- To date, the chloroplast genome sequences of 4 genera of the tribe Senecioneae have been uploaded to GenBank.. - The internal relationships within the genus Gynura and the relationship of the genus Gynura with other genera in the tribe Senecioneae need further research.. - Results: The chloroplast genomes of 4 Gynura species were sequenced, assembled and annotated. - In comparison with those of 12 other Senecioneae species, the Gynura chloroplast genome features were analysed in detail. - Subsequently, differences in the microsatellite and repeat types in the tribe were found. - From the comparison, it was found that IR expansion and contraction are conserved in the genera Gynura, Dendrosenecio and Ligularia. - The whole chloroplast genome sequences of 16 Senecioneae species were used to build a phylogenetic tree. - Conclusions: Sequencing the chloroplast genomes of 4 Gynura species helps us to solve many problems. - The phylogenetic relationship of the genera Gynura and Ligularia was different from that observed previous work. - In a previous phylogenetic tree, the genus Ligularia belonged to the Tussilagininae subtribe, which was in a lineage that diverged earlier than other genera. - Further morphology and genome-wide analyses are needed to clarify the genus relationships.. - Gynura is a genus of flowering plants in the tribe Senecio- neae of the family Asteraceae endemic to Asia, which con- tains 44 species in total [1]. - Many species of the genus Gynura have been reported to have medicinal value for diabetes mellitus, such as G. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Some plants in the genus Gynura have also been used as vegetables and tea in people’s daily lives in East and South Asia. - thus, there is value in studying the genus Gynura. - The phylogenetic relationship is useful information for interspecific hybridizations, but the phylo- genetic relationship of the species in the genus Gynura is, as yet, unclear.. - The chloroplast genomes of angiosperms generally show slow substitution rates under adaptive evolution [12]. - Considering its small size, conserved gene content and simple structure, the chloroplast genome is valid and cost-effective for studying phylogenetic relationships and the evolution of plants in different taxa. - In the present study, we sequenced, assembled and an- notated the chloroplast genomes of four Gynura species.. - Combined with chloroplast genomes of the genus Den- drosenecio, genus Jacobaea, genus Ligularia and genus Pericallis of the tribe Senecioneae, the structure features, repeat motifs, adaptive selection, phylogenetic relation- ships and divergence time were analysed.. - Chloroplast genome features of 16 Senecioneae species In this study, we sequenced and assembled the chloro- plast genome of Gynura bicolor, Gynura divaricata, Gynura formosana and Gynura pseudochina. - The genus Gynura belongs to the tribe Senecioneae, which is the largest tribe of the family Asteraceae. - Although the tribe comprises approximately 500 genera and 3000 species [17], we found that only 4 genera of the tribe Senecio- neae had published chloroplast genomes in GenBank, and their IDs are listed in the methods. - Five species of the genus Dendrosenecio, one species of the genus Jacobaea, five species of the genus Ligularia, one species of the genus Pericallis and four species of the genus Gynura were used to find their similarities and differ- ences. - With the typical quadripartite parts, such as most land plants, the chloroplast genome has one large single copy (LSC), one short single copy (SSC), and two inverted regions (IRa and IRb) (Fig. - vulgaris has the shortest chloroplast genome length, but its SSR region is longer than that of 4 Gynura species. - In addition, there are 95 coding genes in the chloroplast genome of P. - Only the rRNA number is conserved in the chloroplast genome of the tribe Senecioneae, which is the same as that of the families Adoxaceae and Caprifoliaceae [16] but different from that of the genera Oresitrophe and Mukdenia [15].. - The number of microsatellites with mono-, di- and trinu- cleotide repeat motifs varies in the tribe. - Han et al. - is consistent with that in the four Gynura species, but the number of each repeat type is different. - In addition, the forward and palindromic repeat numbers are similar in the Oresitrophe species.. - The chloroplast genome is highly conserved in land plants, but IR expansion and contraction lead to different genome sizes in different plants [18]. - IRa/LSC border and adjacent genes of 16 species of the Senecioneae tribe were carefully analysed to find similar- ities and differences (Fig. - The rps19 and rpl2 genes are located in the LSC/IRb and IRa/LSC borders in pairs. - The start position is just on the border in the four Gynura species, but the others also span the border of IRa/LSC. - By comparison, IR expansion and contraction are conserved in the genera Gynura, Dendrosenecio and Ligularia.. - The whole chloroplast genome sequences of 16 Senecio- neae species were aligned by the MAFFT program to find sequence variation. - This result shows that this region is not conserved, similar to other regions of the chloroplast genome. - For further analysis of the re- sults, the chloroplast genome sequences of four Gynura. - 1 Chloroplast genome map of Gynura divaricata. - The colors of different genes correspond to different functional groups in the legend. - b Frequency of unit size Table 2 Overview of chloroplast genome of 16 Senecioneae species. - The four Gynura species are conserved in the 25,000 bp to 50,000 bp region and are similar to L.. - The function of the NAD(P) H dehydrogen- ase (NDH) complex is well known in photosystem I (PSI) cyclic electron flow (CEF) and chlororespiration [19, 20], so the substitution of ndh genes was further studied. - The ratio of the non-synonymous (dN)/synonymous substitution (dS) rate was calculated by the PAML program. - In the ML tree, two major clades were constructed with a 100% bootstrap value. - In the genus Gynura, G. - The former systemic phylogenies of the tribe Senecioneae based on the ITS region (nuclear) and plastid fragment sequences show a significant difference from the phylogenetic tree [17]. - In a previous phylogenetic tree, the genus Ligularia belongs to the Tussilagininae subtribe, which was in lineage that diverged earlier than other genera. - The sequence is relatively conserved among four Gynura species and five Ligularia species, and the Pi value of most sequence locations is below 0.1 (Additional file 3: Figure S3), which is significantly lower than that of the 16 species alignment. - From the perspective of whole chloroplast genomes, the genus Ligularia is close to the genus Gynura.. - 3 Schematic representation of the border positions of LSC, IRs and SSC in the chloroplast genome of 16 Senecioneae species. - For the divergence time estimation of the 16 Senecio- neae species, Artemisia gmelinii and Chrysanthemum boreale (tribe Anthemideae) were selected as the out- group due to the oldest Artemisia fossil pollen [21, 22].. - The divergence time of the tribes Senecioneae and Anthemideae was 51.39 mya (early Eocene), and the result was consistent. - with that of a previous study on the evolution and phylogenetic of the family Asteroideae based on plastid fragment sequences [22]. - The traditional view on diver- gence time of the genus Gynura is in the Old World after the Atlantic opening. - The divergence time of Gynura spe- cies was approximately 0.3 mya, and the result showed that the divergence time of the genus Gynura was much earlier than that of the traditional view. - The divergence time of the genus Gynura could not start at hundreds or thousands of years ago [23], and the divergence time es- timated by the BEAST program was in the same time period as that of other genera of land plants [13–16].. - 4 Sequence divergence of chloroplast genome sequences in 7 Senecioneae species. - a The Pi value (nucleotide diversity) of the 7 chloroplast genome sequences. - This study analyses the chloroplast genome of four Gynura species used as herbal medicine in parts of Asia.. - By comparing with other plants in the tribe Senecioneae, the repeat motifs, detailed structure features, phylogenetic. - The phylogenetic relationships of the genera Gynura and Ligularia and others are still in doubt. - 5 Maximum-likelihood (ML) phylogenetic tree obtained for 16 Senecioneae species based on the whole chloroplast genome sequences.. - Morphology and genome-wide analyses are needed to further clarify the genus relationships. - Bingru Ren, and the specimens were de- posited in the Herbarium of Institute of Botany, Jiangsu Province and Chinese Academy of Sciences.. - Approximately 1.5 μg of the DNA sample was frag- mented by sonication to a size of 350 bp. - The Perl script NOVOPlasty was used to as- semble the chloroplast genome sequence with a 50 K-mer.. - The chloroplast genome sequence of Dendrosenecio cher- anganiensis (tribe Senecioneae) was selected as the refer- ence genome. - The family Asteraceae plant sequences used in the study were downloaded from GenBank as follows:. - Chloroplast genome annotation. - The whole chloroplast genome sequences were anno- tated by Dual Organellar Genome Annotator [26] and GeSeq [27] with default parameters. - Chloroplast genome sequences of tribe Senecioneae plants Dendrosenecio cheranganiensis and Pericallis hybrida were used as ref- erence sequences. - A schematic diagram of the chloroplast genome with an- notations was obtained by OGDRAW [30].. - The Perl script Microsatellite identification tool (MISA, http://pgrc.ipk-gatersleben.de/misa/misa.html) was used to find the microsatellite regions of the chloroplast genome.. - Chloroplast genome analysis. - All the chloroplast genome sequences were aligned by MAFFT7.427 [34] on the FFT-NS-2 module. - The differ- ent chloroplast genome sequences (LSC, SSC, IRa and IRb) concatenated together to make one sequence per species. - The 16 chloroplast genome sequences of the tribe Sene- cioneae (family Asteraceae) were aligned by MAFFT, and the results were used to analyse the phylogenetic relation- ships. - Both the re- sults of the ML tree and BI tree were visualized by FigTree V1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).. - Bayesian inference (BI) phylogenetic tree obtained for 16 Senecioneae species based on the whole chloroplast genome sequences. - The Pi value (nucleotide diversity) of chloroplast genome sequences between four Gynura species and five Ligularia species.. - 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