Genome editing in angiosperm chloroplasts: targeted DNA double-strand break and base editing.

Chloroplasts are organelles that are derived from a photosynthetic bacterium and have their own genome. Genome editing is a recently developing technology that allows for specific modifications of target sequences. The first successful application of genome editing in chloroplasts was reported in 2021, and since then, this research field has been expanding. Although the chloroplast genome of several dicot species can be stably modified by a conventional method, which involves inserting foreign DNAs into the chloroplast genome via homologous recombination, genome editing offers several advantages over this method. In this review, we introduce genome editing methods targeting the chloroplast genome and describe their advantages and limitations. So far, CRISPR/Cas systems are inapplicable for editing the chloroplast genome because guide RNAs, unlike proteins, cannot be efficiently delivered into chloroplasts. Therefore, protein-based enzymes are used to edit the chloroplast genome. These enzymes contain a chloroplast-transit peptide, the DNA-binding domain of transcription activator-like effector nuclease (TALEN), or a catalytic domain that induces DNA modifications. To date, genome editing methods can cause DNA double-strand break or introduce C:G-to-T:A and A:T-to-G:C base edits at or near the target sequence. These methods are expected to contribute to basic research on the chloroplast genome in many species and to be fundamental methods of plant breeding utilizing the chloroplast genome.

Phylogenomics analysis of Scutellaria (Lamiaceae) of the world.

BACKGROUND: Scutellaria, a sub-cosmopolitan genus, stands as one of the Lamiaceae family's largest genera, encompassing approximately 500 species found in both temperate and tropical montane regions. Recognized for its significant medicinal properties, this genus has garnered attention as a research focus, showcasing anti-cancer, anti-inflammatory, antioxidant, and hepatoprotective qualities. Additionally, it finds application in agriculture and horticulture. Comprehending Scutellaria's taxonomy is pivotal for its effective utilization and conservation. However, the current taxonomic frameworks, primarily based on morphological characteristics, are inadequate. Despite several phylogenetic studies, the species relationships and delimitations remain ambiguous, leaving the genus without a stable and reliable classification system. RESULTS: This study analyzed 234 complete chloroplast genomes, comprising 220 new and 14 previously published sequences across 206 species, subspecies, and varieties worldwide. Phylogenetic analysis was conducted using six data matrices through Maximum Likelihood and Bayesian Inference, resulting in a robustly supported phylogenetic framework for Scutellaria. We propose three subgenera, recommending the elevation of Section Anaspis to subgeneric rank and the merging of Sections Lupulinaria and Apeltanthus. The circumscription of Subgenus Apeltanthus and Section Perilomia needs to be reconsidered. Comparative analysis of chloroplast genomes highlighted the IR/SC boundary feature as a significant taxonomic indicator. We identified a total of 758 SSRs, 558 longer repetitive sequences, and ten highly variable regions, including trnK-rps16, trnC-petN, petN-psbM, accD-psaI, petA-psbJ, rpl32-trnL, ccsA-ndhD, rps15-ycf1, ndhF, and ycf1. These findings serve as valuable references for future research on species identification, phylogeny, and population genetics. CONCLUSIONS: The phylogeny of Scutellaria, based on the most comprehensive sample collection to date and complete chloroplast genome analysis, has significantly enhanced our understanding of its infrageneric relationships. The extensive examination of chloroplast genome characteristics establishes a solid foundation for the future development and utilization of Scutellaria, an important medicinal plant globally.

A comparative plastome approach enhances the assessment of genetic variation in the Melilotus genus.

BACKGROUND: Melilotus, a member of the Fabaceae family, is a pivotal forage crop that is extensively cultivated in livestock regions globally due to its notable productivity and ability to withstand abiotic stress. However, the genetic attributes of the chloroplast genome and the evolutionary connections among different Melilotus species remain unresolved. RESULTS: In this study, we compiled the chloroplast genomes of 18 Melilotus species and performed a comprehensive comparative analysis. Through the examination of protein-coding genes, we successfully established a robust phylogenetic tree for these species. This conclusion is further supported by the phylogeny derived from single-nucleotide polymorphisms (SNPs) across the entire chloroplast genome. Notably, our findings revealed that M. infestus, M. siculus, M. sulcatus, and M. speciosus formed a distinct subgroup within the phylogenetic tree. Additionally, the chloroplast genomes of these four species exhibit two shared inversions. Moreover, inverted repeats were observed to have reemerged in six species within the IRLC. The distribution patterns of single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within protein-coding genes indicated that ycf1 and ycf2 accumulated nonconservative alterations during evolutionary development. Furthermore, an examination of the evolutionary rate of protein-coding genes revealed that rps18, rps7, and rpl16 underwent positive selection specifically in Melilotus. CONCLUSIONS: We present a comparative analysis of the complete chloroplast genomes of Melilotus species. This study represents the most thorough and detailed exploration of the evolution and variability within the genus Melilotus to date. Our study provides valuable chloroplast genomic information for improving phylogenetic reconstructions and making biogeographic inferences about Melilotus and other Papilionoideae species.

Comparative analysis of complete chloroplast genomes of Synotis species (Asteraceae, Senecioneae) for identification and phylogenetic analysis.

BACKGROUND: The Synotis (C. B. Clarke) C. Jeffrey & Y. L. Chen is an ecologically important genus of the tribe Senecioneae, family Asteraceae. Because most species of the genus bear similar morphology, traditional morphological identification methods are very difficult to discriminate them. Therefore, it is essential to develop a reliable and effective identification method for Synotis species. In this study, the complete chloroplast (cp.) genomes of four Synotis species, S. cavaleriei (H.Lév.) C. Jeffrey & Y.L. Chen, S. duclouxii (Dunn) C. Jeffrey & Y.L. Chen, S. nagensium (C.B. Clarke) C. Jeffrey & Y.L. Chen and S. erythropappa (Bureau & Franch.) C. Jeffrey & Y. L. Chen had been sequenced using next-generation sequencing technology and reported here. RESULTS: These four cp. genomes exhibited a typical quadripartite structure and contained the large single-copy regions (LSC, 83,288 to 83,399 bp), the small single-copy regions (SSC, 18,262 to 18,287 bp), and the inverted repeat regions (IR, 24,837 to 24,842 bp). Each of the four cp. genomes encoded 134 genes, including 87 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 2 pseudogenes (ycf1 and rps19). The highly variable regions (trnC-GCA-petN, ccsA-psaC, trnE-UUC-rpoB, ycf1, ccsA and petN) may be used as potential molecular barcodes. The complete cp. genomes sequence of Synotis could be used as the potentially effective super-barcode to accurately identify Synotis species. Phylogenetic analysis demonstrated that the four Synotis species were clustered into a monophyletic group, and they were closed to the Senecio, Crassocephalum and Dendrosenecio in tribe Senecioneae. CONCLUSIONS: This study will be useful for further species identification, evolution, genetic diversity and phylogenetic studies within this genus Synotis and the tribe Senecioneae.

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.

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.

Solanum aculeatissimum and Solanum torvum chloroplast genome sequences: a comparative analysis with other Solanum chloroplast genomes.

BACKGROUND: Solanum aculeatissimum and Solanum torvum belong to the Solanum species, and they are essential plants known for their high resistance to diseases and adverse conditions. They are frequently used as rootstocks for grafting and are often crossbred with other Solanum species to leverage their resistance traits. However, the phylogenetic relationship between S. aculeatissimum and S. torvum within the Solanum genus remains unclear. Therefore, this paper aims to sequence the complete chloroplast genomes of S. aculeatissimum and S. torvum and analyze them in comparison with 29 other previously published chloroplast genomes of Solanum species. RESULTS: We observed that the chloroplast genomes of S. aculeatissimum and S. torvum possess typical tetrameric structures, consisting of one Large Single Copy (LSC) region, two reverse-symmetric Inverted Repeats (IRs), and one Small Single Copy (SSC) region. The total length of these chloroplast genomes ranged from 154,942 to 156,004 bp, with minimal variation. The highest GC content was found in the IR region, while the lowest was in the SSC region. Regarding gene content, the total number of chloroplast genes and CDS genes remained relatively consistent, ranging from 128 to 134 and 83 to 91, respectively. Nevertheless, there was notable variability in the number of tRNA genes and rRNAs. Relative synonymous codon usage (RSCU) analysis revealed that both S. aculeatissimum and S. torvum preferred codons that utilized A and U bases. Analysis of the IR boundary regions indicated that contraction and expansion primarily occurred at the junction between SSC and IR regions. Nucleotide polymorphism analysis and structural variation analysis demonstrated that chloroplast variation in Solanum species mainly occurred in the LSC and SSC regions. Repeat sequence analysis revealed that A/T was the most frequent base pair in simple repeat sequences (SSR), while Palindromic and Forward repeats were more common in long sequence repeats (LSR), with Reverse and Complement repeats being less frequent. Phylogenetic analysis indicated that S. aculeatissimum and S. torvum belonged to the same meristem and were more closely related to Cultivated Eggplant. CONCLUSION: These findings enhance our comprehension of chloroplast genomes within the Solanum genus, offering valuable insights for plant classification, evolutionary studies, and potential molecular markers for species identification.

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.

Comparative and phylogenetic analysis of the complete chloroplast genomes of 10 Artemisia selengensis resources based on high-throughput sequencing.

BACKGROUND: Artemisia selengensis, classified within the genus Artemisia of the Asteraceae family, is a perennial herb recognized for its dual utility in culinary and medicinal domains. There are few studies on the chloroplast genome of A. selengensis, and the phylogeographic classification is vague, which makes phylogenetic analysis and evolutionary studies very difficult. RESULTS: The chloroplast genomes of 10 A. selengensis in this study were highly conserved in terms of gene content, gene order, and gene intron number. The genome lengths ranged from 151,148 to 151,257 bp and were typical of a quadripartite structure with a total GC content of approximately 37.5%. The chloroplast genomes of all species encode 133 genes, including 88 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Due to the contraction and expansion of the inverted repeats (IR), the overlap of ycf1 and ndhF genes occurred at the inverted repeats B (IRB) and short single copy sequence (SSC) boundaries. According to a codon use study, the frequent base in the chloroplast genome of A. selengensis' third codon position was A/T. The number of SSR repeats was 42-44, most of which were single nucleotide A/T repeats. Sequence alignment analysis of the chloroplast genome showed that variable regions were mainly distributed in single copy regions, nucleotide diversity values of 0 to 0.009 were calculated by sliding window analysis, 8 mutation hotspot regions were detected, and coding regions were more conserved than non-coding regions. Analysis of non-synonymous substitution (Ka) and synonymous substitution (Ks) revealed that accD, rps12, petB, and atpF genes were affected by positive selection and no genes were affected by neutral selection. Based on the findings of the phylogenetic analysis, Artemisia selengensis was sister to the genus Artemisia Chrysanthemum and formed a monophyletic group with other Artemisia genera. CONCLUSIONS: In this research, the present study systematically compared the chloroplast genomic features of A. selengensis and provided important information for the study of the chloroplast genome of A. selengensis and the evolutionary relationships among Asteraceae species.

Complete chloroplast genome structural characterization of two Aerides (Orchidaceae) species with a focus on phylogenetic position of Aerides flabellata.

BACKGROUND: The disputed phylogenetic position of Aerides flabellata Rolfe ex Downie, due to morphological overlaps with related species, was investigated based on evidence of complete chloroplast (cp) genomes. The structural characterization of complete cp genomes of A. flabellata and A. rosea Lodd. ex Lindl. & Paxton were analyzed and compared with those of six related species in "Vanda-Aerides alliance" to provide genomic information on taxonomy and phylogeny. RESULTS: The cp genomes of A. flabellata and A. rosea exhibited conserved quadripartite structures, 148,145 bp and 147,925 bp in length, with similar GC content (36.7 ~ 36.8%). Gene annotations revealed 110 single-copy genes, 18 duplicated in inverted regions, and ten with introns. Comparative analysis across related species confirmed stable sequence identity and higher variation in single-copy regions. However, there are notable differences in the IR regions between two Aerides Lour. species and the other six related species. The phylogenetic analysis based on CDS from complete cp genomes indicated that Aerides species except A. flabellata formed a monophyletic clade nested in the subtribe Aeridinae, being a sister group to Renanthera Lour., consistent with previous studies. Meanwhile, a separate clade consisted of A. flabellata and six Vanda R. Br. species was formed, as a sister taxon to Holcoglossum Schltr. CONCLUSIONS: This research was the first report on the complete cp genomes of A. flabellata. The results provided insights into understanding of plastome evolution and phylogenetic relationships of Aerides. The phylogenetic analysis based on complete cp genomes showed that A. flabellata should be placed in Vanda rather than in Aerides.

Comparative structure and evolution of the organellar genomes of Padina usoehtunii (Dictyotales) with the brown algal crown radiation clade.

BACKGROUND: Organellar genomes have become increasingly essential for studying genetic diversity, phylogenetics, and evolutionary histories of seaweeds. The order Dictyotales (Dictyotophycidae), a highly diverse lineage within the Phaeophyceae, is long-term characterized by a scarcity of organellar genome datasets compared to orders of the brown algal crown radiation (Fucophycidae). RESULTS: We sequenced the organellar genomes of Padina usoehtunii, a representative of the order Dictyotales, to investigate the structural and evolutionary differences by comparing to five other major brown algal orders. Our results confirmed previously reported findings that the rate of structural rearrangements in chloroplast genomes is higher than that in mitochondria, whereas mitochondrial sequences exhibited a higher substitution rate compared to chloroplasts. Such evolutionary patterns contrast with land plants and green algae. The expansion and contraction of the inverted repeat (IR) region in the chloroplast correlated with the changes in the number of boundary genes. Specifically, the size of the IR region influenced the position of the boundary gene rpl21, with complete rpl21 genes found within the IR region in Dictyotales, Sphacelariales and Ectocarpales, while the rpl21 genes in Desmarestiales, Fucales, and Laminariales span both the IR and short single copy (SSC) regions. The absence of the rbcR gene in the Dictyotales may indicate an endosymbiotic transfer from the chloroplast to the nuclear genome. Inversion of the SSC region occurred at least twice in brown algae. Once in a lineage only represented by the Ectocarpales in the present study and once in a lineage only represented by the Fucales. Photosystem genes in the chloroplasts experienced the strongest signature of purifying selection, while ribosomal protein genes in both chloroplasts and mitochondria underwent a potential weak purifying selection. CONCLUSIONS: Variations in chloroplast genome structure among different brown algal orders are evolutionarily linked to their phylogenetic positions in the Phaeophyceae tree. Chloroplast genomes harbor more structural rearrangements than the mitochondria, despite mitochondrial genes exhibiting faster mutation rates. The position and the change in the number of boundary genes likely shaped the IR regions in the chloroplast, and the produced structural variability is important mechanistically to create gene diversity in brown algal chloroplast.

Thirteen complete chloroplast genomes of the costaceae family: insights into genome structure, selective pressure and phylogenetic relationships.

BACKGROUND: Costaceae, commonly known as the spiral ginger family, consists of approximately 120 species distributed in the tropical regions of South America, Africa, and Southeast Asia, of which some species have important ornamental, medicinal and ecological values. Previous studies on the phylogenetic and taxonomic of Costaceae by using nuclear internal transcribed spacer (ITS) and chloroplast genome fragments data had low resolutions. Additionally, the structures, variations and molecular evolution of complete chloroplast genomes in Costaceae still remain unclear. Herein, a total of 13 complete chloroplast genomes of Costaceae including 8 newly sequenced and 5 from the NCBI GenBank database, representing all three distribution regions of this family, were comprehensively analyzed for comparative genomics and phylogenetic relationships. RESULT: The 13 complete chloroplast genomes of Costaceae possessed typical quadripartite structures with lengths from 166,360 to 168,966 bp, comprising a large single copy (LSC, 90,802 - 92,189 bp), a small single copy (SSC, 18,363 - 20,124 bp) and a pair of inverted repeats (IRs, 27,982 - 29,203 bp). These genomes coded 111 - 113 different genes, including 79 protein-coding genes, 4 rRNA genes and 28 - 30 tRNAs genes. The gene orders, gene contents, amino acid frequencies and codon usage within Costaceae were highly conservative, but several variations in intron loss, long repeats, simple sequence repeats (SSRs) and gene expansion on the IR/SC boundaries were also found among these 13 genomes. Comparative genomics within Costaceae identified five highly divergent regions including ndhF, ycf1-D2, ccsA-ndhD, rps15-ycf1-D2 and rpl16-exon2-rpl16-exon1. Five combined DNA regions (ycf1-D2 + ndhF, ccsA-ndhD + rps15-ycf1-D2, rps15-ycf1-D2 + rpl16-exon2-rpl16-exon1, ccsA-ndhD + rpl16-exon2-rpl16-exon1, and ccsA-ndhD + rps15-ycf1-D2 + rpl16-exon2-rpl16-exon1) could be used as potential markers for future phylogenetic analyses and species identification in Costaceae. Positive selection was found in eight protein-coding genes, including cemA, clpP, ndhA, ndhF, petB, psbD, rps12 and ycf1. Maximum likelihood and Bayesian phylogenetic trees using chloroplast genome sequences consistently revealed identical tree topologies with high supports between species of Costaceae. Three clades were divided within Costaceae, including the Asian clade, Costus clade and South American clade. Tapeinochilos was a sister of Hellenia, and Parahellenia was a sister to the cluster of Tapeinochilos + Hellenia with strong support in the Asian clade. The results of molecular dating showed that the crown age of Costaceae was about 30.5 Mya (95% HPD: 14.9 - 49.3 Mya), and then started to diverge into the Costus clade and Asian clade around 23.8 Mya (95% HPD: 10.1 - 41.5 Mya). The Asian clade diverged into Hellenia and Parahellenia at approximately 10.7 Mya (95% HPD: 3.5 - 25.1 Mya). CONCLUSION: The complete chloroplast genomes can resolve the phylogenetic relationships of Costaceae and provide new insights into genome structures, variations and evolution. The identified DNA divergent regions would be useful for species identification and phylogenetic inference in Costaceae.

Complete chloroplast genome and comparison of herbicides toxicity on Aeschynomene indica (Leguminosae) in upland direct-seeding paddy field.

BACKGROUND: Indian jointvetch (Aeschynomene indica) is a common and pernicious weed found in the upland direct-seeding rice fields in the lower reaches of the Yangtze River in China. However, there are few reports on the degree of harm, genetic characteristics, and management methods of this weed. The purpose of this study is to clarify the harm of Indian jointvetch to upland direct-seeding rice, analyze the genetic characteristics of this weed based on chloroplast genomics and identify its related species, and screen herbicides that are effective in managing this weed in upland direct-seeding rice fields. RESULTS: In a field investigation in upland direct-seeding rice paddies in Shanghai and Jiangsu, we determined that the plant height and maximum lateral distance of Indian jointvetch reached approximately 134.2 cm and 57.9 cm, respectively. With Indian jointvetch present at a density of 4/m2 and 8/m2, the yield of rice decreased by approximately 50% and 70%, respectively. We further obtained the first assembly of the complete chloroplast (cp.) genome sequence of Indian jointvetch (163,613 bp). There were 161 simple sequence repeats, 166 long repeats, and 83 protein-encoding genes. The phylogenetic tree and inverted repeat region expansion and contraction analysis based on cp. genomes demonstrated that species with closer affinity to A. indica included Glycine soja, Glycine max, and Sesbania cannabina. Moreover, a total of 3281, 3840, and 3838 single nucleotide polymorphisms were detected in the coding sequence regions of the cp. genomes of S. cannabina voucher IBSC, G. soja, and G. max compared with the A. indica sequence, respectively. A greenhouse pot experiment indicated that two pre-emergence herbicides, saflufenacil and oxyfluorfen, and two post-emergence herbicides, florpyrauxifen-benzyl and penoxsulam, can more effectively manage Indian jointvetch than other common herbicides in paddy fields. The combination of these two types of herbicides is recommended for managing Indian jointvetch throughout the entire growth period of upland direct-seeding rice. CONCLUSIONS: This study provides molecular resources for future research focusing on the identification of the infrageneric taxa, phylogenetic resolution, and biodiversity of Leguminosae plants, along with recommendations for reliable management methods to control Indian jointvetch.

Comparative analysis of the organelle genomes of Aconitum carmichaelii revealed structural and sequence differences and phylogenetic relationships.

In this study, we conducted an assembly and analysis of the organelle genomes of Aconitum carmichaelii. Our investigation encompassed the examination of organelle genome structures, gene transfer events, and the environmental selection pressures affecting A. carmichaelii. The results revealed distinct evolutionary patterns in the organelle genomes of A. carmichaelii. Especially, the plastome exhibited a more conserved structure but a higher nucleotide substitution rate (NSR), while the mitogenome displayed a more complex structure with a slower NSR. Through homology analysis, we identified several instances of unidirectional protein-coding genes (PCGs) transferring from the plastome to the mitogenome. However, we did not observe any events which genes moved from the mitogenome to the plastome. Additionally, we observed multiple transposable element (TE) fragments in the organelle genomes, with both organelles showing different preferences for the type of nuclear TE insertion. Divergence time estimation suggested that rapid differentiation occurred in Aconitum species approximately 7.96 million years ago (Mya). This divergence might be associated with the reduction in CO2 levels and the significant uplift of the Qinghai-Tibet Plateau (QTP) during the late Miocene. Selection pressure analysis indicated that the dN/dS values of both organelles were less than 1, suggested that organelle PCGs were subject to purification selection. However, we did not detect any positively selected genes (PSGs) in Subg. Aconitum and Subg. Lycoctonum. This observation further supports the idea that stronger negative selection pressure on organelle genes in Aconitum results in a more conserved amino acid sequence. In conclusion, this study contributes to a deeper understanding of organelle evolution in Aconitum species and provides a foundation for future research on the genetic mechanisms underlying the structure and function of the Aconitum plastome and mitogenome.

Genetic Insights into the Extremely Dwarf Hibiscus syriacus var. micranthus: Complete Chloroplast Genome Analysis and Development of a Novel dCAPS Marker.

This study explored the chloroplast (cp) genomes of three Hibiscus syriacus (HS) specimens endemic to Korea possessing unique ornamental and conservation values: the dwarf H. syriacus var. micranthus (HSVM), renowned for its small stature and breeding potential; HS 'Tamra', a cultivar from Korea's southernmost islands, noteworthy for its distinctive beauty; and HS Natural Monument no. 521 (N.M.521), a specimen of significant lifespan and height. Given the scarcity of evolutionary studies on these specimens, we assembled and analyzed their cp genomes. We successfully assembled genomes spanning 160,000 to 160,100 bp and identified intraspecific variants. Among these, a unique ATA 3-mer insertion in the trnL-UAA region was identified in HSVM, highlighting its value as a genetic resource. Leveraging this finding, we developed a novel InDel dCAPS marker, which was validated across 43 cultivars, enhancing our ability to distinguish HSVM and its derivatives from other HS cultivars. Phylogenetic analysis involving 23 Malvaceae species revealed that HSVM forms a clade with woody Hibiscus species, closely associating with N.M.520, which may suggest a shared ancestry or parallel evolutionary paths. This investigation advances our understanding of the genetic diversity in Korean HS and offers robust tools for accurate cultivar identification, aiding conservation and breeding efforts.

Comparative and phylogenetic analysis of the complete chloroplast genomes of ten Pittosporum species from East Asia.

Pittosporum (Pittosporaceae) is famous as the ornamental and medical values, which is distributed tropical and subtropical regions of Eastern Hemisphere. The few phylogenetic studies have included samples from the Pacific Island, but the phylogenetic relationships of Asian species has not been studied. Here, the complete chloroplast (cp) genomes of ten Pittosporum species from East Asia were first sequenced and compared with those of the published species of this genus. Our results indicated that cp genomes of these species had a typical and conserved quadripartite structure. 131 genes were identical in order and orientation and no changes of inverted repeat (IR) occurred. However, the comparative analysis of cp genomes suggested that sequence divergence mainly appeared in non-coding or intergenic regions, in which several divergence hotspots were identified. By contrast, protein-coding genes showed the lowest variance under strong purifying selection. Phylogenetic analysis based on the cp genome sequences showed that the tested Pittosporum species were clustered into two major clades, in which the Asian species formed Clade I and the remaining species from Australia and New Zealand formed Clade II with high support values, which was consistent with the results of ITS data with low support values. These results suggested that cp genome is a robust phylogenetic indicator for deep nodes in the phylogeny of Pittosporum. Meanwhile, these results will provide the valuable information to better understand the phylogeny and biogeography of Pittosporum.

Analysis of chloroplast genome characteristics and codon usage bias in 14 species of Annonaceae.

For the study of species evolution, chloroplast gene expression, and transformation, the chloroplast genome is an invaluable resource. Codon usage bias (CUB) analysis is a tool that is utilized to improve gene expression and investigate evolutionary connections in genetic transformation. In this study, we analysed chloroplast genome differences, codon usage patterns and the sources of variation on CUB in 14 Annonaceae species using bioinformatics tools. The study showed that there was a significant variation in both gene sizes and numbers between the 14 species, but conservation was still maintained. It's worth noting that there were noticeable differences in the IR/SC sector boundary and the types of SSRs among the 14 species. The mono-nucleotide repeat type was the most common, with A/T repeats being more prevalent than G/C repeats. Among the different types of repeats, forward and palindromic repeats were the most abundant, followed by reverse repeats, and complement repeats were relatively rare. Codon composition analysis revealed that all 14 species had a frequency of GC lower than 50%. Additionally, it was observed that the proteins in-coding sequences of chloroplast genes tend to end with A/T at the third codon position. Among these species, 21 codons exhibited bias (RSCU > 1), and there were 8 high-frequency (HF) codons and 5 optimal codons that were identical across the species. According to the ENC-plot and Neutrality plot analysis, natural selection had less impact on the CUB of A. muricate and A. reticulata. Based on the PR2-plot, it was evident that base G had a higher frequency than C, and T had a higher frequency A. The correspondence analysis (COA) revealed that codon usage patterns different in Annonaceae.

Complete chloroplast genome of the Malus baccata var. gracilis provides insights into the evolution and phylogeny of Malus species.

Malus baccata (L.) var. gracilis (Rehd.) has high ornamental value and breeding significance, and comparative chloroplast genome analysis was applied to facilitate genetic breeding for desired traits and resistance and provide insight into the phylogeny of this genus. Using data from whole-genome sequencing, a tetrameric chloroplast genome with a length of 159,992 bp and a total GC content of 36.56% was constructed. The M. baccata var. gracilis chloroplast genome consists of a large single-copy sequence (88,100 bp), a short single-copy region (19,186 bp), and two inverted repeat regions, IRa (26,353 bp) and IRb (26,353 bp). This chloroplast genome contains 112 annotated genes, including 79 protein-coding genes (nine multicopy), 29 tRNA genes (eight multicopy), and four rRNA genes (all multicopy). Calculating the relative synonymous codon usage revealed a total of 32 high-frequency codons, and the codons exhibited a biased usage pattern towards A/U as the ending nucleotide. Interspecific sequence comparison and boundary analysis revealed significant sequence variation in the vast single-copy region, as well as generally similar expansion and contraction of the SSC and IR regions for 10 analyzed Malus species. M. baccata var. gracilis and Malus hupehensis were grouped together into one branch based on phylogenetic analysis of chloroplast genome sequences. The chloroplast genome of Malus species provides an important foundation for species identification, genetic diversity analysis, and Malus chloroplast genetic engineering. Additionally, the results can facilitate the use of pendant traits to improve apple tree shape.

Comparative chloroplast genomics of 24 species shed light on the genome evolution and phylogeny of subtribe Coelogyninae (Orchidaceae).

BACKGROUND: The orchids of the subtribe Coelogyninae are among the most morphologically diverse and economically important groups within the subfamily Epidendroideae. Previous molecular studies have revealed that Coelogyninae is an unambiguously monophyletic group. However, intergeneric and infrageneric relationships within Coelogyninae are largely unresolved. There has been long controversy over the classification among the genera within the subtribe. RESULTS: The complete chloroplast (cp.) genomes of 15 species in the subtribe Coelogyninae were newly sequenced and assembled. Together with nine available cp. genomes in GenBank from representative clades of the subtribe, we compared and elucidated the characteristics of 24 Coelogyninae cp. genomes. The results showed that all cp. genomes shared highly conserved structure and contained 135 genes arranged in the same order, including 89 protein-coding genes, 38 tRNAs, and eight rRNAs. Nevertheless, structural variations in relation to particular genes at the IR/SC boundary regions were identified. The diversification pattern of the cp. genomes showed high consistency with the phylogenetic placement of Coelogyninae. The number of different types of SSRs and long repeats exhibited significant differences in the 24 Coelogyninae cp. genomes, wherein mononucleotide repeats (A/T), and palindromic repeats were the most abundant. Four mutation hotspot regions (ycf1a, ndhF-rp132, psaC-ndhE, and rp132-trnL) were determined, which could serve as effective molecular markers. Selection pressure analysis revealed that three genes (ycf1a, rpoC2 and ycf2 genes) might have experienced apparent positive selection during the evolution. Using the alignments of whole cp. genomes and protein-coding sequences, this study presents a well-resolved phylogenetic framework of Coelogyninae. CONCLUSION: The inclusion of 55 plastid genome data from a nearly complete generic-level sampling provide a comprehensive view of the phylogenetic relationships among genera and species in subtribe Coelogyninae and illustrate the diverse genetic variation patterns of plastid genomes in this species-rich plant group. The inferred relationships and informally recognized major clades within the subtribe are presented. The genetic markers identified here will facilitate future studies on the genetics and phylogeny of subtribe Coelogyninae.

Twelve newly assembled jasmine chloroplast genomes: unveiling genomic diversity, phylogenetic relationships and evolutionary patterns among Oleaceae and Jasminum species.

BACKGROUND: Jasmine (Jasminum), renowned for its ornamental value and captivating fragrance, has given rise to numerous species and accessions. However, limited knowledge exists regarding the evolutionary relationships among various Jasminum species. RESULTS: In the present study, we sequenced seven distinct Jasminum species, resulting in the assembly of twelve high-quality complete chloroplast (cp) genomes. Our findings revealed that the size of the 12 cp genomes ranged from 159 to 165 kb and encoded 134-135 genes, including 86-88 protein-coding genes, 38-40 tRNA genes, and 8 rRNA genes. J. nudiflorum exhibited a larger genome size compared to other species, mainly attributed to the elevated number of forward repeats (FRs). Despite the typically conservative nature of chloroplasts, variations in the presence or absence of accD have been observed within J. sambac. The calculation of nucleotide diversity (Pi) values for 19 cp genomes indicated that potential mutation hotspots were more likely to be located in LSC regions than in other regions, particularly in genes ycf2, rbcL, atpE, ndhK, and ndhC (Pi > 0.2). Ka/Ks values revealed strong selection pressure on the genes rps2, atpA, rpoA, rpoC1, and rpl33 when comparing J. sambac with the three most closely related species (J. auriculatum, J. multiflorum, and J. dichotomum). Additionally, SNP identification, along with the results of Structure, PCA, and phylogenetic tree analyses, divided the Jasminum cp genomes into six groups. Notably, J. polyanthum showed gene flow signals from both the G5 group (J. nudiflorum) and the G3 group (J. tortuosum and J. fluminense). Phylogenetic tree analysis reflected that most species from the same genus clustered together with robust support in Oleaceae, strongly supporting the monophyletic nature of cp genomes within the genus Jasminum. CONCLUSION: Overall, this study provides comprehensive insights into the genomic composition, variation, and phylogenetic relationships among various Jasminum species. These findings enhance our understanding of the genetic diversity and evolutionary history of Jasminum.