Effects of Klebsiella michiganensis LDS17 on Codonopsis pilosula growth, rhizosphere soil enzyme activities, and microflora, and genome-wide analysis of plant growth-promoting genes.

Codonopsis pilosula is a perennial herbaceous liana with medicinal value. It is critical to promote Codonopsis pilosula growth through effective and sustainable methods, and the use of plant growth-promoting bacteria (PGPB) is a promising candidate. In this study, we isolated a PGPB, Klebsiella michiganensis LDS17, that produced a highly active 1-aminocyclopropane-1-carboxylate deaminase from the Codonopsis pilosula rhizosphere. The strain exhibited multiple plant growth-promoting properties. The antagonistic activity of strain LDS17 against eight phytopathogenic fungi was investigated, and the results showed that strain LDS17 had obvious antagonistic effects on Rhizoctonia solani, Colletotrichum camelliae, Cytospora chrysosperma, and Phomopsis macrospore with growth inhibition rates of 54.22%, 49.41%, 48.89%, and 41.11%, respectively. Inoculation of strain LDS17 not only significantly increased the growth of Codonopsis pilosula seedlings but also increased the invertase and urease activities, the number of culturable bacteria, actinomycetes, and fungi, as well as the functional diversity of microbial communities in the rhizosphere soil of the seedlings. Heavy metal (HM) resistance tests showed that LDS17 is resistant to copper, zinc, and nickel. Whole-genome analysis of strain LDS17 revealed the genes involved in IAA production, siderophore synthesis, nitrogen fixation, P solubilization, and HM resistance. We further identified a gene (koyR) encoding a plant-responsive LuxR solo in the LDS17 genome. Klebsiella michiganensis LDS17 may therefore be useful in microbial fertilizers for Codonopsis pilosula. The identification of genes related to plant growth and HM resistance provides an important foundation for future analyses of the molecular mechanisms underlying the plant growth promotion and HM resistance of LDS17. IMPORTANCE: We comprehensively evaluated the plant growth-promoting characteristics and heavy metal (HM) resistance ability of the LDS17 strain, as well as the effects of strain LDS17 inoculation on the Codonopsis pilosula seedling growth and the soil qualities in the Codonopsis pilosula rhizosphere. We conducted whole-genome analysis and identified lots of genes and gene clusters contributing to plant-beneficial functions and HM resistance, which is critical for further elucidating the plant growth-promoting mechanism of strain LDS17 and expanding its application in the development of plant growth-promoting agents used in the environment under HM stress.

[Screening and expression analysis of transcription factors involved in genuineness of Codonopsis pilosula in Shanxi].

This study aims to mine the transcription factors that affect the genuineness of Codonopsis pilosula in Shanxi based on the transcriptome data of C. pilosula samples collected from Shanxi and Gansu, and then analyze the gene expression patterns, which will provide a theoretical basis for the molecular assisted breeding of C. pilosula. Gene ontology(GO) functional annotation, conserved motif prediction, and gene expression pattern analysis were performed for the differential transcription factors predicted based on the transcriptome data of C. pilosula from different habitats. A total of 61 differentially expressed genes(DEGs) were screened out from the transcriptome data. Most of the DEGs belonged to AP2/ERF-ERF family, with the conserved motif of [2X]-[LG]-[3X]-T-[3X]-[AARAYDRAA]-[3X]-[RG]-[2X]-A-[2X]-[NFP]. Forty-three of the DEGs showed significantly higher gene expression in C. pilosula samples from Shanxi than in the samples from Gansu, including 11 genes in the AP2/ERF-ERF family, 5 genes in the NAC fa-mily, 1 gene in the bHLH family, and 2 genes in the RWP-RK family, while 18 transcription factors showed higher expression levels in the samples from Gansu. GO annotation predicted that most of the DEGs were enriched in GO terms related to transcriptional binding activity(103), metabolic process(26), and stress response(23). The expression of transcription factor genes, CpNAC92, CpNAC100, CpbHLH128, and CpRAP2-7 was higher in the samples from Shanxi and in the roots of C. pilosula. CpNAC92, CpbHLH128, and CpRAP2-7 responded to the low temperature, temperature difference, and iron stresses, while CpNAC100 only responded to low temperature and iron stresses. The screening and expression analysis of the specific transcription factors CpNAC92, CpNAC100, CpbHLH128, and CpRAP2-7 in C. pilosula in Shanxi laid a theoretical foundation for further research on the mechanism of genuineness formation of C. pilosula.

De Novo Transcriptome Sequencing of Codonopsis lanceolata for Identification of Triterpene Synthase and Triterpene Acetyltransferase.

Codonopsis lanceolata (Campanulaceae) is a perennial plant commonly known as the bonnet bellflower. This species is widely used in traditional medicine and is considered to have multiple medicinal properties. In this study, we found that shoots and roots of C. lanceolata contained various types of free triterpenes (taraxerol, ß-amyrin, α-amyrin, and friedelin) and triterpene acetates (taraxerol acetate, ß-amyrin acetate, and α-amyrin acetate). The content of triterpenes and triterpene acetates by GC analysis was higher in the shoot than in the roots. To investigate the transcriptional activity of genes involved in triterpenes and triterpene acetate biosynthesis, we performed de novo transcriptome analysis of shoots and roots of C. lanceolata by sequencing using the Illumina platform. A total of 39,523 representative transcripts were obtained. After functional annotation of the transcripts, the differential expression of genes involved in triterpene biosynthetic pathways was investigated. Generally, the transcriptional activity of unigenes in the upstream region (MVA and MEP pathway) of triterpene biosynthetic pathways was higher in shoots than in roots. Various triterpene synthases (2,3-oxidosqualene cyclase, OSC) participate to produce triterpene skeletons by the cyclization of 2,3-oxidosqualene. A total of fifteen contigs were obtained in annotated OSCs in the representative transcripts. Functional characterization of four OSC sequences by heterologous expression in yeast revealed that ClOSC1 was determined as taraxerol synthase, and ClOSC2 was a mixed-amyrin synthase producing α-amyrin and ß-amyrin. Five putative contigs of triterpene acetyltransferases showed high homology to the lettuce triterpene acetyltransferases. Conclusively, this study provides the basis of molecular information, particularly for the biosynthesis of triterpenes and triterpene acetates in C. lanceolata.

In vitro propagation of Codonopsis pilosula (Franch.) Nannf. using apical shoot segments and phytochemical assessments of the maternal and regenerated plants.

BACKGROUND: Codonopsis pilosula (Franch.) Nannf. is a medicinal plant traditionally used in China, Korea, and Japan to treat many diseases including poor gastrointestinal function, low immunity, gastric ulcers, and chronic gastritis. The increasing therapeutic and preventive use of C. pilosula has subsequently led to depletion of the natural populations of this species thus necessitating propagation of this important medicinal plant. Here, we developed an efficient and effective in vitro propagation protocol for C. pilosula using apical shoot segments. We tested various plant tissue culture media for the growth of C. pilosula and evaluated the effects of plant growth regulators on the shoot proliferation and rooting of regenerated C. pilosula plants. Furthermore, the tissues (roots and shoots) of maternal and in vitro-regenerated C. pilosula plants were subjected to Fourier-transform near-infrared (FT-NIR) spectrometry, Gas chromatography-mass spectrometry (GC-MS), and their total flavonoids, phenolics, and antioxidant capacity were determined and compared. RESULTS: Full-strength Murashige and Skoog (MS) medium augmented with vitamins and benzylaminopurine (1.5 mg·L-1) regenerated the highest shoot number (12 ± 0.46) per explant. MS medium augmented with indole-3-acetic acid (1.0 mg·L-1) produced the highest root number (9 ± 0.89) and maximum root length (20.88 ± 1.48 mm) from regenerated C. pilosula shoots. The survival rate of in vitro-regenerated C. pilosula plants was 94.00% after acclimatization. The maternal and in vitro-regenerated C. pilosula plant tissues showed similar FT-NIR spectra, total phenolics, total flavonoids, phytochemical composition, and antioxidant activity. Randomly amplified polymorphic DNA (RAPD) test confirmed the genetic fidelity of regenerated C. pilosula plants. CONCLUSIONS: The proposed in vitro propagation protocol may be useful for the rapid mass multiplication and production of high quality C. pilosula as well as for germplasm preservation to ensure sustainable supply amidst the ever-increasing demand.

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.

Astragalus polysaccharides combined with Codonopsis pilosula polysaccharides modulates the physiological characteristics of trophoblasts via miR-92a-1-5p/CCR7 axis.

BACKGROUND: When the abnormality occurs in proliferation, differentiation and apoptosis of trophoblasts, the invasion ability of placental trophoblast is weakened, which is prone to trigger the occurrence of various pregnancy diseases such as repeated spontaneous abortion (RSA). Clinically, Astragalus and Codonopsis pilosula polysaccharides (APS and CPPS) are used for the treatment of unexplained recurrent spontaneous abortion (URSA). Therefore, we aimed to probe into the roles of APS and CPPS in biological behaviors of placental trophoblasts. METHODS: The trophoblasts were treated with APS and CPPS, and transfected with miR-92a-1-5p mimic and CCR7 plasmid to explore the roles of APS and CPPS. Cell viability and apoptosis were determined by CCK-8 and flow cytometry, respectively. The levels of miRNA/mRNA and protein were measured by qRT-PCR and western blot, respectively. The interaction between miR-92a-1-5p and CCR7 was analyzed by TargetScan and dual-luciferase reporter assay. Invasion and migration rates were assessed by Transwell and wound healing assays, respectively. RESULTS: APS combined with CPPS enhanced viability, Bcl-2 expression, and migration and invasion capabilities, while suppressing apoptosis, and expressions of Bax, Bim and miR-92a-1-5p in trophoblasts. Nevertheless, miR-92a-1-5p mimic produced the inverse modulations in trophoblasts, and partially reversed the effects of APS and CPPS. Furthermore, overexpression of CCR7, the target of miR-92a-1-5p, partially offset the effect of miR-92a-1-5p mimic in trophoblasts. CONCLUSION: Astragalus combined with Codonopsis pilosula polysaccharides modulates the biological behaviors of trophoblasts via miR-92a-1-5p/CCR7 axis. The regulatory axis we studied will be helpful for the treatment of URSA in the future.

Genome-Wide Characterization of SPL Gene Family in Codonopsis pilosula Reveals the Functions of CpSPL2 and CpSPL10 in Promoting the Accumulation of Secondary Metabolites and Growth of C. pilosula Hairy Root.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors play critical roles in regulating diverse aspects of plant growth and development, including vegetative phase change, plant architecture, anthocyanin accumulation, lateral root growth, etc. In the present study, 15 SPL genes were identified based on the genome data of Codonopsis pilosula, a well-known medicinal plant. Phylogenetic analysis clustered CpSPLs into eight groups (G1-G8) along with SPLs from Arabidopsis thaliana, Solanum lycopersicum, Oryza sativa and Physcomitrella patens. CpSPLs in the same group share similar gene structure and conserved motif composition. Cis-acting elements responding to light, stress and phytohormone widely exist in their promoter regions. Our qRT-PCR results indicated that 15 CpSPLs were differentially expressed in different tissues (root, stem, leaf, flower and calyx), different developmental periods (1, 2 and 3 months after germination) and various conditions (NaCl, MeJA and ABA treatment). Compared with the control, overexpression of CpSPL2 or CpSPL10 significantly promoted not only the growth of hairy roots, but also the accumulation of total saponins and lobetyolin. Our results established a foundation for further investigation of CpSPLs and provided novel insights into their biological functions. As far as we know, this is the first experimental research on gene function in C. pilosula.

[Cloning and expression analysis of CpPMM gene in Codonopsis pilosula].

GDP-mannose is an important precursor for the synthesis of Codonopsis pilosula polysaccharides and involved in the synthesis of sugar chains. Phosphomannomutase(PMM)catalyzes the conversion of mannose-6-phosphate(Man-6-P)to mannose-1-phosphate(Man-1-P)to synthesize GDP-mannose. In this study, specific primers were designed based on the PMM gene sequence information in transcriptome data, and the full length of the C. pilosula PMM gene was cloned and named CpPMM. The correlation between the CpPMM gene expression and C. pilosula polysaccharide synthesis was analyzed by a series of bioinformatics analysis, prokaryotic expression and qRT-PCR. The results show that the CpPMM gene contains a 741 bp open reading frame(ORF), encoding 246 amino acids, which is highly similar to the PMM of other species and highly homologous to the Helianthus annuus from the Asteraceae family. It was predicted to be a hydrophilic non-transmembrane protein without signal peptide, which was predicted to be located in the cytoplasm with multiple phosphorylation sites. Combined with predictive analysis of conserved domains, this protein belongs to the HAD(haloacid dehalogenase)superfamily; prokaryotic expression studies show that the size of the CpPMM fusion protein is about 29 kDa, which is consistent with the relative molecular mass predicted. The target protein is an inclusion body and is partially soluble. The qRT-PCR results showed that the CpPMM gene exerted spatiotemporal expression patterns, and the expression level in fruiting period was significantly higher than that in the other three periods such as the flowering period. Along with the growth period of C. pilosula, the polysaccharide content of C. pilosula showed a gradual increase trend, reaching the highest during the harvest time. And there are significant differences in the polysaccharide content of C. pilosula in each period. In this study, the CpPMM gene was cloned from the root of C. pilosula, at the same time, the prokaryotic expression system was constructed. In addition, its gene expression level is highly correlated with the polysaccharide content of C. pilosula. It lays the foundation for further studying the function of CpPMM gene and the analysis of biosynthetic pathways of polysaccharides in medicinal plants.

The application of DNA molecular markers in the study of Codonopsis species genetic variation, a review.

Codonopsis genus is comprised of species that are perennial plants primarily distributed across all east, southeast, and Central Asia. The most famous species of Codonopsis are C. tangshen, C. lanceolate, and C. pilosula. The records showed that they have a long story usage as traditional Chinese medicines, as they were alleged to be able to intensify the spleen and the lung as well as enriching blood and engendering liquid. Certain species have a culinary value in southern China and Southeast Asia, where they are considered as tea, wine, soup, plaster, and porridge. Codonopsis species were shown to be of great importance in medicine, due to their broad biological activity. Therefore, a clear understanding of their genetic diversity is needed.  Adequate distinctions and descriptions of those species are necessary to preserve plant reservoir, investigations of genes associated with desirable traits, and understanding of evolutionary relationships. Subsequently, various molecular marker techniques such as Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeats (SSR), and Inter Simple Sequence Repeat (ISSR), Single Nucleotide Polymorphism (SNP), internal transcribed spacer (ITS), and Sequence-Characterized Amplified Region (SCAR) have been improved to provide  detailed informations about genomes, that historically were  not possible to obtain based on only phenotypic methods. This review represents the usage of DNA molecular markers for molecular diversity analysis of medically important species belonging to the genus Codonopsis.

Selection of suitable reference genes for qRT-PCR expression analysis of Codonopsis pilosula under different experimental conditions.

Codonopsis pilosula is a well-known medicinal plant. Although its transcriptome sequence has been published, suitable reference genes have not been systematically identified for conducting expression analyses via quantitative real-time polymerase chain reaction (qRT-PCR). To screen appropriate genes for use with this species, we applied four different methods-GeNorm, NormFinder, BestKeeper, and RefFinder-to evaluate the stability of 13 candidates: CpiEF1Bb, CpiCACS, CpiF-Box, Cpiß-Tubulin, CpiGAPDH, CpiActin2, CpiAPT1, CpiActin7, CpiActin8, CpiRPL6, CpiHAF1, CpiTubulin6, and CpiUBQ12. Expression was examined by qRT-PCR for various tissue types, chemical treatments, and developmental stages. For all tested samples, CpiGAPDH proved to be the most stable. Comprehensive analysis indicated that the most stable internal reference genes were CpiF-Box and CpiCACS in different tissues and at different developmental stages, respectively. Under NaCl stress, CpiAPT1 was the best internal reference gene. For methyl jasmonate and abscisic acid treatments, CpiGAPDH and CpiF-Box, respectively, presented the highest degree of expression stability. Based on these findings, we chose CpiSPL9 as the target gene for validating the suitability of these selected reference genes. All of these results provide a foundation for accurate quantification of expression levels by genes of interest in C. pilosula.

Selection and Validation of Reference Genes for Gene Expression Studies in Codonopsis pilosula Based on Transcriptome Sequence Data.

Relative gene expression analyses by RT-qPCR (reverse transcription-quantitative PCR) are highly dependent on the reference genes in normalizing the expression data of target genes. Therefore, inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, and the selection and validation of suitable internal reference genes becomes essential. In this study, we retrieved the commonly used reference genes in transcriptome datasets of Codonopsis pilosula by RNA-Seq (unpublished data), and selected 15 candidate reference genes according to the coefficient of variation (CV) and fold change (FC) value of gene expression. The expression levels of candidate reference genes, which is at different growth stages, undergoing cold stress and drought stress, was determined by RT-qPCR. The expression stability of these genes was evaluated using software packages and algorithms including ΔCt, geNorm, NormFinder and Bestkeeper. Then appropriate reference genes were screened and validated by target gene-UDGPase (UDP glucose pyrophosphorylase). The optimal RGs combinations of C. pilosula, including PP2A59γ, CPY20-1, UBCE32, RPL5B and UBC18 for developmental stage, RPL5B, RPL13 and PP2A59γ for cold treatment, RPL13 and PP2A59γ for drought treatment, were found and proposed as reference genes for future work. This paper laid foundations for both the selection of reference genes and exploration in metabolic mechanism of C. pilosula.

Transcriptome analysis reveals novel insights into the continuous cropping induced response in Codonopsis tangshen, a medicinal herb.

Codonopsis tangshen Oliv. (C. tangshen Oliv.), a famous medicinal herb in China, is seriously affected by continuous cropping (C-cro). The physiological and biochemical results indicated that C-cro significantly affected the malonaldehyde (MDA) and chlorophyll content, as well as activities of catalase (CAT) and superoxide dismutase (SOD) when compared with the non-continuous cropping (NC-cro) group. Transcriptome profiling found 762 differentially expressed genes, including 430 up-regulated and 332 down-regulated genes by C-cro. In addition, pathway enrichment analysis revealed that genes related to 'Tyrosine degradation I', 'Glycogen synthesis' and 'Phenylalanine and tyrosine catabolism' were up-regulated, and genes associated with 'Signal transduction', 'Immune system', etc. were down-regulated by C-cro. The expression of target genes was further validated by Q-PCR. In this study, we demonstrated the effects of C-cro on C. tangshen at the transcriptome level, and found possible C-cro responsive candidate genes. These findings could be further beneficial for improving the continuous cropping tolerance.

Response of Bioactive Metabolite and Biosynthesis Related Genes to Methyl Jasmonate Elicitation in Codonopsis pilosula.

Bioactive metabolites in Codonopsis pilosula are of particular interest as an immunostimulant. Methyl jasmonate (MeJA) plays an important role in the elicitation of metabolite biosynthesis. Here, we explored the response of metabolites to MeJA elicitation in C. pilosula adventitious roots and multiple shoots. The results showed that the biomass, polysaccharide, and lobetyolin content of adventitious roots exhibited the highest increases with 100 µmol·L-1 MeJA at the 16th day of subculture, whereas the atractylenolide III (a terpenoid) content increased extremely with 50 µmol·L-1 MeJA treatment at the 7th day of subculture. In addition, the biomass and lobetyolin content significantly increased at the 4th day after treatment. Similarly, the polysaccharide and lobetyolin content increased in multiple shoots. Further identification of different metabolites responding to MeJA by ¹H-NMR showed an extremely significant increase of the lobetyolinin level, which coincided with lobetyolin. Accordingly, the precursor, fatty acids, showed a highly significant decrease in their levels. Furthermore, a significant increase in ß-d-fructose-butanol glycoside was detected, which was accompanied by a decrease in the sucrose level. Accordingly, the enzyme genes responsible for terpenoid and carbohydrate biosynthesis, CpUGPase, and CpPMK, were up regulated. In conclusion, MeJA promoted culture growth and accelerated bioactive metabolite accumulation by regulating the expression of the metabolite biosynthesis related genes, CpUGPase and CpPMK in C. pilosula.

Study on Hybrid Characteristics of Medicinally Used Cultivated Codonopsis Species Using Ribosomal Internal Transcribed Spacer (ITS) Sequencing.

Codonopsis taxa, as a traditional Chinese medicinal and edible plant, has found expanding domestic and foreign applications in recent decades. However, the poor management in germplasm resources market has inevitably caused an unnecessary hybrid of the provenances. In order to clarify the hybrid characteristics of germplasm resources in the main production area, the Codonopsis cultivars collected from the provinces Gansu, Shannxi, Shanxi, and Hubei of China were researched, using internal transcribed spacer (ITS) sequence technology. The confirmation of additive nucleotides based on the ITS sequencing of polymerase chain reaction (PCR) mixture was optimized and used to study the hybrid of Codonopsis cultivars. The results showed that when the ratio of PCR mixture increased up to 15 percent, the presence of a double peak in the sequencing electrophoresis map could be confirmed, suggesting the existence of additive nucleotides. According to the method above, 46 samples of Codonopsis cultivars collected during 2016 and 2017 were studied and compared with the samples collected from the year 2009 to 2010. All of the samples collected during 2016 and 2017 were hybridized and no genetic pure lines were found. In addition, the sites of variable base reduced greatly, concentrating at positions 122 and/or 226. These phenomena suggested that the genetic diversity of Codonopsis cultivars declined and the germplasm resources gradually converged. More attention should be paid to the reasonable exploitation and genetic breeding of Codonopsis taxa.

[Clone and expression of CpGAPDH gene in Codonopsis pilosula].

GAPDH(glyceraldehyde-3-phosphate dehydrogenase) gene is a key enzyme gene in carbohydrate metabolism and always used as reference gene. To clarify and complete the biosynthetic pathway of polysaccharide, the GAPDH gene in Codonopsis pilosula, named CpGAPDH, was cloned according to the transcriptome of pilosula, using the GAPDH gene in potato as query. The CpGAPDH contained a 1 014 bp open reading frame(ORF) and encoded a protein with 337 amino acids. Bioinformatic analysis clearly suggested that CpGAPDH shared high similarity with GAPDH among other plants, and had the closest relatives to potato and danshen. The predicted protein did not have signal peptide, which indicated that it might be located in the cytoplasm. According to the existing of several phosphorylation sites and the conserved domains analysis, we predicted that it belonged to Gp_dh_N superfamily. Prokaryotic expression showed that the recombinant expressed a 44.3 kDa protein, which was corresponding to the theoretical relative molecular mass. However, the relative transcript level of the CpGAPDH did not have significant differences in different tissues and roots at different developmental stages of pilosula. Moreover, the stability of the CpGAPDH was analyzed by BestKeeper, geNorm, and NormFinder and RefFinder software, which showed that the CpGAPDH was more stable and could be used as a new reference gene. All these lay a foundation for the expression analysis of the gene relative to the polysaccharide synthesis.

Evaluation of DNA barcodes in Codonopsis (Campanulaceae) and in some large angiosperm plant genera.

DNA barcoding is expected to be one of the most promising tools in biological taxonomy. However, there have been no agreements on which core barcode should be used in plants, especially in species-rich genera with wide geographical distributions. To evaluate their discriminatory power in large genera, four of the most widely used DNA barcodes, including three plastid regions (matK, rbcL, trnH-psbA) and nuclear internal transcribed spacer (nrITS), were tested in seven species-rich genera (Ficus, Pedicularis, Rhodiola, Rhododendron,Viburnum, Dendrobium and Lysimachia) and a moderate size genus, Codonopsis. All of the sequences from the aforementioned seven large genera were downloaded from NCBI. The related barcodes for Codonopsis were newly generated in this study. Genetics distances, DNA barcoding gaps and phylogenetic trees of the four single barcodes and their combinations were calculated and compared in the seven genera. As for single barcode, nrITS has the most variable sites, the clearest intra- and inter-specific divergences and the highest discrimination rates in the seven genera. Among the combinations of barcodes, ITS+matK performed better than all the single barcodes in most cases and even the three- and four-loci combinations in the seven genera. Therefore, we recommend ITS+matK as the core barcodes for large plant genera.

The abiotic and biotic drivers of rapid diversification in Andean bellflowers (Campanulaceae).

The tropical Andes of South America, the world's richest biodiversity hotspot, are home to many rapid radiations. While geological, climatic, and ecological processes collectively explain such radiations, their relative contributions are seldom examined within a single clade. We explore the contribution of these factors by applying a series of diversification models that incorporate mountain building, climate change, and trait evolution to the first dated phylogeny of Andean bellflowers (Campanulaceae: Lobelioideae). Our framework is novel for its direct incorporation of geological data on Andean uplift into a macroevolutionary model. We show that speciation and extinction are differentially influenced by abiotic factors: speciation rates rose concurrently with Andean elevation, while extinction rates decreased during global cooling. Pollination syndrome and fruit type, both biotic traits known to facilitate mutualisms, played an additional role in driving diversification. These abiotic and biotic factors resulted in one of the fastest radiations reported to date: the centropogonids, whose 550 species arose in the last 5 million yr. Our study represents a significant advance in our understanding of plant evolution in Andean cloud forests. It further highlights the power of combining phylogenetic and Earth science models to explore the interplay of geology, climate, and ecology in generating the world's biodiversity.

Transcriptome sequencing of Codonopsis pilosula and identification of candidate genes involved in polysaccharide biosynthesis.

BACKGROUND: Codonopsis pilosula (Franch.) Nannf. is one of the most widely used medicinal plants. Although chemical and pharmacological studies have shown that codonopsis polysaccharides (CPPs) are bioactive compounds and that their composition is variable, their biosynthetic pathways remain largely unknown. Next-generation sequencing is an efficient and high-throughput technique that allows the identification of candidate genes involved in secondary metabolism. PRINCIPAL FINDINGS: To identify the components involved in CPP biosynthesis, a transcriptome library, prepared using root and other tissues, was assembled with the help of Illumina sequencing. A total of 9.2 Gb of clean nucleotides was obtained comprising 91,175,044 clean reads, 102,125 contigs, and 45,511 unigenes. After aligning the sequences to the public protein databases, 76.1% of the unigenes were annotated. Among these annotated unigenes, 26,189 were assigned to Gene Ontology categories, 11,415 to Clusters of Orthologous Groups, and 18,848 to Kyoto Encyclopedia of Genes and Genomes pathways. Analysis of abundance of transcripts in the library showed that genes, including those encoding metallothionein, aquaporin, and cysteine protease that are related to stress responses, were in the top list. Among genes involved in the biosynthesis of CPP, those responsible for the synthesis of UDP-L-arabinose and UDP-xylose were highly expressed. SIGNIFICANCE: To our knowledge, this is the first study to provide a public transcriptome dataset prepared from C. pilosula and an outline of the biosynthetic pathway of polysaccharides in a medicinal plant. Identified candidate genes involved in CPP biosynthesis provide understanding of the biosynthesis and regulation of CPP at the molecular level.

Genetic polymorphism of medicinally-used Codonopsis species in an internal transcribed spacer sequence of nuclear ribosomal DNA and its application to authenticate Codonopsis Radix.

Codonopsis Radix has been prescribed as the roots of Codonopsis pilosula, C. pilosula var. modesta and C. tangshen in Chinese Pharmacopoeia. In order to find out genetic markers for identifying the 3 taxa and to authenticate Codonopsis Radix, the molecular analysis of the internal transcribed spacer sequence of nuclear ribosomal DNA was conducted on Codonopsis plants collected widely from Gansu Prov. and Chongqing city of China, the main producing areas of Codonopsis Radix. Significant genetic polymorphism was observed, represented by 11 types of ITS sequences in C. pilosula, 5 types in C. pilosula var. modesta and 5 types in C. tangshen. Among the determined sequences, 1, 1 and 2 types were thought to be of pure lines of each taxon, respectively, designated as types P0, PM0, T1 and T3, and the rest might be derived from hybridization. Hybrid lines were inferred to be resulting from the combination of these pure lines. The informative sites for discriminating the 3 taxa were detected at the nucleotide positions 122nd, 226th, 441st and 489th from upstream of the ITS sequence. For discrimination of the types of C. tangshen, including one type T0 registered in GenBank, the nucleotides at positions 135th, 489th and 500th were informative. Botanical sources of the crude drugs produced in a wide range of the southeast Gansu Prov. were C. pilosula, just those from Wenxian of Gansu Prov. were C. pilosula var. modesta. The crude drugs produced in Chongqing were derived from C. tangshen.

Phylogenetic analysis with the iPlant discovery environment.

The iPlant Collaborative's Discovery Environment is a unified Web portal to many bioinformatics applications and analytical workflows, including various methods of phylogenetic analysis. This unit describes example protocols for phylogenetic analyses, starting at sequence retrieval from the GenBank sequence database, through to multiple sequence alignment inference and visualization of phylogenetic trees. Methods for extracting smaller sub-trees from very large phylogenies, and the comparative method of continuous ancestral character state reconstruction based on observed morphology of extant species related to their phylogenetic relationships, are also presented.