diplandrorchis was deeply nested within Neottia, forming a clade. Aphyllorchis and Limodorum formed another clade, sister to Epipactis. Within the Cephalanthera clade, C. nanchuanica was sister to C. falcata with a strong support., Conclusions: This study demonstrated that the cp genome characters of C. nanchuanica are highly similar to those of other Cephalanthera species, except for the mycoheterotrophic species C. humilis. Although the cp genomes of Cephalanthera species (excluding C. humilis) exhibited conservation in genome structure and sequence, SSR repeats and mutational hotspots were identified, which could potentially serve as as molecular markers for distinguishing Cephalanthera species. The phylogenetic analysis based on the protein-coding genes provided high-resolution support for the infrageneric classification. Therefore, cp genome data will be instrumental in resolving the phylogeny of the genus Cephalanthera., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Phanera Lour., a genus in the subfamily Cercidoideae of the family Leguminosae, is characterized by woody liana habit, tendrils, and distinctive bilobate or bifoliolate leaves. The genus holds important medicinal value and constitutes a complex group characterized by morphological diversity and unstable taxonomic boundaries. However, limited information on the chloroplast genomes of this genus currently available constrains our understanding of its species diversity. Hence, it is necessary to obtain more chloroplast genome information to uncover the genetic characteristics of this genus., Methods: We collected and assembled the complete chloroplast genomes of nine representative Phanera plants, including Phanera erythropoda , Phanera vahlii , Phanera aureifolia , Phanera bidentata , Phanera japonica , Phanera saigonensis , Phanera championii , Phanera yunnanensis , and Phanera apertilobata . We then conducted a comparative analysis of these genomes and constructed phylogenetic trees., Results: These species are each characterized by a typical quadripartite structure. A total of 130-135 genes were annotated, and the GC content ranged from 39.25-42.58%. Codon usage analysis indicated that codons encoding alanine were dominant. We found 82-126 simple sequence repeats, along with 5448 dispersed repeats, mostly in the form of forward repeats. Phylogenetic analysis revealed that 16 Phanera species form a well-supported monophyletic group, suggesting a possible monophyletic genus. Furthermore, 10 hypervariable regions were detected for identification and evolutionary studies., Conclusions: We focused on comparing chloroplast genome characteristics among nine Phanera species and conducted phylogenetic analyses, laying the foundation for further phylogenetic research and species identification of Phanera .

Background: Capsicum (Solanaceae) is a globally important vegetable crop and is also used therapeutically in traditional medicine systems. However, little is known of the genetic variation within the commonly grown cultivars, the evolutionary relationships and differences in the chloroplast (cp.) genomes between Capsicum species remain unclear., Results: The cp. genomes of 32 Capsicum varieties in three species from 6 countries were investigated. The cp. genome of Capsicum was found to be ~ 156 kb in length and to contain 113 unique genes, of which 79 encoded proteins, 30 encoded transfer tRNAs, and 4 were for ribosomal RNAs. The 32 varieties that we chose for study represented 13 genotypes, containing a total of 608 indels, 83 SNPs, 47 SSRs and 281-306 repeat sequences. We then included several previously sequenced Capsicum cp. genomes, and found that the nine investigated species showed a number of differences in the characteristics of the four IR boundaries, and it was the non-coding regions that contained the most variable regions. We conducted a phylogenetic reconstruction using the cp. genomes of 43 representative species of Solanaceae, and the resulting phylogeny generally reflected the currently accepted classification, with the species of the pungent group having close relationship with one another., Conclusions: This study provides a comprehensive analysis of Capsicum chloroplast genomes, revealing significant variations in IR boundaries and other genomic features. These findings enhance our understanding of Capsicum evolution and genetic diversity., (© 2024. The Author(s).)

Background: Habenaria, a genus in the family Orchidaceae, are the nearly cosmopolitan orchids, and most species have significant medicinal and ornamental values. Despite the morphological and molecular data that have been studied in recent years, the phylogenetic relationship is still unclear., Results: We sequenced, assembled, and annotated the chloroplast (cp) genomes of two species (Habenaria aitchisonii Rchb.f. and Habenaria tibetica Schltr.ex Limpricht) of Habenaria grown on the Qinghai-Tibetan Plateau (QTP), and compared them with seven previously published cp genomes which may aid in the genomic profiling of these species. The two genomes ranged from 155,259-155,269 bp in length and both included 132 genes, encoding 86 proteins, 38 tRNAs and 8 rRNAs. In the cp genomes, the tandem repeats (797), SSRs (2195) and diverse loci (3214) were identified. Comparative analyses of codon usage, amino frequency, microsatellite, oligo repeats and transition and transversion substitutions revealed similarities between the species. Moreover, we identified 16 highly polymorphic regions with a nucleotide diversity above 0.02, which may be suitable for robust authentic barcoding and inferring in the phylogeny of Habenaria species. Among the polymorphic regions, positive selection was significantly exerted on several genes, such as cemA, petA, and ycf1. This finding may suggest an important adaptation strategy for the two Habenaria species on the QTP. The phylogenetic relationship revealed that H. aitchisonii and H. tibetica were more closely related to each other than to the other species, and the other seven species were clustered in three groups. In addition, the estimated divergence time suggested that the two species separated from the others approximately 0.39 Mya in the Neogene period. Our findings also suggest that Habenaria can be divided into different sections., Conclusions: The results of this study enriched the genomics resources of Habenaria, and SSR marker may aid in the conservation management of two endangered species., (© 2024. The Author(s).)

Targeting heterologous multi-transmembrane domain (TMD) proteins to plant chloroplasts requires sequences in addition to the chloroplast transit peptide (cTP). The N-terminal domain (N-region), located C-terminal to the cTP in chloroplast inner envelope membrane proteins, is an essential region for import. However, it was unclear if the N-region functions solely as a spacer sequence to facilitate cTP access or if it plays an active role in the import process. This study addresses the N-region's role by using combinations of cTPs and N-regions from Arabidopsis chloroplast inner envelope membrane proteins to direct the cyanobacterial protein SbtA to the chloroplast. We find that the sequence context of the N-region affects the chloroplast import efficiency of SbtA, with particular sequences mis-targeting the protein to different cellular sub-compartments. Additionally, specific cTP and N-region pairs exhibit varying targeting efficiencies for different heterologous proteins. Substituting individual N-region motifs did not significantly alter the chloroplast targeting efficiency of a particular cTP and N-region pair. We conclude that the N-region exhibits contextual functioning and potentially functional redundancy in motifs., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Although the chloroplast genome (cpDNA) of higher plants is known to exist as a large protein-DNA complex called 'plastid nucleoid', researches on its DNA state and regulatory elements are limited. In this study, we performed the assay for transposase-accessible chromatin sequencing (ATAC-seq) on five common tissues across five grasses, and found that the accessibility of different regions in cpDNA varied widely, with the transcribed regions being highly accessible and accessibility patterns around gene start and end sites varying depending on the level of gene expression. Further analysis identified a total of 3970 putative protein binding footprints on cpDNAs of five grasses. These footprints were enriched in intergenic regions and co-localized with known functional elements. Footprints and their flanking accessibility varied dynamically among tissues. Cross-species analysis showed that footprints in coding regions tended to overlap non-degenerate sites and contain a high proportion of highly conserved sites, indicating that they are subject to evolutionary constraints. Taken together, our results suggest that the accessibility of cpDNA has biological implications and provide new insights into the transcriptional regulation of chloroplasts., (© 2024. The Author(s).)

Background: Caryophyllaceae contains 100 genera and 3000 species, many of which are valuable both ecologically and economically. However, as past research has shown, the fundamental phylogenetic relationships of Caryophyllaceae are still debatable, and molecular dating based on chloroplast genomes has not been thoroughly examined for the entire family., Methods: In this study, the complete chloroplast genome sequences of Arenaria kansuensis Maxim., A. roborowskii Maxim., A. przewalskii Maxim., and Silene aprica Turcz were described. Additionally, the four newly generated along with eighteen published chloroplast genomes of Caryophyllaceae were included for comparative genomics analysis., Results: These 22 chloroplast genomes had typical quadripartite structure, with 128-134 distinct genes and lengths ranging from 133,621 bp to 153,957 bp. The 22 Caryophyllaceae chloroplast genomes showed significant variations in the number of long repeats and SSR types; mononucleotide repeats (A/T) and palindromic repeats were the most common types. Three substantially divergent areas containing atpB-rbcL, rbcL-accD, and accD were found by further comparative study, which could serve as effective molecular markers. The codon bias of chloroplast genomes in Caryophyllaceae were mainly affected by natural selection, but other factors such as mutation pressure could also affect the codon bias to some extent. Fourteen optimal codons were identified in the chloroplast genome of Caryophyllaceae. Phylogenetic analysis demonstrated that the monophyly of any of the three recognized subfamilies within Caryophyllaceae was not supported by our data. Meanwhile, seven well-supported clades correspond to 8 tribes were found in phylogenetic trees. The results of molecular dating demonstrated that the divergence between Caryophyllaceae and Amaranthaceae was estimated to occur in 69 Ma. The Paronychieae was the oldest tribe of the eight tribes included in this study, diverged at 59.92 Ma., Conclusion: This study provides resources for further investigations on the identification, genetic engineering, evolution, and phylogeny of Caryophyllaceae species., Competing Interests: Declarations. Ethics approval and consent to participate: This study including the collection of plant samples complies with relevant institutional, national, and international guidelines and legislation. All the necessary permissions have been granted for this research. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Vernicia montana and V. fordii are economically important woody oil species in the Euphorbiaceae that have great industrial oil and ornamental greening properties, however, the wild resources of Vernicia trees have been reduced because of their habitat destruction. Considering the diverse economic and ecological importance of Vernicia species, it is important to collect more molecular data to determine the genetic differences between V. montana and V. fordii., Results: We sequenced, assembled, and annotated the complete chloroplast (CP) genome of two tung trees based on the genome skimming approach. The whole CP genomes of V. montana and V. fordii were 163,518 bp and 161,495 bp in length, both including a pair of inverted repeats separated by a large single-copy and a small single-copy region. We detected a total number of 311 tandem repeats, 100 dispersed repeats, and 255 simple repeats from V. montana and V. fordii CP genomes. The mean value of nucleotide diversity between the two species was 0.0122, and the average Ka/Ks ratio across all coding genes was 0.3483. Comparative chloroplast genome analysis showed that the coding regions were more conserved than the non-coding regions. The phylogenetic relationships yielded by the complete genome sequences showed that V. montana was closely related to V. fordii and is considered as a sister group., Conclusions: We sequenced, assembled, annotated, and analyzed the CP genome of two tung trees, which will be useful in investigating the conservation genetics and potential breeding applications of this oil shrub., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. The plant was collected in non protected area; no any legal authorization/license is required. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)