Sequence characteristics, genetic diversity and phylogenetic analysis of the Cucurbita ficifolia (Cucurbitaceae) chloroplasts genome.

BACKGROUND: Curcubita ficifolia Bouché (Cucurbitaceae) has high value as a food crop and medicinal plant, and also has horticultural value as rootstock for other melon species. China is home to many different cultivars, but the genetic diversity of these resources and the evolutionary relationships among them, as well as the differences between C. ficifolia and other Cucurbita species, remain unclear. RESULTS: We investigated the chloroplast (cp) genomes of 160 C. ficifolia individuals from 31 populations in Yunnan, a major C. ficifolia production area in China. We found that the cp genome of C. ficifolia is ~151 kb and contains 128 genes, of which 86 are protein coding genes, 34 encode tRNA, and eight encode rRNAs. We also identified 64 SSRs, mainly AT repeats. The cp genome was found to contain a total of 204 SNP and 57 indels, and a total of 21 haplotypes were found in the 160 study individuals. The reverse repeat (IR) region of C. ficifolia contained a few differences compared with this region in the six other Cucurbita species. Sequence difference analysis demonstrated that most of the variable regions were concentrated in the single copy (SC) region. Moreover, the sequences of the coding regions were found to be more similar among species than those of the non-coding regions. The phylogenies reconstructed from the cp genomes of 61 representative species of Cucurbitaceae reflected the currently accepted classification, in which C. ficifolia is sister to the other Cucurbita species, however, different interspecific relationships were found between Cucurbita species. CONCLUSIONS: These results will be valuable in the classification of C. ficifolia genetic resources and will contribute to our understanding of evolutionary relationships within the genus Cucurbita.

The complete chloroplast genome of Mussaenda pubescens and phylogenetic analysis.

The chloroplast (cp) genome sequence of Mussaenda pubescens, a promising resource that is used as a traditional medicine and drink, is important for understanding the phylogenetic relationships among the Mussaenda family and genetic improvement and reservation. This research represented the first comprehensive description of the morphological characteristics of M. pubescens, as well as an analysis of the complete cp genome and phylogenetic relationship. The results indicated a close relationship between M. pubescens and M. hirsutula based on the morphological characteristics of the flower and leaves. The cp was sequenced using the Illumina NovaSeq 6000 platform. The results indicated the cp genome of M. pubescens spanned a total length of 155,122 bp, including a pair of inverted repeats (IRA and IRB) with a length of 25,871 bp for each region, as well as a large single-copy (LSC) region and a small single-copy (SSC) region with lengths of 85,370 bp and 18,010 bp, respectively. The results of phylogenetic analyses demonstrated that species within the same genus displayed a tendency to group closely together. It was suggested that Antirhea, Cinchona, Mitragyna, Neolamarckia, and Uncaria might have experienced an early divergence. Furthermore, M. hirsutula showed a close genetic connection to M. pubescens, with the two species having partially overlapping distributions in China. This study presents crucial findings regarding the identification, evolution, and phylogenetic research on Mussaenda plants, specifically targeting M. pubescens.

Extraction and analysis of high-quality chloroplast DNA with reduced nuclear DNA for medicinal plants.

BACKGROUND: Obtaining high-quality chloroplast genome sequences requires chloroplast DNA (cpDNA) samples that meet the sequencing requirements. The quality of extracted cpDNA directly impacts the efficiency and accuracy of sequencing analysis. Currently, there are no reported methods for extracting cpDNA from Erigeron breviscapus. Therefore, we developed a suitable method for extracting cpDNA from E. breviscapus and further verified its applicability to other medicinal plants. RESULTS: We conducted a comparative analysis of chloroplast isolation and cpDNA extraction using modified high-salt low-pH method, the high-salt method, and the NaOH low-salt method, respectively. Subsequently, the number of cpDNA copies relative to the nuclear DNA (nDNA ) was quantified via qPCR. As anticipated, chloroplasts isolated from E. breviscapus using the modified high-salt low-pH method exhibited intact structures with minimal cell debris. Moreover, the concentration, purity, and quality of E. breviscapus cpDNA extracted through this method surpassed those obtained from the other two methods. Furthermore, qPCR analysis confirmed that the modified high-salt low-pH method effectively minimized nDNA contamination in the extracted cpDNA. We then applied the developed modified high-salt low-pH method to other medicinal plant species, including Mentha haplocalyx, Taraxacum mongolicum, and Portulaca oleracea. The resultant effect on chloroplast isolation and cpDNA extraction further validated the generalizability and efficacy of this method across different plant species. CONCLUSIONS: The modified high-salt low-pH method represents a reliable approach for obtaining high-quality cpDNA from E. breviscapus. Its universal applicability establishes a solid foundation for chloroplast genome sequencing and analysis of this species. Moreover, it serves as a benchmark for developing similar methods to extract chloroplast genomes from other medicinal plants.

The complete Chloroplast genome of Stachys geobombycis and comparative analysis with related Stachys species.

Herb genomics, at the forefront of traditional Chinese medicine research, combines genomics with traditional practices, facilitating the scientific validation of ancient remedies. This integration enhances public understanding of traditional Chinese medicine's efficacy and broadens its scope in modern healthcare. Stachys species encompass annual or perennial herbs or small shrubs, exhibiting simple petiolate or sessile leaves. Despite their wide-ranging applications across various fields, molecular data have been lacking, hindering the precise identification and taxonomic elucidation of Stachys species. To address this gap, we assembled the complete chloroplast (CP) genome of Stachys geobombycis and conducted reannotation and comparative analysis of seven additional species within the Stachys genus. The findings demonstrate that the CP genomes of these species exhibit quadripartite structures, with lengths ranging from 14,523 to 150,599 bp. Overall, the genome structure remains relatively conserved, hosting 131 annotated genes, including 87 protein coding genes, 36 tRNA genes, and 8 rRNA genes. Additionally, 78 to 98 SSRs and long repeat sequences were detected , and notably, 6 highly variable regions were identified as potential molecular markers in the CP genome through sequence alignment. Phylogenetic analysis based on Bayesian inference and maximum likelihood methods strongly supported the phylogenetic position of the genus Stachys as a member of Stachydeae tribe. Overall, this comprehensive bioinformatics study of Stachys CP genomes lays the groundwork for phylogenetic classification, plant identification, genetic engineering, evolutionary studies, and breeding research concerning medicinal plants within the Stachys genus.

Plastid genome data provide new insights into the dynamic evolution of the tribe Ampelopsideae (Vitaceae).

BACKGROUND: Ampelopsideae J. Wen & Z.L. Nie is a small-sized tribe of Vitaceae Juss., including ca. 47 species from four genera showing a disjunct distribution worldwide across all the continents except Antarctica. There are numerous species from the tribe that are commonly used as medicinal plants with immune-modulating, antimicrobial, and anti-hypertensive properties. The tribe is usually recognized into three clades, i.e., Ampelopsis Michx., Nekemias Raf., and the Southern Hemisphere clade. However, the relationships of the three clades differ greatly between the nuclear and the plastid topologies. There has been limited exploration of the chloroplast phylogenetic relationships within Ampelopsideae, and studies on the chloroplast genome structure of this tribe are only available for a few individuals. In this study, we aimed to investigate the evolutionary characteristics of plastid genomes of the tribe, including their genome structure and evolutionary insights. RESULTS: We sequenced, assembled, and annotated plastid genomes of 36 species from the tribe and related taxa in the family. Three main clades were recognized within Ampelopsideae, corresponding to Ampelopsis, Nekemias, and the Southern Hemisphere lineage, respectively, and all with 100% bootstrap supports. The genome sequences and content of the tribe are highly conserved. However, comparative analyses suggested that the plastomes of Nekemias demonstrate a contraction in the large single copy region and an expansion in the inverted repeat region, and possess a high number of forward and palindromic repeat sequences distinct from both Ampelopsis and the Southern Hemisphere taxa. CONCLUSIONS: Our results highlighted plastome variations in genome length, expansion or contraction of the inverted repeat region, codon usage bias, and repeat sequences, are corresponding to the three lineages of the tribe, which probably faced with different environmental selection pressures and evolutionary history. This study provides valuable insights into understanding the evolutionary patterns of plastid genomes within the Ampelopsideae of Vitaceae.

Comparative analysis of medicinal plants Scutellaria baicalensis and common adulterants based on chloroplast genome sequencing.

BACKGROUND: Scutellaria baicalensis Georgi has been extensively used as a medicinal herb in China for over 2000 years. They may be intentionally or inadvertently substituted or blended with comparable species in the local market, threatening clinical medication safety. Molecular markers are effective tools to prevent misidentification and eliminate doping and falsification among Scutellaria plants. This study screened four highly variable regions to identify Scutellaria and its adulterants. In addition, a phylogenetic analysis was performed using the complete cp genome combined with published Scutellaria species samples. Moreover, a comparative analysis of the cp genomes was conducted to investigate the cp genome evolution of S. baicalensis. RESULTS: The complete cp genome of five species of Scutellaria was sequenced for the first time, and four previously published Scutellaria species were re-sequenced. They all exhibited a conserved quadripartite structure in their cp genomes, including two distinct regions, namely a small and large single copy region, respectively, and two inverted repeats encompassing the majority of ribosomal RNA genes. Furthermore, the nine species exhibited high conservation from aspects of the genome structure, codon usage, repeat sequences, and gene content. Four highly variable regions (matK-rps16, ndhC-trnV-UAC, psbE-petL, and rps16-trnQ-UUG) may function as potential molecular markers for differentiating S. baicalensis from its adulterants. Additionally, the monophyly of Scutellaria was ascertained and could be reclassified into two subgenera, subgenus Anaspis and subgenus Scutellaria, as evidenced by the phylogenetic analyses on sequences of cp genome and shared protein-coding sequences. According to the molecular clock analysis, it has been inferred that the divergence of Scutellaria occurred at approximately 4.0 Mya during the Pliocene Epoch. CONCLUSION: Our study provides an invaluable theoretical basis for further Scutellaria species identification, phylogenetics, and evolution analysis.

The complete chloroplast genome of the halophyte flowering plant Suaeda monoica from Jeddah, Saudi Arabia.

The complete chloroplast genome (plastome) of the annual flowering halophyte herb Suaeda monoica Forssk. ex J. F. Gmel. family (Amaranthaceae) that grows in Jeddah, Saudi Arabia, was identified for the first time in this study. Suaeda monoica is a medicinal plant species whose taxonomic classification remains controversial. Further, studying the species is useful for current conservation and management efforts. In the current study, the full chloroplast genome S. monoica was reassembled using whole-genome next-generation sequencing and compared with the previously published chloroplast genomes of Suaeda species. The chloroplast genome size of Suaeda monoica was 151,789 bp, with a single large copy of 83,404 bp, a small single copy of 18,007 bp and two inverted repeats regions of 25,189 bp. GC content in the whole genome was 36.4%. The cp genome included 87 genes that coded for proteins, 37 genes coding for tRNA, 8 genes coding for rRNA and one non-coding pseudogene. Five chloroplast genome features were compared between S. monoica and S. japonica, S. glauca, S. salsa, S. malacosperma and S. physophora. Among Suaeda genus and equal to most angiosperms chloroplast genomes, the RSCU values were conservative. Two pseudogenes (accD and ycf1), rpl16 intron and ndhF-rpl32 intergenic spacer, were highlighted as suitable DNA barcodes for different Suaeda species. Phylogenetic analyses show Suaeda cluster into three main groups; one in which S. monoica was closer to S. salsa. The obtained result provided valuable information on the characteristics of the S. monoica chloroplast genome and the phylogenetic relationships.

Decoding the complete organelle genomic architecture of Stewartia gemmata: an early-diverging species in Theaceae.

BACKGROUND: Theaceae, comprising 300 + species, holds significance in biodiversity, economics, and culture, notably including the globally consumed tea plant. Stewartia gemmata, a species of the earliest diverging tribe Stewartieae, is critical to offer insights into Theaceae's origin and evolutionary history. RESULT: We sequenced the complete organelle genomes of Stewartia gemmata using short/long reads sequencing technologies. The chloroplast genome (158,406 bp) exhibited a quadripartite structure including the large single-copy region (LSC), a small single-copy region (SSC), and a pair of inverted repeat regions (IRs); 114 genes encoded 80 proteins, 30 tRNAs, and four rRNAs. The mitochondrial genome (681,203 bp) exhibited alternative conformations alongside a monocyclic structure: 61 genes encoding 38 proteins, 20 tRNAs, three rRNAs, and RNA editing-impacting genes, including ATP6, RPL16, COX2, NAD4L, NAD5, NAD7, and RPS1. Comparative analyses revealed frequent recombination events and apparent rRNA gene gains and losses in the mitochondrial genome of Theaceae. In organelle genomes, the protein-coding genes exhibited a strong A/U bias at codon endings; ENC-GC3 analysis implies selection-driven codon bias. Transposable elements might facilitate interorganelle sequence transfer. Phylogenetic analysis confirmed Stewartieae's early divergence within Theaceae, shedding light on organelle genome characteristics and evolution in Theaceae. CONCLUSIONS: We studied the detailed characterization of organelle genomes, including genome structure, composition, and repeated sequences, along with the identification of lateral gene transfer (LGT) events and complexities. The discovery of a large number of repetitive sequences and simple sequence repeats (SSRs) has led to new insights into molecular phylogenetic markers. Decoding the Stewartia gemmata organellar genome provides valuable genomic resources for further studies in tea plant phylogenomics and evolutionary biology.

Complete chloroplast genomes of eight Artemisia species: Comparative analysis, molecular identification, and phylogenetic analysis.

Artemisia L. is the largest genus in the Asteraceae, and well known for its high medicinal value. The morphological features of Artemisia species are similar, making taxonomic identification and evolutionary research difficult. We sequenced chloroplast genomes of eight Artemisia species, all of which are common adulterants of A. argyi. We used novel genetic data and compared these data to the published A. argyi chloroplast genome in to develop molecular markers for species identification and reconstructing phylogenetic relationships between Artemisia species. The eight chloroplast sequences were highly similar in gene order, content, and structure, encoding a total of 114 genes (82 protein-coding genes, 28 tRNAs, and four rRNAs). All species harboured similar repeat sequences and simple sequence repeats (SSRs), ranging from 47 to 49 and 38 to 40 repeats, respectively. In addition, we identified five hypervariable regions (rpl32-trnL, rps16-trnQ, petN-psbM, trnE-rpoB, and atpA-trnR) and ten variable coding genes (ycf1, psbG, rpl36, psaC, psaI, accD, psbT, ndhD, ndhE, and psbH), which can be used to develop chloroplast molecular markers. Finally, phylogenetic reconstructions based on six datasets produced similar topologies, revealing A. argyi is closely related to species often found as adulterants, as expected. Our research provides valuable new information on the evolution and phylogenetic relationships between Artemisia chloroplast genomes and identifies valuable molecular makers to distinguish it from closely related species.

Comparative complete chloroplast genome of Geum japonicum: evolution and phylogenetic analysis.

Geum japonicum (Rosaceae) has been widely used in China as a traditional herbal medicine due to its high economic and medicinal value. However, the appearance of Geum species is relatively similar, making identification difficult by conventional phenotypic methods, and the studies of genomics and species evolution are lacking. To better distinguish the medicinal varieties and fill this gap, we carried out relevant research on the chloroplast genome of G. japonicum. Results show a typical quadripartite structure of the chloroplast genome of G. japonicum with a length of 156,042 bp. There are totally 131 unique genes in the genome, including 87 protein-coding genes, 36 tRNA genes, and 8 rRNA genes, and there were also 87 SSRs identified and mostly mononucleotide Adenine-Thymine. We next compared the plastid genomes among four Geum species and obtained 14 hypervariable regions, including ndhF, psbE, trnG-UCC, ccsA, trnQ-UUG, rps16, psbK, trnL-UAA, ycf1, ndhD, atpA, petN, rps14, and trnK-UUU. Phylogenetic analysis revealed that G. japonicum is most closely related to Geum aleppicum, and possibly has some evolutionary relatedness with an ancient relic plant Taihangia rupestris. This research enriched the genome resources and provided fundamental insights for evolutionary studies and the phylogeny of Geum.

Chloroplast genome of Justicia procumbens: genomic features, comparative analysis, and phylogenetic relationships among Justicieae species.

Justicia procumbens L. is a traditional medicinal plant that is widely distributed in China. However, little is known about the genetic diversity and evolution of this genus, and no genomic studies have been carried out on J. procumbens previously. In this study, we aimed to assemble and annotate the first complete chloroplast genome (cpDNA) of J. procumbens and compare it with all previously published cpDNAs within the tribe Justicieae. Genome structure and comparative and phylogenetic analyses were performed. The 150,454 bp-long J. procumbens cpDNA has a circular and quadripartite structure consisting of a large single copy, a small single copy, and two inverted repeat regions. It contains 133 genes, of which 88 are protein-coding genes, 37 are tRNA genes, and eight are rRNA genes. Twenty-four simple sequence repeats (SSRs) and 81 repeat sequences were identified. Comparative analyses with other Justicieae species revealed that the non-coding regions of J. procumbens cpDNA showed greater variation than did the coding regions. Moreover, phylogenetic analysis based on 14 cpDNA sequences from Justicieae species showed that J. procumbens and J. flava were most closely related. This study provides valuable genetic information to support further research on the genetic diversity and evolutionary development of the tribe Justicieae.

The complete chloroplast genome sequence of Nepeta bracteata and comparison with congeneric species.

Nepeta bracteata (N. bracteata) is an important medicinal plant used by Chinese ethnic minorities. However, the lack of knowledge regarding the chloroplast genome of N. bracteata has imposed current limitations on our study. Here, we used Next-generation sequencing to obtain the chloroplast genome of N. bracteata. The findings suggested that the 151,588 bp cp genome of N. bracteata comprises 130 genes, including 35 tRNA genes and 87 protein-coding genes. And its chloroplast genome exhibits a typical quadripartite structure, the largest single copy (LSC; 82,819 bp) and the smallest single copy (SSC; 17,557 bp) separate a pair of inverted repeats IR regions (IRa and IRb; 25,606 bp) from one another. Interestingly, palindromic repeats are more common, as shown by the examination of repetition. In the interim, 18 SSRs were discovered in the interim, the bulk of which were Adenine-Thymine (A-T) mononucleotides. Meanwhile, we compared it with five other species from the Nepeta genus. Five hypervariable areas were found by the study, including ndhH-rps15, accD-psal, ndhG-ndhl, trnH-GUG-psbA, and rpoC1-rpoB. Furthermore, the phylogenetic study revealed that N. bracteata and Nepeta stewartiana (N. stewartiana) were linked to each other most closely. In summary, our findings enrich the resources available for chloroplast genomes in the Nepeta genus. Moreover, these hypervariable regions have the potential to be developed into molecular markers, enabling the rapid identification of species within the Nepeta genus. Comparative analysis of species within the Nepeta genus can help enhance our study of their phylogenetic relationships, potential medicinal properties and bioprospecting.

Comparatively analyzing of chloroplast genome and new insights into phylogenetic relationships regarding the genus Stephania.

The medicinal plant of the genus Stephania holds significant economic importance in the pharmaceutical industry. However, accurately classifying and subdividing this genus remains a challenge. Herein, the chloroplast (cp) genomes of Stephania and Cyclea were sequenced, and the primary characteristics, repeat sequences, inverted repeats regions, simple sequence repeats, and codon usage bias of 17 species were comparatively analyzed. Twelve markers were identified through genome alignment and sliding window analysis. Moreover, a molecular clock analysis revealed the divergence between subgenus (subg.) Botryodiscia and the combined Cyclea, subg. Stephania and Tuberiphania during the early Oligocene epoch. Notably, the raceme-type inflorescence represents the ancestral state of the Stephania and Cyclea. The genetic relationships inferred from the cp genome and protein-coding genes exhibited similar topologies. Additionally, the paraphyletic relationship between the genera Cyclea and Stephania was confirmed. Bayesian inference, maximum likelihood, and neighbor-joining trees consistently showed that section Tuberiphania and Transcostula were non-monophyletic. In conclusion, this research provides valuable insights for further investigations into species identification, evolution, and phylogenetics within the Stephania genus.

Insight into chloroplast genome structural variation of the Mongolian endemic species Adonis mongolica (Ranunculaceae) in the Adonideae tribe.

Adonis mongolica is a threatened species that is endemic to Mongolia. It is a medicinal plant from the Adonis genus and has been used to treat heart diseases. However, the genomics and evolution of this species have not been thoroughly studied. We sequenced the first complete plastome of A. mongolica and compared it with ten Adonideae species to describe the plastome structure and infer phylogenetic relationships. The complete plastome of A. mongolica was 157,521 bp long and had a typical quadripartite structure with numerous divergent regions. The plastomes of Adonideae had relatively constant genome structures and sizes, except for those of Adonis. The plastome structure was consistent across Adonis. We identified a 44.8 kb large-scale inversion within the large single-copy region and rpl32 gene loss in the Adonis plastomes compared to other members of the Adonideae tribe. Additionally, Adonis had a smaller plastome size (156,917-157,603 bp) than the other genera within the tribe (159,666-160,940 bp), which was attributed to deletions of intergenic regions and partial and complete gene losses. These results suggested that an intramolecular mutation occurred in the ancestor of the Adonis genus. Based on the phylogenetic results, Adonis separated earlier than the other genera within the Adonideae tribe. The genome structures and divergences of specific regions in the Adonis genus were unique to the Adonideae tribe. This study provides fundamental knowledge for further genomic research in Mongolia and a better understanding of the evolutionary history of endemic plants.

New Insights into Phylogenetic Relationship of Hydrocotyle (Araliaceae) Based on Plastid Genomes.

Hydrocotyle, belonging to the Hydrocotyloideae of Araliaceae, consists of 95 perennial and 35 annual species. Due to the lack of stable diagnostic morphological characteristics and high-resolution molecular markers, the phylogenetic relationships of Hydrocotyle need to be further investigated. In this study, we newly sequenced and assembled 13 whole plastid genomes of Hydrocotyle and performed comparative plastid genomic analyses with four previously published Hydrocotyle plastomes and phylogenomic analyses within Araliaceae. The plastid genomes of Hydrocotyle exhibited typical quadripartite structures with lengths from 152,659 bp to 153,669 bp, comprising a large single-copy (LSC) region (83,958-84,792 bp), a small single-copy (SSC) region (18,585-18,768 bp), and a pair of inverted repeats (IRs) (25,058-25,145 bp). Each plastome encoded 113 unique genes, containing 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Comparative analyses showed that the IR boundaries of Hydrocotyle plastomes were highly similar, and the coding and IR regions exhibited more conserved than non-coding and single-copy (SC) regions. A total of 2932 simple sequence repeats and 520 long sequence repeats were identified, with specificity in the number and distribution of repeat sequences. Six hypervariable regions were screened from the SC region, including four intergenic spacers (IGS) (ycf3-trnS, trnS-rps4, petA-psbJ, and ndhF-rpl32) and two coding genes (rpl16 and ycf1). Three protein-coding genes (atpE, rpl16, and ycf2) were subjected to positive selection only in a few species, implying that most protein-coding genes were relatively conserved during the plastid evolutionary process. Plastid phylogenomic analyses supported the treatment of Hydrocotyle from Apiaceae to Araliaceae, and topologies with a high resolution indicated that plastome data can be further used in the comprehensive phylogenetic research of Hydrocotyle. The diagnostic characteristics currently used in Hydrocotyle may not accurately reflect the phylogenetic relationships of this genus, and new taxonomic characteristics may need to be evaluated and selected in combination with more comprehensive molecular phylogenetic results.

[Characterization and phylogenetic analysis of complete chloroplast genome of cultivated Qinan agarwood].

"Tangjie" leaves of cultivated Qinan agarwood were used to obtain the complete chloroplast genome using high-throughput sequencing technology. Combined with 12 chloroplast genomes of Aquilaria species downloaded from NCBI, bioinformatics method was employed to determine the chloroplast genome characteristics and phylogenetic relationships. The results showed that the chloroplast genome sequence length of cultivated Qinan agarwood "Tangjie" leaves was 174 909 bp with a GC content of 36.7%. A total of 136 genes were annotated, including 90 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Sequence repeat analysis detected 80 simple sequence repeats(SSRs) and 124 long sequence repeats, with most SSRs composed of A and T bases. Codon preference analysis revealed that AUU was the most frequently used codon, and codons with A and U endings were preferred. Comparative analysis of Aquilaria chloroplast genomes showed relative conservation of the IR region boundaries and identified five highly variable regions: trnD-trnY, trnT-trnL, trnF-ndhJ, petA-cemA, and rpl32, which could serve as potential DNA barcodes specific to the Aquilaria genus. Selection pressure analysis indicated positive selection in the rbcL, rps11, and rpl32 genes. Phylogenetic analysis revealed that cultivated Qinan agarwood "Tangjie" and Aquilaria agallocha clustered together(100% support), supporting the Chinese origin of Qinan agarwood from Aquilaria agallocha. The chloroplast genome data obtained in this study provide a foundation for studying the genetic diversity of cultivated Qinan agarwood and molecular identification of the Aquilaria genus.

Complete Chloroplast Genome of Hypericum perforatum and Dynamic Evolution in Hypericum (Hypericaceae).

Hypericum perforatum (St. John's Wort) is a medicinal plant from the Hypericaceae family. Here, we sequenced the whole chloroplast genome of H. perforatum and compared the genome variation among five Hypericum species to discover dynamic changes and elucidate the mechanisms that lead to genome rearrangements in the Hypericum chloroplast genomes. The H. perforatum chloroplast genome is 139,725 bp, exhibiting a circular quadripartite structure with two copies of inverted repeats (IRs) separating a large single-copy region and a small single-copy region. The H. perforatum chloroplast genome encodes 106 unique genes, including 73 protein-coding genes, 29 tRNAs, and 4 rRNAs. Hypericum chloroplast genomes exhibit genome rearrangement and significant variations among species. The genome size variation among the five Hypericum species was remarkably associated with the expansion or contraction of IR regions and gene losses. Three genes-trnK-UUU, infA, and rps16-were lost, and three genes-rps7, rpl23, and rpl32-were pseudogenized in Hypericum. All the Hypericum chloroplast genomes lost the two introns in clpP, the intron in rps12, and the second intron in ycf3. Hypericum chloroplast genomes contain many long repeat sequences, suggesting a role in facilitating rearrangements. Most genes, according to molecular evolution assessments, are under purifying selection.

Complete plastid genome structure of 13 Asian Justicia (Acanthaceae) species: comparative genomics and phylogenetic analyses.

BACKGROUND: Justicia L. is the largest genus in Acanthaceae Juss. and widely distributed in tropical and subtropical regions of the world. Previous phylogenetic studies have proposed a general phylogenetic framework for Justicia based on several molecular markers. However, their studies were mainly focused on resolution of phylogenetic issues of Justicia in Africa, Australia and South America due to limited sampling from Asia. Additionally, although Justicia plants are of high medical and ornamental values, little research on its genetics was reported. Therefore, to improve the understanding of its genomic structure and relationships among Asian Justicia plants, we sequenced complete chloroplast (cp.) genomes of 12 Asian plants and combined with the previously published cp. genome of Justicia leptostachya Hemsl. for further comparative genomics and phylogenetic analyses. RESULTS: All the cp. genomes exhibit a typical quadripartite structure without genomic rearrangement and gene loss. Their sizes range from 148,374 to 151,739 bp, including a large single copy (LSC, 81,434-83,676 bp), a small single copy (SSC, 16,833-17,507 bp) and two inverted repeats (IR, 24,947-25,549 bp). GC contents range from 38.1 to 38.4%. All the plastomes contain 114 genes, including 80 protein-coding genes, 30 tRNAs and 4 rRNAs. IR variation and repetitive sequences analyses both indicated that Justicia grossa C. B. Clarke is different from other Justicia species because its lengths of ndhF and ycf1 in IRs are shorter than others and it is richest in SSRs and dispersed repeats. The ycf1 gene was identified as the candidate DNA barcode for the genus Justicia. Our phylogenetic results showed that Justicia is a polyphyletic group, which is consistent with previous studies. Among them, J. grossa belongs to subtribe Tetramerinae of tribe Justicieae while the other Justicia members belong to subtribe Justiciinae. Therefore, based on morphological and molecular evidence, J. grossa should be undoubtedly recognized as a new genus. Interestingly, the evolutionary history of Justicia was discovered to be congruent with the morphology evolution. CONCLUSION: Our study not only elucidates basic features of Justicia whole plastomes, but also sheds light on interspecific relationships of Asian Justicia plants for the first time.

Assembly and analysis of the complete mitochondrial genome of Forsythia suspensa (Thunb.) Vahl.

BACKGROUND: Forsythia suspensa (Thunb.) Vahl is a valuable ornamental and medicinal plant. Although the nuclear and chloroplast genomes of F. suspensa have been published, its complete mitochondrial genome sequence has yet to be reported. In this study, the genomic DNA of F. suspensa yellowish leaf material was extracted, sequenced by using a mixture of Illumina Novaseq6000 short reads and Oxford Nanopore PromethION long reads, and the sequencing data were assembled and annotated. RESULT: The F. suspensa mitochondrial genome was obtained in the length of 535,692 bp with a circular structure, and the GC content was 44.90%. The genome contains 60 genes, including 36 protein-coding genes, 21 tRNA genes, and three rRNA genes. We further analyzed RNA editing of the protein-coding genes, relative synonymous codon usage, and sequence repeats based on the genomic data. There were 25 homologous sequences between F. suspensa mitochondria and chloroplast genome, which involved the transfer of 8 mitochondrial genes, and 9473 homologous sequences between mitochondrial and nuclear genomes. Analysis of the nucleic acid substitution rate, nucleic acid diversity, and collinearity of protein-coding genes of the F. suspensa mitochondrial genome revealed that the majority of genes may have undergone purifying selection, exhibiting a slower rate of evolution and a relatively conserved structure. Analysis of the phylogenetic relationships among different species revealed that F. suspensa was most closely related to Olea europaea subsp. Europaea. CONCLUSION: In this study, we sequenced, assembled, and annotated a high-quality F. suspensa mitochondrial genome. The results of this study will enrich the mitochondrial genome data of Forsythia, lay a foundation for the phylogenetic development of Forsythia, and promote the evolutionary analysis of Oleaceae species.

Engineering of insecticidal hybrid gene into potato chloroplast genome exhibits promising control of Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The potato chloroplast was transformed with codon optimized synthetic hybrid cry gene (SN19) to mitigate crop losses by Colorado potato beetle (CPB). The bombarded explants (leaves and internode) were cultured on MS medium supplemented with BAP (2.0 mg/l), NAA (0.2 mg/l), TDZ (2.0 mg/l) and GA3 (0.1 mg/l); spectinomycin 50 mg/l was used as a selection agent in the medium. Leaf explants of cultivar Kuroda induced highest percentage (92%) of callus where cultivar Santae produced the highest percentage (85.7%) of transplastomic shoots. Sante and Challenger showed 9.6% shoot regeneration efficiency followed by cultivar Simply Red (8.8%). PCR amplification yielded 16 postive transplastomic plantlets out of 21 spectinomycin resistant ones. Target gene integration was confirmed by PCR and Southern blot, whereas RT-qPCR was used to assess the expression level of transgene. The localization of visual marker gene gfp was tracked by laser scanning confocal microscopy which confirmed its expression in chloroplasts of leaf cells. The transplastomic plants ensured high mortality to both larvae and adult CPB. Foliage consumption and weight gain of CPB fed on transplastomic leaves were lower compared to the control plants. Sucessful implementation of current research findings can lead to a viable solution to CPB mediated potato losses globally.