Comparative Chloroplast Genomics Research Articles

Comparative chloroplast genome analyses of cultivated and wild Capsicum species shed light on evolution and phylogeny

The chloroplast (cp.) genome, also known as plastome, plays crucial roles in plant survival, adaptation, and evolution. The stable genetic structure of cp. genomes provides an ideal system for investigating species evolution. We sequenced three complete cp. genome sequences of Capsicum species and analyzed them using sequences of various Capsicum species retrieved from the NCBI database. The cp. genome of Capsicum species maintains a well-preserved quadripartite structure consisting of two inverted repeats (IRs) flanked by a large single copy (LSC) region and a small single copy (SSC) region. The sizes of cp. genome sequences ranged from 156,583 bp (C. lycianthoides) to 157,390 bp (C.pubescens). A total of 127–132 unique genes, including 83–87 protein-coding, 36–37 tRNA, and eight rRNA genes, were predicted. Comparison of cp. genomes of 10 Capsicum species revealed high sequence similarity in genome-wide organization and gene arrangements. Fragments of trnT-UGU/trnL-UAA, ccsA, ndhD, rps12, and ycf1 were identified as variable regions, and nucleotide variability of LSC and SSC was higher than that of IR. Phylogenetic speciation analysis showed that the major domesticated C. annuum species were the most extensively divergent species and closely related to C. tovarii and C. frutescens. Analysis of divergent times suggested that a substantial range of speciation events started occurring ~ 25.79 million years ago (Mya). Overall, comparative analysis of cp. genomes of Capsicum species not only offers new insights into their genetic variation and phylogenetic relationships, but also lays a foundation for evolutionary history, genetic diversity, conservation, and biological breeding of Capsicum species.

Comparative chloroplast genomics, phylogenetic relationships and molecular markers development of Aglaonema commutatum and seven green cultivars of Aglaonema

Aglaonema commutatum is a famous species in the Aglaonema genus, which has important ornamental and economic value. However, its chloroplast genome information and phylogenetic relationships among popular green cultivars of Aglaonema in southern China have not been reported. Herein, chloroplast genomes of one variety of A. commutatum and seven green cultivars of Aglaonema, namely, A. commutatum ‘San Remo’, ‘Kai Sa’, ‘Pattaya Beauty’, ‘Sapphire’, ‘Silver Queen’, ‘Snow White’, ‘White Gem’, and ‘White Horse Prince’, were sequenced and assembled for comparative analysis and phylogeny. These eight genomes possessed a typical quadripartite structure that consisted of a LSC region (90,799–91,486 bp), an SSC region (20,508–21,137 bp) and a pair of IR regions (26,661–26,750 bp). Each genome contained 112 different genes, comprising 79 protein-coding genes, 29 tRNA genes and 4 rRNA genes. The gene orders, GC contents, codon usage frequency, and IR/SC boundaries were highly conserved among these eight genomes. Long repeats, SSRs, SNPs and indels were analyzed among these eight genomes. Comparative analysis of 15 Aglaonema chloroplast genomes identified 7 highly variable regions, including trnH-GUG-exon1-psbA, trnS-GCU-trnG-UCC-exon1, trnY-GUA-trnE-UUC, psbC-trnS-UGA, trnF-GAA-ndhJ, ccsA-ndhD, and rps15-ycf1-D2. Reconstruction of the phylogenetic trees based on chloroplast genomes, strongly supported that Aglaonema was a sister to Anchomanes, and that the Aglaonema genus was classified into two sister clades including clade I and clade II, which corresponded to two sections, Aglaonema and Chamaecaulon, respectively. One variety and five cultivars, including A. commutatum ‘San Remo’, ‘Kai Sa’, ‘Pattaya Beauty’, ‘Silver Queen’, ‘Snow White’, and ‘White Horse Prince’, were classified into clade I; and the rest of the two cultivars, including ‘Sapphire’ and ‘White Gem’, were classified into clade II. Positive selection was observed in 34 protein-coding genes at the level of the amino acid sites among 77 chloroplast genomes of the Araceae family. Based on the highly variable regions and SSRs, 4 DNA markers were developed to differentiate the clade I and clade II in Aglaonema. In conclusion, this study provided chloroplast genomic resources for Aglaonema, which were useful for its classification and phylogeny.

Comparative chloroplast genomes: insights into the identification and phylogeny of rapid radiation genus Rhodiola.

Rhodiola L. is a genus exhibiting rapid radiation and represents a typical case for studying plastid gene adaptation in species that spread from high altitudes to low altitudes. In this study, 23 samples of 18 Rhodiola species were collected from the Qinghai-Tibetan Plateau and five scattered alpine areas, and the plastid genomes (plastomes) of these species were sequenced, annotated, and compared between high-altitude and widely distributed groups. The plastomes of Rhodiola were found to be highly conserved in terms of gene size, content, and order but highly variable in several lineage-specific features, such as codon usage bias, IR boundary shifting, and distinct repeat sequence structures binding to SSRs. Codon usage in the genes of photosystem II exhibited an obvious preference, reflecting significant environmental adaptation pressures. In this study, three repeat regions compounded with trinucleotide and mononucleotide repeats were found for the first time in R. forrestii, R. himalensis, and R. yunnanensis. High-variability regions such as ndhF, ycf1, trnH-psbA, and rpoC1-rpoB were screened, laying the foundation for the precise identification of these species. The phylogenetic analysis revealed the occurrence of cyto-nuclear discordance, likely originating from the frequent interspecific hybridization events observed within Rhodiola species during rapid radiation. Dioecious and hermaphrodite species can be broadly categorized into two subclades, probably they have different environmental adaptation strategies in response to climate change. In addition, the phylogenetic tree supported the monophyly of R. forrestii and R. yunnanensis, which compose R. Sect. Pseudorhodiola. In conclusion, plastome data enrich the genetic information available for the Rhodiola genus and may provide insight into species migration events during climate change.

Comparative chloroplast genomes of Argentina species: genome evolution and phylogenomic implications.

The genus Argentina Hill belongs to the tribe Potentilleae Sweet and contains approximately 75 species predominantly distributed in the Sino-Himalayan region and the Malesian archipelago. So far we have less knowledge on the phylogenetic relationships within Argentina owing to limited sampling of Argentina taxa or gene fragments in previous studies. Moreover, to date there is no phylogenetic study on Argentina from the perspective of comparative chloroplast (cp) genomics. Here we performed comparative genomic analyses on the cp genomes of 39 accessions representing 18 taxa of Argentina. The Argentina cp genomes presented the typical quadripartite structure, with the sizes ranging from 155 096 bp to 157 166 bp. The 39 Argentina cp genomes contained a set of 112 unique genes, comprising four ribosomal RNA (rRNA) genes, 30 transfer RNA (tRNA) genes, as well as 78 protein-coding genes (PCGs). The cp genome organization, gene content and order in Argentina were highly conserved, but some visible divergences were present in IR/SC boundary regions. Ten regions (trnH-GUG-psbA, trnG-GCC-trnfM-CAU, trnD-GUC-trnY-GUA, rpl32-trnL-UAG, atpH-atpI, rps16-trnQ-UUG, trnS-GCU-trnG-UCC, ndhF-rpl32, trnR-UCU-atpA, and accD-psaI) were identified as excellent candidate DNA markers for future studies on species identification, population genetics and phylogeny of Argentina. Our results indicated that Argentina is monophyletic. In the current sampling, the A. smithiana - A. anserina clade was sister to the remainder of Argentina. Our results corroborated the previous taxonomic treatments to transfer A. phanerophlebia and A. micropetala from the genus Sibbaldia L. to Argentina. Our results showed close relationships among A. stenophylla, A. microphylla, A. taliensis, and A. tatsienluensis, congruent with previous studies based on the morphology of these species. Twenty-six genes (rps3, rps15, rps16, rps19, rpl16, rpl20, rpl22, rpoA, rpoB, rpoC1, rpoC2, atpA, atpF, psbB, psbF, ndhA, ndhB, ndhC, ndhD, ndhF, rbcL, accD, ccsA, matK, ycf1, ycf2) were with sites under positive selection, and adaptive evolution of these genes might have played crucial roles in Argentina species adaptation to the harsh mountain environment. This study will facilitate future work on taxonomy, phylogenetics, and adaptive evolution of Argentina.

Comparative chloroplast genomes of Dactylicapnos species: insights into phylogenetic relationships

BackgroundDactylicapnos is a climbing herbaceous vine, distributed from the Himalayas to southwestern China, and some of the species have important medicinal values. However, the chloroplast genomes of Dactylicapnos have never been investigated. In this study, chloroplast genomes of seven Dactylicapnos species covering all three sections and one informal group of Dactylicapnos were sequenced and assembled, and the detailed comparative analyses of the chloroplast genome structure were provided for the first time.ResultsThe results showed that the chloroplast genomes of Dactylicapnos have a typical quadripartite structure with lengths from 172,344 bp to 176,370 bp, encoding a total of 133–140 genes, containing 88–94 protein-coding genes, 8 rRNAs and 37–39 tRNAs. 31 codons were identified as relative synonymous codon usage values greater than one in the chloroplast genome of Dactylicapnos genus based on 80 protein-coding genes. The results of the phylogenetic analysis showed that seven Dactylicapnos species can be divided into three main categories. Phylogenetic analysis revealed that seven species form three major clades which should be treated as three sections.ConclusionsThis study provides the initial report of the chloroplast genomes of Dactylicapnos, their structural variation, comparative genomic and phylogenetic analysis for the first time. The results provide important genetic information for development of medical resources, species identification, infrageneric classification and diversification of Dactylicapnos.

Comparative chloroplast genomes study of five officinal Ardisia Species: Unraveling interspecific diversity and evolutionary insights in Ardisia

Ardisia S.W. (Primulaceae), naturally distributed in tropical and subtropical regions, has edible and medicinal values and is prevalent in clinical and daily use in China. More genetic information for distinct species delineation is needed to support the development and utilization of the genus Ardisia. We sequenced, annotated, and compared the chloroplast genomes of five Ardisia species: A. brunnescens, A. pusilla, A. squamulosa, A. crenata, and A. brevicaulis in this study. We found a typical quadripartite structure in all five chloroplast genomes, with lengths ranging from 155,045 to 156,943 bp. Except for A. pusilla, which lacked the ycf15 gene, the other four Ardisia species contained 114 unique genes, including 79 protein-coding genes, 30 tRNAs, and four rRNAs. In addition, the rps19 pseudogene gene was present only in A. brunnescens. Five highly variable DNA barcodes were identified for five Ardisia species, including trnT-GGU-psbD, trnT-UGU-trnL-UAA, rps4-trnT-UGU, rpl32-trnL-UAG, and rpoB-trnC-GAA. The RNA editiing sites of protein-coding genes in the five Ardisia plastome were characterized and compared, and 274 (A. crenata)-288 (A. brevicaulis) were found. The results of the phylogenetic analysis were consistent with the morphological classification. Sequence alignment and phylogenetic analysis showed that ycf15 genes were highly divergent in Primulaceae. Reconstructions of ancestral character states indicated that leaf margin morphology is critical for classifying the genus Ardisia, with a rodent-like character being the most primitive. These results provide valuable information on the taxonomy and evolution of Ardisia plants.

Comparative chloroplast genome analysis of six millet species along with related Poaceae family members

Comparative chloroplast genome analysis of six millet species along with related Poaceae family members

Comparative chloroplast genome analyses provide insights into evolutionary history of Rhizophoraceae mangroves.

The Rhizophoraceae family comprises crucial mangrove plants that inhabit intertidal environments. In China, eight Rhizophoraceae mangrove species exist. Although complete chloroplast (Cp) genomes of four Rhizophoraceae mangrove plants have been reported, the Cp genomes of the remaining four species remain unclear, impeding a comprehensive understanding of the evolutionary history of this family. Illumina high-throughput sequencing was employed to obtain the DNA sequences of Rhizophoraceae species. Cp genomes were assembled by NOVOPlasty and annotated using CpGAVAS software. Phylogenetic and divergence time analyses were conducted using MEGA and BEAST 2 software. Four novel Cp genomes of Rhizophoraceae mangrove species (Bruguiera sexangula, Bruguiera gymnorrhiza, Bruguiera × rhynchopetala and Rhizophora apiculata) were successfully assembled. The four Cp genomes ranged in length from 163,310 to 164,560 bp, with gene numbers varying from 124 to 128. The average nucleotide diversity (Pi) value of the eight Rhizophoraceae Cp genomes was 0.00596. Phylogenetic trees constructed based on the complete Cp genomes supported the monophyletic origin of Rhizophoraceae. Divergence time estimation based on the Cp genomes of representative species from Malpighiales showed that the origin of Rhizophoraceae occurred at approximately 58.54-50.02 million years ago (Mya). The divergence time within the genus Rhizophora (∼4.51 Mya) was much earlier than the divergence time within the genus Bruguiera (∼1.41 Mya), suggesting recent speciation processes in these genera. Our data provides new insights into phylogenetic relationship and evolutionary history of Rhizophoraceae mangrove plants.

Comparative chloroplast genome analyses provide new insights into phylogeny of Taraxacum and molecular markers for distinguishing rubber producing dandelions from their weedy relatives in China

Taraxacum species are utilized as traditional herbal medicine or as industrial crops to producing natural rubber. However, the classification of Taraxacum species has long been a challenge, thereby limiting their industrial application. To facilitate the correct screening rubber-producing dandelions from the weedy relatives, 38 chloroplast (Cp) genomes of Taraxacum spp. mainly gathered in Xinjiang, China, were assembled, including both rubber- and non-rubber-producing ones, and were used to reconstruct the phylogeny of Taraxacum. The results reveal that T. mongolicum and T. dealbatum are the primitive species in Taraxacum studied here, while T. erythrospermum and T. compactum are the closest relatives to the rubber-producing dandelion T. kok-saghyz. The discrepancies among the phylogenetic relationships inferred from Cp genomes, nuclear ribosomal internal transcribed spacer and morphology traits reflect chloroplast capture events due to putative ancient cross-species hybridization within Taraxacum. Comparative genomic analysis showed that, although highly conserved for most areas, some regions exhibit relatively high levels of variation in Cp genomes among different Taraxacum species, such as ndhD-ccsA and trnL-trnF regions. A total of 37 loci in Taraxacum Cp genomes were identified as novel putative molecular markers that might assist to distinguish rubber producing dandelions from their weedy relatives in their distribution center. Our results provide new insights into the phylogenetic relationships within the genus Taraxacum. In addition, the Cp genomic resources reported in this study provide genetic foundation for distinguishing rubber producing dandelions from their weedy relatives and future exploration of these industrial important species.

Comparative chloroplast genomics reveals the phylogeny and the adaptive evolution of Begonia in China

BackgroundThe Begonia species are common shade plants that are mostly found in southwest China. They have not been well studied despite their medicinal and decorative uses because gene penetration, decreased adaptability, and restricted availability are all caused by frequent interspecific hybridization.ResultTo understand the patterns of mutation in the chloroplast genomes of different species of Begonia, as well as their evolutionary relationships, we collected seven Begonia species in China and sequenced their chloroplast genomes. Begonia species exhibit a quadripartite structure of chloroplast genomes (157,634 − 169,694 bp), consisting of two pairs of inverted repeats (IR: 26,529 − 37,674 bp), a large single copy (LSC: 75,477 − 86,500 bp), and a small single copy (SSC: 17,861 − 18,367 bp). 128–143 genes (comprising 82–93 protein-coding genes, 8 ribosomal RNAs, and 36–43 transfer RNAs) are found in the chloroplast genomes. Based on comparative analyses, this taxon has a relatively similar genome structure. A total of six substantially divergent DNA regions (trnT-UGU-trnL-UAA, atpF-atpH, ycf4-cemA, psbC-trnS-UGA, rpl32-trnL-UAG, and ccsA-ndhD) are found in the seventeen chloroplast genomes. These regions are suitable for species identification and phylogeographic analysis. Phylogenetic analysis shows that Begonia species that were suited to comparable environments grouped in a small clade and that all Begonia species formed one big clade in the phylogenetic tree, supporting the genus’ monophyly. In addition, positive selection sites were discovered in eight genes (rpoC1, rpoB, psbE, psbK, petA, rps12, rpl2, and rpl22), the majority of which are involved in protein production and photosynthesis.ConclusionUsing these genome resources, we can resolve deep-level phylogenetic relationships between Begonia species and their families, leading to a better understanding of evolutionary processes. In addition to enhancing species identification and phylogenetic resolution, these results demonstrate the utility of complete chloroplast genomes in phylogenetically and taxonomically challenging plant groupings.

Comparative Chloroplast Genome Analyses of Six Hemlock Trees in East Asia: Insights into Their Genomic Characterization and Phylogenetic Relationship

Hemlocks (Pinaceae: Tsuga) are widely distributed in North America and East Asia, forming a reticulate evolutionary structure in East Asia with significant ecological importance. To clarify the chloroplast genome characteristics and phylogenetic relationships among Tsuga species, we analyzed the chloroplast genomes of T. chinensis var. tchekiangensis, T. chinensis, T. diversifolia, T. dumosa, T. forrestii, and T. sieboldii, performing associated phylogenetic analyses. The results reveal that the chloroplast genome lengths among the six Tsuga species vary from 120,520 to 121,010 bp, encompassing about 108 to 112 genes, including 35/32 tRNA genes and 4 rRNA genes. A codon usage analysis highlighted a preference for A/U-ending codons, and all six nucleotide types have A/T bases and a prevalence of mononucleotides. Notably, all Tsuga species exhibit inverted repeat (IR) contractions and possess unique hexanucleotides absent in the other species of Pinaceae, potentially making them more susceptible to gene recombination or rearrangement during evolution. While most variations are observed in non-coding regions, particularly in intergenic fragments, substantial variation sites are also present within the genes. The phylogenetic tree, constructed using chloroplast genomes, substantiates the sister taxa relationship between Tsuga and Nothotsuga. Furthermore, it confirms that T. chinensis var. tchekiangensis exhibits a closer relationship with T. forrestii than with T. chinensis. These findings not only provide partial evidence that T. chinensis may not constitute a monophyletic species but also underscore the necessity of reevaluating the taxonomic status of T. chinensis var. tchekiangensis. In addition, while the RSCU cluster analysis is basically consistent with the phylogenetic analysis, it also highlights a distinct differentiation between Nothotsuga and Tsuga. This study not only provides molecular-level phylogenetic classification evidence of Pinaceous genera via chloroplast genome analyses but also offers compelling evidence for further exploring the relationships and species delimitation among the hemlocks of East Asia.

Comparative chloroplast genomes provided insights into the evolution and species identification on the Datureae plants.

Generally, chloroplast genomes of angiosperms are always highly conserved but carry a certain number of variation among species. In this study, chloroplast genomes of 13 species from Datureae tribe that are of importance both in ornamental gardening and medicinal usage were studied. In addition, seven chloroplast genomes from Datureae together with two from Solanaceae species retrieved from the National Center for Biotechnology Information (NCBI) were integrated into this study. The chloroplast genomes ranged in size from 154,686 to 155,979 and from 155,497 to 155,919 bp for species of Datura and Brugmansia, respectively. As to Datura and Brugmansia, a total of 128 and 132 genes were identified, in which 83 and 87 protein coding genes were identified, respectively; Furthermore, 37 tRNA genes and 8 rRNA genes were both identified in Datura and Brugmansia. Repeats analysis indicated that the number and type varied among species for Simple sequence repeat (SSR), long repeats, and tandem repeats ranged in number from 53 to 59, 98 to 99, and 22 to 30, respectively. Phylogenetic analysis based on the plastid genomes supported the monophyletic relationship among Datura and Brugmansia and Trompettia, and a refined phylogenic relationships among each individual was resolved. In addition, a species-specific marker was designed based on variation spot that resulted from a comparative analysis of chloroplast genomes and verified as effective maker for identification of D. stramonium and D. stramonium var. inermis. Interestingly, we found that 31 genes were likely to be under positive selection, including genes encoding ATP protein subunits, photosystem protein subunit, ribosome protein subunits, NAD(P)H dehydrogenase complex subunits, and clpP, petB, rbcL, rpoCl, ycf4, and cemA genes. These genes may function as key roles in the adaption to diverse environment during evolution. The diversification of Datureae members was dated back to the late Oligocene periods. These chloroplast genomes are useful genetic resources for taxonomy, phylogeny, and evolution for Datureae.

Comparative Chloroplast Genomics of 21 Species in Zingiberales with Implications for Their Phylogenetic Relationships and Molecular Dating.

Zingiberales includes eight families and more than 2600 species, with many species having important economic and ecological value. However, the backbone phylogenetic relationships of Zingiberales still remain controversial, as demonstrated in previous studies, and molecular dating based on chloroplast genomes has not been comprehensively studied for the whole order. Herein, 22 complete chloroplast genomes from 21 species in Zingiberales were sequenced, assembled, and analyzed. These 22 genomes displayed typical quadripartite structures, which ranged from 161,303 bp to 163,979 bp in length and contained 111-112 different genes. The genome structures, gene contents, simple sequence repeats, long repeats, and codon usage were highly conserved, with slight differences among these genomes. Further comparative analysis of the 111 complete chloroplast genomes of Zingiberales, including 22 newly sequenced ones and the remaining ones from the national center for biotechnology information (NCBI) database, identified three highly divergent regions comprising ccsA, psaC, and psaC-ndhE. Maximum likelihood and Bayesian inference phylogenetic analyses based on chloroplast genome sequences found identical topological structures and identified a strongly supported backbone of phylogenetic relationships. Cannaceae was sister to Marantaceae, forming a clade that was collectively sister to the clade of (Costaceae, Zingiberaceae) with strong support (bootstrap (BS) = 100%, and posterior probability (PP) = 0.99-1.0); Heliconiaceae was sister to the clade of (Lowiaceae, Strelitziaceae), then collectively sister to Musaceae with strong support (BS = 94-100%, and PP = 0.93-1.0); the clade of ((Cannaceae, Marantaceae), (Costaceae, Zingiberaceae)) was sister to the clade of (Musaceae, (Heliconiaceae, (Lowiaceae, Strelitziaceae))) with robust support (BS = 100%, and PP = 1.0). The results of divergence time estimation of Zingiberales indicated that the crown node of Zingiberales occurred approximately 85.0 Mya (95% highest posterior density (HPD) = 81.6-89.3 million years ago (Mya)), with major family-level lineages becoming from 46.8 to 80.5 Mya. These findings proved that chloroplast genomes could contribute to the study of phylogenetic relationships and molecular dating in Zingiberales, as well as provide potential molecular markers for further taxonomic and phylogenetic studies of Zingiberales.

Comparative analysis of switchgrass chloroplast genomes provides insights into identification, phylogenetic relationships and evolution of different ecotypes

Switchgrass (Panicum virgatum L.) plays a crucial role as a valuable forage and bioenergy resource. There are two ecotypes of switchgrass, upland and lowland, which exhibit significant differences in their adaptability to various environmental stressors. To enhance ecotype identification and gain a comprehensive understanding of the evolutionary relationships between these two ecotypes at the population level, we assembled 20 switchgrass chloroplast genomes (10 uplands and 10 lowlands) collected from different latitudinal regions. All chloroplast genomes displayed the typical quadripartite structure, with sizes ranging from 139,497 bp (J170. A) to 139,746 bp (J440. A and J636. B). The annotation of these 20 switchgrass chloroplast genomes revealed a consistent number of genes, including 40 transfer RNAs, eight ribosomal RNAs, and 86 protein-coding genes. Through multiscale analyses of the chloroplast genomes, we observed conservation in the upland and lowland chloroplasts in terms of IR boundaries, repetitive sequences, and codon usage. Our comparative chloroplast genome analysis revealed that large single copy regions exhibited the highest nucleotide diversity, followed by small single copy regions, while inverted repeat regions showed a relatively conserved pattern. Additionally, we identified a hotspot, trnG-GCC-trnM-CAU, which was exclusive to the lowland ecotype, suggesting its potential to differentiate between the two ecotypes. In the phylogenetic analysis, we found that some accessions from both ecotypes were intermixed and clustered into one clade, highlighting the complexity of the phylogenetic evolution process. In conclusion, we employed population-level comparative genomic analysis to investigate genetic variations and phylogenetic relationships among different ecotypes, thereby offering novel insights into the evolutionary dynamics and classifications of switchgrass.

Comparative chloroplast genome analysis of four Polygonatum species insights into DNA barcoding, evolution, and phylogeny

The Polygonatum genus represents a perennial herb with the Liliaceae family, boasting substantial economic and medicinal significance. The majority of Polygonatum plants exhibit notable similarity while lacking distinctive identifying characteristics, thus resulting in the proliferation of adulterated medicinal materials within the market. Within this study, we conducted an in-depth analysis of the complete chloroplast (cp) genomes of four Polygonatum plants and compared them with four closely akin species. The primary objectives were to unveil structural variations, species divergence, and the phylogenetic interrelations among taxa. The cp genomes of the four Polygonatum species were typified by a conventional quadripartite structure, incorporating a large single copy region (LSC), a small single copy region (SSC), and a pair of inverted repeat regions. In total, we annotated a range of 131 to 133 genes, encompassing 84 to 86 protein-coding genes, 38 transfer RNA (tRNA) genes, 8 ribosomal RNA (rRNA) genes, and 0 to 2 pseudogenes (ycf1, infA). Our comparative analyses unequivocally revealed a remarkable consistency in gene order and GC content within the Polygonatum genus. Furthermore, we predicted a potential 59 to 64 RNA editing sites distributed across 22 protein-coding genes, with the ndhB gene exhibiting the most prominent propensity for RNA editing sites, boasting a tally of 15 sites. Notably, six regions of substantial potential variability were ascertained, characterized by elevated Pi values. Noteworthy, molecular markers for species identification, population genetic scrutiny, and phylogenetic investigations within the genus were identified in the form of the psaJ-rpl33 and trnS + trnT-psaD barcodes. The resultant phylogenetic tree unequivocally depicted the formation of a monophyletic clade comprising species within the evolutionary framework of Liliaceae, demonstrating closer evolutionary affinities with Maianthemum, Dracaeneae, and Asparageae. This comprehensive compendium of findings collectively contributes to the advancement of molecular species identification, elucidation of phylogenetic interrelationships, and the establishment of DNA barcodes tailored to the Polygonatum species.

Comparative chloroplast genome analysis of seven extant Citrullus species insight into genetic variation, phylogenetic relationships, and selective pressure

Citrullus ecirrhosus, Citrullus rehmii, and Citrullus naudinianus are three important related wild species of watermelon in the genus Citrullus, and their morphological differences are clear, however, their chloroplast genome differences remain unknown. This study is the first to assemble, analyze, and publish the complete chloroplast genomes of C. ecirrhosus, C. rehmii, and C. naudinianus. A comparative analysis was then conducted among the complete chloroplast genomes of seven extant Citrullus species, and the results demonstrated that the average genome sizes of Citrullus is 157,005 bp, a total of 130–133 annotated genes were identified, including 8 rRNA, 37 tRNA and 85–88 protein-encoding genes. Their gene content, order, and genome structure were similar. However, noncoding regions were more divergent than coding regions, and rps16-trnQ was a hypervariable fragment. Thirty-four polymorphic SSRs, 1,271 SNPs and 234 INDELs were identified. Phylogenetic trees revealed a clear phylogenetic relationship of Citrullus species, and the developed molecular markers (SNPs and rps16-trnQ) could be used for taxonomy in Citrullus. Three genes (atpB, clpP1, and rpoC2) were identified to undergo selection and would promote the environmental adaptation of Citrullus.

Whole chloroplast genome sequences contribute to phylogenetic relatedness and cultivar identification in cacao (Theobroma cacao L.)

Cacao (Theobroma cacao L.) is considered as economic importance crop playing a major role as source of chocolate industries for production of both chocolate candy and cocoa butter. Despite the advantages of cacao in industrial sector, understanding the global structure of cacao chloroplast genome plays a crucial role in explaining phylogenetic relationships and cultivar identification. This study aimed to perform phylogenetic analysis of cacao genotypes and develop DNA barcode markers using cacao chloroplast sequences. Chloroplast genome sequencing of two cacao genotypes (DR-1 and Sca-12) was conducted by the Illumina Miseq platform. Phylogenetic analysis of 12 cacao genotypes including two genotypes sequenced in this study (DR-1 and Sca-12) and ten genotypes previously sequenced (HQ336404, EET-64, ICS-01, ICS-06, ICS-39, Pentagonum, Sca-6, Stahel, Amelonado, and Criollo-22) showed a clear separation between bulk and fine types. This result demonstrated the usefulness of chloroplast sequences in revealing phylogenetic relatedness. Based on comparative chloroplast genome analysis of two cacao genotypes, DR-1 and Sca-12, three insertion/deletion (InDel) markers named as Theca_indel01, Theca_indel02, and Theca_indel03 which designed from the regions of trnA-UGC-rrn23, trnK-UUU-rps16, and rps16 intron, respectively were successfully developed to reveal barcode system for cacao genetic discrimination. Overall, these findings would provide valuable insight into the chloroplast genome structure of cacao plant, as well as information about the benefits of chloroplast sequences for constructing phylogenetic relationships and developing DNA barcode markers.

Identification of three cultivated varieties of Scutellaria baicalensis using the complete chloroplast genome as a super-barcode

Scutellaria baicalensis has been one of the most commonly used traditional Chinese medicinal plants in China for more than 2000 years. The three new varieties cultivated could not be distinguished by morphology before flowering. It will hinder the promotion of later varieties. Chloroplast DNA has been widely used in species identification. Moreover, previous studies have shown that complete chloroplast genome sequences have been suggested as super barcodes for identifying plants. Therefore, we sequenced and annotated the complete chloroplast genomes of three cultivated varieties. The chloroplast genomes of SBW, SBR, and SBP were 151,702 bp, 151,799 bp, and 151,876 bp, which contained 85 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. The analysis of the repeat sequences, codon usage, and comparison of chloroplast genomes shared a high degree of conservation. However, the sliding window results show significant differences among the three cultivated varieties in matK-rps16 and petA-psbJ. And we found that the matK-rps16 sequence can be used as a barcode for the identification of three varieties. In addition, the complete chloroplast genome contains more variations and can be used as a super-barcode to identify these three cultivated varieties. Based on the protein-coding genes, the phylogenetic tree demonstrated that SBP was more closely related to SBW, in the three cultivated varieties. Interestingly, we found that S. baicalensis and S. rehderiana are closely related, which provides new ideas for the development of S. baicalensis. The divergence time analysis showed that the three cultivated varieties diverged at about 0.10 Mya. Overall, this study showed that the complete chloroplast genome could be used as a super-barcode to identify three cultivated varieties of S. baicalensis and provide biological information, and it also contributes to bioprospecting.

Comparative chloroplast genomics of 34 species in subtribe Swertiinae (Gentianaceae) with implications for its phylogeny

BackgroundSubtribe Swertiinae, a medicinally significant and highly speciose Subtribe of family Gentianaceae. Despite previous extensive studies based on both morphology and molecular data, intergeneric and infrageneric relationships within subtribe Swertiinae remain controversial.MethodsHere, we employed four newly generated Swertia chloroplast genomes with thirty other published genomes to elucidate their genomic characteristics.ResultsThe 34 chloroplast genomes were small and ranged in size from 149,036 to 154,365 bp, each comprising two inverted repeat regions (size range 25,069–26,126 bp) that separated large single-copy (80,432–84,153 bp) and small single-copy (17,887–18,47 bp) regions, and all the chloroplast genomes showed similar gene orders, contents, and structures. These chloroplast genomes contained 129–134 genes each, including 84–89 protein-coding genes, 37 tRNAs, and 8 rRNAs. The chloroplast genomes of subtribe Swertiinae appeared to have lost some genes, such as rpl33, rpl2 and ycf15 genes. Comparative analyses revealed that two mutation hotspot regions (accD-psaI and ycf1) could serve as effective molecular markers for further phylogenetic analyses and species identification in subtribe Swertiinae. Positive selection analyses showed that two genes (ccsA and psbB) had high Ka/Ks ratios, indicating that chloroplast genes may have undergone positive selection in their evolutionary history. Phylogenetic analysis showed that the 34 subtribe Swertiinae species formed a monophyletic clade, with Veratrilla, Gentianopsis and Pterygocalyx located at the base of the phylogenetic tree. Some genera of this subtribe, however, were not monophyletic, including Swertia, Gentianopsis, Lomatogonium, Halenia, Veratrilla and Gentianopsis. In addition, our molecular phylogeny was consistent with taxonomic classification of subtribe Swertiinae in the Roate group and Tubular group. The results of molecular dating showed that the divergence between subtrib Gentianinae and subtrib Swertiinae was estimated to occur in 33.68 Ma. Roate group and Tubular group in subtribe Swertiinae approximately diverged in 25.17 Ma.ConclusionOverall, our study highlighted the taxonomic utility of chloroplast genomes in subtribe Swertiinae, and the genetic markers identified here will facilitate future studies on the evolution, conservation, population genetics, and phylogeography of subtribe Swertiinae species.

Complete chloroplast genome sequences of Myristicaceae species with the comparative chloroplast genomics and phylogenetic relationships among them.

Myristicaceae was widly distributed from tropical Asia to Oceania, Africa, and tropical America. There are 3 genera and 10 species of Myristicaceae present in China, mainly distributed in the south of Yunnan Province. Most research on this family focuses on fatty acids, medicine, and morphology. Based on the morphology, fatty acid chemotaxonomy, and a few of molecular data, the phylogenetic position of Horsfieldia pandurifolia Hu was controversial. In this study, the chloroplast genomes of two Knema species, Knema globularia (Lam.) Warb. and Knema cinerea (Poir.) Warb., were characterized. Comparing the genome structure of these two species with those of other eight published species, including three Horsfieldia species, four Knema species, and one Myristica species, it was found that the chloroplast genomes of these species were relatively conserved, retaining the same gene order. Through sequence divergence analysis, there were 11 genes and 18 intergenic spacers were subject to positive selection, which can be used to analyze the population genetic structure of this family. Phylogenetic analysis showed that all Knema species were clustered in the same group and formed a sister clade with Myristica species support by both high maximum likelihood bootstrap values and Bayesian posterior probabilities; among Horsfieldia species, Horsfieldia amygdalina (Wall.) Warb., Horsfieldia kingii (Hook.f.) Warb., Horsfieldia hainanensis Merr. and Horsfieldia tetratepala C.Y.Wu. were grouped together, but H. pandurifolia formed a single group and formed a sister clade with genus Myristica and Knema. Through the phylogenetic analysis, we support de Wilde' view that the H. pandurifolia should be separated from Horsfieldia and placed in the genus Endocomia, namely Endocomia macrocoma subsp. prainii (King) W.J.de Wilde. The findings of this study provide a novel genetic resources for future research in Myristicaceae and provide a molecular evidence for the taxonomic classification of Myristicaceae.