Analysis of the complete plastomes of Albizia kalkora (Roxb.) Prain 1897 (Fabaceae).

Albizia kalkora (Roxb.) Prain 1897, belonging to the family Fabaceae, is not only a landscape tree but also a medicinal plant. At present, few plastomes have been reported from Albizia, which delays the in-depth phylogenomic studies and the development of high-resolution discriminating markers for this genus. Herein, we sequenced the first plastome of A. kalkora by NGS technology. The genome is a circular structure (176,158 bp), containing a large single-copy (LSC) region (91,521 bp), a small copy (SSC) region (5237 bp), and two inverted repeat (IR) regions (39,700 bp each). It has 35.45% GC content and encodes 109 unique genes, which are 76 protein-coding, 4 rRNA, and 29 tRNA genes. The genetic distance analysis of the intergenic spacer regions for A. kalkora, A. odoratissima and A. bracteate shows four intergenic regions with very high K2p values, namely, ccsA-ndhD (15.04), matK-rps16 (10.77), rps11-rpl36 (17.63) and rps3-rps19 (20.08), which can discriminate the three Albizia species. In addition, we identified ten pairs of regions that could be utilized to design primers to discriminate the three Albizia species. The phylogenetic analysis showed Albizia was closely related to Samanea. The results in this study will provide valuable information to elucidate the classification, identification and evolutionary history of Albizia.

Mitochondrial genome of Artemisia argyi L. suggested conserved mitochondrial protein-coding genes among genera Artemisia, Tanacetum and Chrysanthemum.

BACKGROUND: Artemisia argyi L., also known as mugwort, is a perennial herb whose leaves are commonly used as a source of traditional medicines. However, the evolution and structure of the mitochondrial genome (mitogenome) in A. argyi remain unclear. In this study, the mitogenome of A. argyi was assembled and characterized for the first time. RESULTS: The mitogenome of A. argyi was a circular molecule of 229,354 bp. It encodes 56 genes, including 33 protein-coding genes (PCGs), 20 tRNA genes, and three rRNA genes, and three pseudogenes. Five trans-spliced introns were observed in three PCGs namely, nad1, nad2 and nad5. Repeat analysis identified 65 SSRs, 14 tandem repeats, and 167 dispersed repeats. The A. argyi mitogenome contains 12 plastid transfer sequences from 79 bp to 2552 bp. Five conserved MTPTs were identified in all 18 Asteraceae species. Comparison of mitogenome between A. argyi and one Artemisia specie and two Chrysanthemum species showed 14 conserved gene clusters. Phylogenetic analysis with organelle genomes of A. argyi and 18 other Anthemideae plants showed inconsistent phylogenetic trees, which implied that the evolutionary rates of PCGs and rrna genes derived from mitochondrion and plastid were incongruent. The Ka/Ks ratio of the 27 shared protein-coding genes in the 18 Anthemideae species are all less than 1 indicating that these genes were under the effect of purifying selection. Lastly, a total of 568 RNA editing sites in PCGs were further identified. The average editing frequency of non-synonymous changes was significantly higher than that of synonymous changes (one-sample Student's t-test, p-values ≤ 0.05) in three tissues (root, leaf and stem). CONCLUSIONS: In this study, the gene content, genome size, genome comparison, mitochondrial plastid sequences, dN/dS analysis of mitochondrial protein-coding genes, and RNA-editing events in A. argyi mitogenome were determined, providing insights into the phylogenetic relationships of Asteraceae plant.

De Novo Hybrid Assembly of the Salvia miltiorrhiza Mitochondrial Genome Provides the First Evidence of the Multi-Chromosomal Mitochondrial DNA Structure of Salvia Species.

Salvia miltiorrhiza has been an economically important medicinal plant. Previously, an S. miltiorrhiza mitochondrial genome (mitogenome) assembled from Illumina short reads, appearing to be a single circular molecule, has been published. Based on the recent reports on the plant mitogenome structure, we suspected that this conformation does not accurately represent the complexity of the S. miltiorrhiza mitogenome. In the current study, we assembled the mitogenome of S. miltiorrhiza using the PacBio and Illumina sequencing technologies. The primary structure of the mitogenome contained two mitochondrial chromosomes (MC1 and MC2), which corresponded to two major conformations, namely, Mac1 and Mac2, respectively. Using two approaches, including (1) long reads mapping and (2) polymerase chain reaction amplification followed by Sanger sequencing, we observed nine repeats that can mediate recombination. We predicted 55 genes, including 33 mitochondrial protein-coding genes (PCGs), 3 rRNA genes, and 19 tRNA genes. Repeat analysis identified 112 microsatellite repeats and 3 long-tandem repeats. Phylogenetic analysis using the 26 shared PCGs resulted in a tree that was congruent with the phylogeny of Lamiales species in the APG IV system. The analysis of mitochondrial plastid DNA (MTPT) identified 16 MTPTs in the mitogenome. Moreover, the analysis of nucleotide substitution rates in Lamiales showed that the genes atp4, ccmB, ccmFc, and mttB might have been positively selected. The results lay the foundation for future studies on the evolution of the Salvia mitogenome and the molecular breeding of S. miltiorrhiza.

Characterization of Codonopsis pilosula subsp. tangshen plastome and comparative analysis of Codonopsis species.

Codonopsis pilosula subsp. tangshen is one of the most important medicinal herbs used in traditional Chinese medicine. Correct identification of materials from C. pilosula subsp. tangshen is critical to ensure the efficacy and safety of the associated medicines. Traditional DNA molecular markers could distinguish Codonopsis species well, so we need to develop super or specific molecular markers. In this study, we reported the plastome of Codonopsis pilosula subsp. tangshen (Oliv.) D.Y. Hong conducted phylogenomic and comparative analyses in the Codonopsis genus for the first time. The entire length of the Codonopsis pilosula subsp. tangshen plastome was 170,672 bp. There were 108 genes in the plastome, including 76 protein-coding genes, 28 transfer RNA (tRNA), and four ribosomal RNA (rRNA) genes. Comparative analysis indicated that Codonopsis pilosula subsp. tangshen had an unusual large inversion in the large single-copy (LSC) region compared with the other three Codonopsis species. And there were two dispersed repeat sequences at both ends of the inverted regions, which might mediate the generation of this inversion. We found five hypervariable regions among the four Codonopsis species. PCR amplification and Sanger sequencing experiments demonstrated that two hypervariable regions could distinguish three medicinal Codonopsis species. Results obtained from this study will support taxonomic classification, discrimination, and molecular evolutionary studies of Codonopsis species.

The complete chloroplast genome sequence of Clerodendranthus spicatus, a medicinal plant for preventing and treating kidney diseases from Lamiaceae family.

BACKGROUND: Clerodendranthus spicatus (Thunb.) C. Y. Wu ex H. W. Li is one of the most important medicines for the treatment of nephrology in the southeast regions of China. To understand the taxonomic classification of Clerodendranthus species and identify species discrimination markers, we sequenced and characterized its chloroplast genome in the current study. METHODS AND RESULTS: Total genomic DNA were isolated from dried leaves of C. spicatus and sequenced using an Illumina sequencing platform. The data were assembled and annotated by the NOVOPlasty software and CpGAVAS2 web service. The complete chloroplast genome of C. spicatus was 152,155 bp, including a large single-copy region of 83,098 bp, a small single-copy region of 17,665 bp, and a pair of inverted repeat regions of 25,696 bp. The Isoleucine codons are the most abundant, accounting for 4.17% of all codons. The codons of AUG, UUA, and AGA demonstrated a high degree of usage bias. Twenty-eight simple sequence repeats, thirty-six tandem repeats, and forty interspersed repeats were identified. The distribution of the specific rps19, ycf1, rpl2, trnH, psbA genes were analyzed. Analysis of the genetic distance of the intergenic spacer regions shows that ndhG-ndhI, accD-psaI, rps15-ycf1, rpl20-clpP, ccsA-ndhD regions have high K2p values. Phylogenetic analysis showed that C. spicatu is closely related to two Lamiaceae species, Tectona grandis, and Glechoma longituba. CONCLUSIONS: In this study, we sequenced and characterized the chloroplast genome of C. spicatus. Phylogenomic analysis has identified species closely related to C. spicatus, which represent potential candidates for the development of drugs improving renal functions.

Phylogenetic analysis and development of molecular markers for five medicinal Alpinia species based on complete plastome sequences.

BACKGROUND: Alpinia species are widely used as medicinal herbs. To understand the taxonomic classification and plastome evolution of the medicinal Alpinia species and correctly identify medicinal products derived from Alpinia species, we systematically analyzed the plastome sequences from five Alpinia species. Four of the Alpinia species: Alpinia galanga (L.) Willd., Alpinia hainanensis K.Schum., Alpinia officinarum Hance, and Alpinia oxyphylla Miq., are listed in the Chinese pharmacopeia. The other one, Alpinia nigra (Gaertn.) Burtt, is well known for its medicinal values. RESULTS: The four Alpinia species: A. galanga, A. nigra, A. officinarum, and A. oxyphylla, were sequenced using the Next-generation sequencing technology. The plastomes were assembled using Novoplasty and annotated using CPGAVAS2. The sizes of the four plastomes range from 160,590 bp for A. galanga to 164,294 bp for A. nigra, and display a conserved quadripartite structure. Each of the plastomes encodes a total of 111 unique genes, including 79 protein-coding, 28 tRNA, and four rRNA genes. In addition, 293-296 SSRs were detected in the four plastomes, of which the majority are mononucleotides Adenine/Thymine and are found in the noncoding regions. The long repeat analysis shows all types of repeats are contained in the plastomes, of which palindromic repeats occur most frequently. The comparative genomic analyses revealed that the pair of the inverted repeats were less divergent than the single-copy region. Analysis of sequence divergence on protein-coding genes showed that two genes (accD and ycf1) had undergone positive selection. Phylogenetic analysis based on coding sequence of 77 shared plastome genes resolves the molecular phylogeny of 20 species from Zingiberaceae. In particular, molecular phylogeny of four sequenced Alpinia species (A. galanga, A. nigra, A. officinarum, and A. oxyphylla) based on the plastome and nuclear sequences showed congruency. Furthermore, a comparison of the four newly sequenced Alpinia plastomes and one previously reported Alpinia plastomes (accession number: NC_048461) reveals 59 highly divergent intergenic spacer regions. We developed and validated two molecular markers Alpp and Alpr, based on two regions: petN-psbM and psaJ-rpl33, respectively. The discrimination success rate was 100 % in validation experiments. CONCLUSIONS: The results from this study will be invaluable for ensuring the effective and safe uses of Alpinia medicinal products and for the exploration of novel Alpinia species to improve human health.

Comparative genome analysis revealed gene inversions, boundary expansions and contractions, and gene loss in the Stemona sessilifolia (Miq.) Miq. chloroplast genome.

Stemona sessilifolia (Miq.) Miq., commonly known as Baibu, is one of the most popular herbal medicines in Asia. In the Chinese Pharmacopoeia, Baibu has multiple authentic sources and there are many similar herbs sold as Baibu in herbal medicine markets. The existence of counterfeits of Baibu brings challenges to its identification. To assist in its accurate identification, we sequenced and analyzed the complete chloroplast genome of S. sessilifolia using next-generation sequencing technology. The genome was found to be 154,037 bp in length, possessing a typical quadripartite structure consisting of a pair of inverted repeats (IRs: 27,090 bp) separated by a large single copy (LSC: 81,949 bp) and a small single copy (SSC: 17,908 bp). A total of 112 unique genes were identified, including 80 protein-coding, 28 transfer RNA and four ribosomal RNA genes. In addition, 45 tandem, 27 forward, 23 palindromic and 104 simple sequence repeats were detected in the genome by repeated analysis. Compared with its counterfeits (Asparagus officinalis and Carludovica palmata) we found that IR expansion and SSC contraction events of S. sessilifolia resulted in two copies of the rpl22 gene in the IR regions and a partial duplication of the ndhF gene in the SSC region. An approximately 3-kb-long inversion was also identified in the LSC region, leading to the petA and cemA genes being presented in the complementary strand of the chloroplast DNA molecule. Comparative analysis revealed some highly variable regions, including trnF-GAA_ndhJ, atpB_rbcL, rps15_ycf1, trnG-UCC_trnR-UCU, ndhF_rpl32, accD_psaI, rps2_rpoC2, trnS-GCU_trnG-UCC, trnT-UGU_trnL-UAA and rps16_trnQ-UUG. Finally, gene loss events were investigated in the context of phylogenetic relationships. In summary, the complete plastome of S. sessilifolia will provide valuable information for the distinction between Baibu and its counterfeits and assist in elucidating the evolution of S. sessilifolia.

Comparative analysis of the complete chloroplast genomes of Cirsium japonicum from China and Korea.

Cirsium japonicum (C. japonicum) is a traditional Chinese medicine belonging to the family Asteraceae. The previous studies have indicated that the chemical compound content of C. japonicum from different places was different. To distinguish C. japonicum from different geographies, the chloroplast genome of C. japonicum from China was sequenced and compared with that from Korea. The total length of this genome is 152,602 bp, similar to that of Korea (152,606 bp). It has a conservative quartile structure which is composed of a large single-copy (LSC) region, a small single-copy (SSC) region and a pair of inverted repeats (IRs) regions, with lengths of 83,487 bp, 18,721 bp, and 25,197 bp, respectively. It encodes 79 protein-coding, 27 transfer RNAs, and 4 ribosomal RNA genes. The overall GC content of the genome is 37.70%. A total of 20 single nucleotide polymorphisms and 6 insertions and deletions were identified between the chloroplast genome of C. japonicum from China and Korea. These results can be applied to develop molecular markers to distinguish C. japonicum from different geographical origins.

Aconitine induces cardiotoxicity through regulation of calcium signaling pathway in zebrafish embryos and in H9c2 cells.

Fuzi, the processed lateral roots of Aconitum carmichaelii Debx., is a traditional herbal medicine that is well known for its excellent pharmacological effects and acute toxicity. Aconitine is one of the diester-diterpene alkaloids and well-known for its arrhythmogenic effects. However, the effects of aconitine in zebrafish have rarely been studied. Therefore, we investigated the effects of aconitine on zebrafish embryos and H9c2 cells. Zebrafish embryos at 48 hours postfertilization were exposed to aconitine, and then, cardiac function and apoptosis were measured. Through transcriptomic analysis, the cardiotoxicity of aconitine in zebrafish embryos was involved in regulating Ca2+ signal pathways. A reverse transcription-polymerase chain reaction was performed to verify the expression of Ca2+ pathway-related genes after 12, 24, 36 and 48 hours of treatment. Meanwhile, intracellular Ca2+ concentrations and cell apoptosis were observed in H9c2 cells treated with half-maximal inhibitory concentration values of aconitine for 30 minutes. The protein levels of troponin T (TnT), caspase 3, Bcl-2 and Bax were detected by western blot analysis. In vivo, 2.0 and 8.0 µm aconitine decreased the heart rate and inhibited the contraction of ventricles and atria in a dose- and time-dependent manner. Furthermore, aconitine increased expression of cacna1c, RYR2, atp2a2b, Myh6, troponin C, p38, caspase 3, Bcl-2 and Bax for 12 hours. In vitro, 1.5 and 4.5 mm aconitine caused intracellular Ca2+ ion oscillation, increased rates of apoptosis, inhibited TnT and Bcl-2 protein expression, and promoted caspase 3 and Bax protein expression. These data confirmed that aconitine at various concentrations induced cardiac dysfunction and apoptosis were related to the Ca2+ signaling pathway.

Phylogenetic relationships of Atractylodes lancea, A. chinensis and A. macrocephala, revealed by complete plastome and nuclear gene sequences.

Atractylodes lancea, A. chinensis, and A. macrocephala are the three most widely used medicinal species of the Atractylodes genus. Their similar morphological features cause disagreement as whether they are three unique species, leading to their frequent misuses in medical products. Our study aimed to understand their relationships through both the complete plastome sequences and nuclear sequences, to identify molecular markers for their differentiation and explore the evolutionary relationships among three species. We sequenced, annotated, and analyzed the plastomes of these three species. The plastomes are 153,201, 153,258, and 153,265 bps in length for A. lancea, A. chinensis, and A. macrocephaly, respectively. Similar to other Asteraceae species, their plastomes exhibit typical quadripartite structures. Each plastome consists of 119 distinct genes, including 78 protein-coding, 37 tRNA, and 4 rRNA genes. Analyses of indels, single-nucleotide polymorphisms and simple sequence repeats, and comparison of plastomes showed high degree of conservation, leading to difficulty in the discovery of differentiating markers. We identified eleven potential molecular markers using an algorithm based on interspecific and intraspecific nucleotide diversity gaps. Validation experiments with fifty-five individuals from the three species collected from the botanical garden and fields confirmed that the marker cz11 could effectively distinguish samples from the three different species. Analysis of the several nuclear sequences suggests that the species of A. macrocephala may be a hybrid of A. lancea and A. chinensis. In summary, the results from this study highlight the complex relationships among of these three medicinal plants.

High level of intraspecific divergence and low frequency of RNA editing in the chloroplast genome sequence of Tagetes erecta.

Tagetes erecta L. is an important commercial and medicinal plant. In this study, we reported the complete chloroplast genome sequence of T. erecta. The genome has a circular structure of 152,076 bp containing a large single-copy region (LSC) of 83,914 bp, a small copy region (SSC) of 18,064 bp, and two inverted repeats (IR) of 25,049 bp by each. It harbors 111 unique genes, including 79 protein-coding genes, 4 ribosomal RNA genes, and 28 transfer RNA genes. A total of 41 microsatellite, 20 tandem, and 37 interspersed repeats were detected in the genome. The phylogenomic analysis shows that T. erecta is a single phylogenetic cluster. The complete chloroplast genome of T. erecta lays the foundation for the phylogenetic, evolutionary, and conservation studies of the genus Tagetes. Furthermore, the intergenic region of atpB-rbcL was variable among the species T. erecta. This suggests that this region might be a mutation hotspot and will be useful for phylogenetic study and the development of molecular markers. At last, we systematically identified the RNA editing sites in the chloroplast genome of T. erecta based on the transcriptome downloaded from the SRA database. This study identified the characteristics of the T. erecta chloroplast genome, SNPs, and RNA editing sites, which will facilitate species identification and phylogenetic analysis within T. erecta.

The complete chloroplast genome sequence of Hemerocallis fulva.

Hemerocallis fulva L. is a traditional Chinese medicine. The flowers of H. fulva are used in ethnic medicine to treat various diseases, including certain central nervous system diseases. In this study, we characterized the complete chloroplast genome of H. fulva. It is 156,059 bp in length and encodes 87 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. The phylogenomic analysis showed that the H. fulva and species of Anemarrhena asphodeloides Bunge, Liriope muscari, and Liriope spicata were clustered together. This chloroplast genome sequencing offers genetic background for conservation and phylogenetic studies.

[Transcriptomics study on mechanism of Aconiti Lateralis Radix Praeparata in treatment of rats with acute heart failure].

In this study,transcriptomics technique was used to investigate the mechanism of action of Aconiti Lateralis Radix Praeparata on acute heart failure rats induced by propafenone hydrochloride.First,rats were randomly divided into normal group,model group and administration group(1.25,2.5,5 g·kg-1).A rat with acute heart failure was constructed by intravenous femoral administration of proparone hydrochloride.The changes of heart rate,+dp/dtmaxand-dp/dtmaxat 5,10,20,30 and 60 min were recorded.Then another group of rats were given the same drug delivery method.In another group of animals,serum TNF-α could be determined by ELISA with the same dosage method.High-throughput sequencing technology was used to detect all gene expression differences in cardiac tissue samples of rats with acute heart failure.Through functional annotation and enrichment analysis,gene expression signaling pathways of rats with acute heart failure and rats with post-administration heart failure were screened out.The results showed that heart rate and LV+dp/dtmaxand LV-dp/dtmaxwere significantly decreased in the model group(P<0.05),while heart rate and LV+dp/dtmax and LV-dp/dtmaxwere significantly increased in the drug group(P<0.05,P<0.01).Moreover,ANP,BNP and TNF-α in acute heart failure rats was significantly decreased in high-dose aconite decoction group(P<0.05).Transcriptomics analysis showed that the mechanism of action was mainly related to activation of PI3 K-AKT signaling pathway and Jak-STAT pathway.Compared with the model group,aconite decoction up-regulated the expression of phosphatidylinostol 3-kinase(PI3 K),lysophosphatidic acid(LAP3),Bcl-3 and STAT genes,and down-regulated the expression of integrin(ITGA),nuclear orphan receptor(Nur77) genes.It could be concluded that the mechanism of aconite in treating acute heart failure rats may be related to the regulation of the PI3 k-Akt/Jak-STAT pathway.

The complete chloroplast genome sequence of the medicinal plant Siegesbeckia orientalis L., the first in the genus Siegesbeckia.

Siegesbeckia orientalis L. is a plant with important medicinal and economic values. We reported the first complete chloroplast genome sequence of S. orientalis. This genome is 151,821 bp in length and comprises a large single-copy region of 83,540 bp, a small single-copy region of 18,225 bp and a pair of inverted repeat regions of 25,028 bp each. It encodes 138 genes, including 79 protein-coding genes, 27 tRNA genes, and 4 rRNA genes. The phylogenomic analysis showed that S. orientalis and Guizotia abyssinia were clustered together. This genomewill lay the foundation for the molecular discovery and phylogenomic study of this genus.

Understanding the molecular mechanisms underlying the effects of light intensity on flavonoid production by RNA-seq analysis in Epimedium pseudowushanense B.L.Guo.

Epimedium pseudowushanense B.L.Guo, a light-demanding shade herb, is used in traditional medicine to increase libido and strengthen muscles and bones. The recognition of the health benefits of Epimedium has increased its market demand. However, its resource recycling rate is low and environmentally dependent. Furthermore, its natural sources are endangered, further increasing prices. Commercial culture can address resource constraints of it.Understanding the effects of environmental factors on the production of its active components would improve the technology for cultivation and germplasm conservation. Here, we studied the effects of light intensities on the flavonoid production and revealed the molecular mechanism using RNA-seq analysis. Plants were exposed to five levels of light intensity through the periods of germination to flowering, the flavonoid contents were measured using HPLC. Quantification of epimedin A, epimedin B, epimedin C, and icariin showed that the flavonoid contents varied with different light intensity levels. And the largest amount of epimedin C was produced at light intensity level 4 (I4). Next, the leaves under the treatment of three light intensity levels ("L", "M" and "H") with the largest differences in the flavonoid content, were subjected to RNA-seq analysis. Transcriptome reconstruction identified 43,657 unigenes. All unigene sequences were annotated by searching against the Nr, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. In total, 4008, 5260, and 3591 significant differentially expressed genes (DEGs) were identified between the groups L vs. M, M vs. H and L vs. H. Particularly, twenty-one full-length genes involved in flavonoid biosynthesis were identified. The expression levels of the flavonol synthase, chalcone synthase genes were strongly associated with light-induced flavonoid abundance with the highest expression levels found in the H group. Furthermore, 65 transcription factors, including 31 FAR1, 17 MYB-related, 12 bHLH, and 5 WRKY, were differentially expressed after light induction. Finally, a model was proposed to explain the light-induced flavonoid production. This study provided valuable information to improve cultivation practices and produced the first comprehensive resource for E. pseudowushanense transcriptomes.

Iridoids from the roots of Valeriana jatamansi Jones.

Five iridoids, named as chlorovaltrate P-T, together with six known analogues, (4ß,8ß)-8-methoxy-3-methoxy-10-methylene-2,9-dioxatricyclo[4.3.1.03,7]decan-4-ol, chlorovaltrate A, (1R,3R,5R,7S,8R,9S)-3,8-epoxy-1-O-ethyl-5-hydroxyvalechlorine, 8-methoxy-4-acetoxy-3-chlormethyl-10-methylen-2,9-dioxa-tricyclo[4.3.1.03,7]decan, (1S,3R,5R,7S,8R,9S)-3,8-epoxy-1-O-ethyl-5-hydroxyvalechlorine, (1R,3R,5R,7S,8R,9S)-3,8-epoxy-1-O-methyl-5-hydroxyvalechlorine were isolated from the roots of Valeriana jatamansi (syn. Valeriana wallichii). Their structures were elucidated by extensive analysis of 1D, 2D NMR and HRESIMS spectroscopic. The absolute configuration of chlorovaltrate P-T were established by comparing their experimental and calculated electronic circular dichroism (ECD) spectra. 3,8-epoxy iridoids exhibited weak cytotoxicity against the lung adenocarcinoma (A 549) and gastric carcinoma cells (SGC 7901). Some also showed moderate neuroprotective effects against CoCl2-induced neuronal cell death in PC12 cells.

Identification of Symmetrical RNA Editing Events in the Mitochondria of Salvia miltiorrhiza by Strand-specific RNA Sequencing.

Salvia miltiorrhiza is one of the most widely-used medicinal plants. Here, we systematically analyzed the RNA editing events in its mitochondria. We developed a pipeline using REDItools to predict RNA editing events from stand-specific RNA-Seq data. The predictions were validated using reverse transcription, RT-PCR amplification and Sanger sequencing experiments. Putative sequences motifs were characterized. Comparative analyses were carried out between S. miltiorrhiza, Arabidopsis thaliana and Oryza sativa. We discovered 1123 editing sites, including 225 "C to U" sites in the protein-coding regions. Fourteen of sixteen (87.5%) sites were validated. Three putative DNA motifs were identified around the predicted sites. The nucleotides on both strands at 115 of the 225 sites had undergone RNA editing, which we called symmetrical RNA editing (SRE). Four of six these SRE sites (66.7%) were experimentally confirmed. Re-examination of strand-specific RNA-Seq data from A. thaliana and O. sativa identified 327 and 369 SRE sites respectively. 78, 20 and 13 SRE sites were found to be conserved among A. thaliana, O. sativa and S. miltiorrhiza respectively. This study provides a comprehensive picture of RNA editing events in the mitochondrial genome of S. miltiorrhiza. We identified SREs for the first time, which may represent a universal phenomenon.

Intraspecific and heteroplasmic variations, gene losses and inversions in the chloroplast genome of Astragalus membranaceus.

Astragalus membranaceus is an important medicinal plant in Asia. Several of its varieties have been used interchangeably as raw materials for commercial production. High resolution genetic markers are in urgent need to distinguish these varieties. Here, we sequenced and analyzed the chloroplast genome of A. membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao using the next generation DNA sequencing technology. The genome was assembled using Abyss and then subjected to gene prediction using CPGAVAS and repeat analysis using MISA, Tandem Repeats Finder, and REPuter. Finally, the genome was subjected phylogenetic and comparative genomic analyses. The complete genome is 123,582 bp long, containing only one copy of the inverted repeat. Gene prediction revealed 110 genes encoding 76 proteins, 30 tRNAs, and four rRNAs. Five intra-specific hypermutation loci were identified, three of which are heteroplasmic. Furthermore, three gene losses and two large inversions were identified. Comparative genomic analyses demonstrated the dynamic nature of the Papilionoideae chloroplast genomes, which showed occurrence of numerous hypermutation loci, frequent gene losses, and fragment inversions. Results obtained herein elucidate the complex evolutionary history of chloroplast genomes and have laid the foundation for the identification of genetic markers to distinguish A. membranaceus varieties.

Computational Identification and Systematic Classification of Novel Cytochrome P450 Genes in Salvia miltiorrhiza.

Salvia miltiorrhiza is one of the most economically important medicinal plants. Cytochrome P450 (CYP450) genes have been implicated in the biosynthesis of its active components. However, only a dozen full-length CYP450 genes have been described, and there is no systematic classification of CYP450 genes in S. miltiorrhiza. We obtained 77,549 unigenes from three tissue types of S. miltiorrhiza using RNA-Seq technology. Combining our data with previously identified CYP450 sequences and scanning with the CYP450 model from Pfam resulted in the identification of 116 full-length and 135 partial-length CYP450 genes. The 116 genes were classified into 9 clans and 38 families using standard criteria. The RNA-Seq results showed that 35 CYP450 genes were co-expressed with CYP76AH1, a marker gene for tanshinone biosynthesis, using r≥0.9 as a cutoff. The expression profiles for 16 of 19 randomly selected CYP450 obtained from RNA-Seq were validated by qRT-PCR. Comparing against the KEGG database, 10 CYP450 genes were found to be associated with diterpenoid biosynthesis. Considering all the evidence, 3 CYP450 genes were identified to be potentially involved in terpenoid biosynthesis. Moreover, we found that 15 CYP450 genes were possibly regulated by antisense transcripts (r≥0.9 or r≤-0.9). Lastly, a web resource (SMCYP450, http://www.herbalgenomics.org/samicyp450) was set up, which allows users to browse, search, retrieve and compare CYP450 genes and can serve as a centralized resource.

Complex interplay among DNA modification, noncoding RNA expression and protein-coding RNA expression in Salvia miltiorrhiza chloroplast genome.

Salvia miltiorrhiza is one of the most widely used medicinal plants. As a first step to develop a chloroplast-based genetic engineering method for the over-production of active components from S. miltiorrhiza, we have analyzed the genome, transcriptome, and base modifications of the S. miltiorrhiza chloroplast. Total genomic DNA and RNA were extracted from fresh leaves and then subjected to strand-specific RNA-Seq and Single-Molecule Real-Time (SMRT) sequencing analyses. Mapping the RNA-Seq reads to the genome assembly allowed us to determine the relative expression levels of 80 protein-coding genes. In addition, we identified 19 polycistronic transcription units and 136 putative antisense and intergenic noncoding RNA (ncRNA) genes. Comparison of the abundance of protein-coding transcripts (cRNA) with and without overlapping antisense ncRNAs (asRNA) suggest that the presence of asRNA is associated with increased cRNA abundance (p<0.05). Using the SMRT Portal software (v1.3.2), 2687 potential DNA modification sites and two potential DNA modification motifs were predicted. The two motifs include a TATA box-like motif (CPGDMM1, "TATANNNATNA"), and an unknown motif (CPGDMM2 "WNYANTGAW"). Specifically, 35 of the 97 CPGDMM1 motifs (36.1%) and 91 of the 369 CPGDMM2 motifs (24.7%) were found to be significantly modified (p<0.01). Analysis of genes downstream of the CPGDMM1 motif revealed the significantly increased abundance of ncRNA genes that are less than 400 bp away from the significantly modified CPGDMM1motif (p<0.01). Taking together, the present study revealed a complex interplay among DNA modifications, ncRNA and cRNA expression in chloroplast genome.