Yingjie Zhu

Background The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over. Methodology/Principal Findings Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level. Conclusions The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.

Background The internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA is regarded as one of the candidate DNA barcodes because it possesses a number of valuable characteristics, such as the availability of conserved regions for designing universal primers, the ease of its amplification, and sufficient variability to distinguish even closely related species. However, a general analysis of its ability to discriminate species in a comprehensive sample set is lacking. Methodology/Principal Findings In the current study, 50,790 plant and 12,221 animal ITS2 sequences downloaded from GenBank were evaluated according to sequence length, GC content, intra- and inter-specific divergence, and efficiency of identification. The results show that the inter-specific divergence of congeneric species in plants and animals was greater than its corresponding intra-specific variations. The success rates for using the ITS2 region to identify dicotyledons, monocotyledons, gymnosperms, ferns, mosses, and animals were 76.1%, 74.2%, 67.1%, 88.1%, 77.4%, and 91.7% at the species level, respectively. The ITS2 region unveiled a different ability to identify closely related species within different families and genera. The secondary structure of the ITS2 region could provide useful information for species identification and could be considered as a molecular morphological characteristic. Conclusions/Significance As one of the most popular phylogenetic markers for eukaryota, we propose that the ITS2 locus should be used as a universal DNA barcode for identifying plant species and as a complementary locus for CO1 to identify animal species. We have also developed a web application to facilitate ITS2-based cross-kingdom species identification (http://its2-plantidit.dnsalias.org).

A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH–psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that ( i ) the three plastid markers showed high levels of universality (87.1–92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; ( ii ) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9–79.1% of species, compared with only 49.7% with rbcL + matK ; and ( iii ) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.

A novel human organic transporter, OATP2, has been identified that transports taurocholic acid, the adrenal androgen dehydroepiandrosterone sulfate, and thyroid hormone, as well as the hydroxymethylglutaryl-CoA reductase inhibitor, pravastatin. OATP2 is expressed exclusively in liver in contrast to all other known transporter subtypes that are found in both hepatic and nonhepatic tissues. OATP2 is considerably diverged from other family members, sharing only 42% sequence identity with the four other subtypes. Furthermore, unlike other subtypes, OATP2 did not transport digoxin or aldosterone. The rat isoform oatp1 was also shown to transport pravastatin, whereas other members of the OATP family, <i>i.e.</i>rat oatp2, human OATP, and the prostaglandin transporter, did not. Cis-inhibition studies indicate that both OATP2 and roatp1 also transport other statins including lovastatin, simvastatin, and atorvastatin. In summary, OATP2 is a novel organic anion transport protein that has overlapping but not identical substrate specificities with each of the other subtypes and, with its liver-specific expression, represents a functionally distinct OATP isoform. Furthermore, the identification of oatp1 and OATP2 as pravastatin transporters suggests that they are responsible for the hepatic uptake of this liver-specific hydroxymethylglutaryl-CoA reductase inhibitor in rat and man.

The complete sequences of chloroplast genomes provide wealthy information regarding the evolutionary history of species. With the advance of next-generation sequencing technology, the number of completely sequenced chloroplast genomes is expected to increase exponentially, powerful computational tools annotating the genome sequences are in urgent need.We have developed a web server CPGAVAS. The server accepts a complete chloroplast genome sequence as input. First, it predicts protein-coding and rRNA genes based on the identification and mapping of the most similar, full-length protein, cDNA and rRNA sequences by integrating results from Blastx, Blastn, protein2genome and est2genome programs. Second, tRNA genes and inverted repeats (IR) are identified using tRNAscan, ARAGORN and vmatch respectively. Third, it calculates the summary statistics for the annotated genome. Fourth, it generates a circular map ready for publication. Fifth, it can create a Sequin file for GenBank submission. Last, it allows the extractions of protein and mRNA sequences for given list of genes and species. The annotation results in GFF3 format can be edited using any compatible annotation editing tools. The edited annotations can then be uploaded to CPGAVAS for update and re-analyses repeatedly. Using known chloroplast genome sequences as test set, we show that CPGAVAS performs comparably to another application DOGMA, while having several superior functionalities.CPGAVAS allows the semi-automatic and complete annotation of a chloroplast genome sequence, and the visualization, editing and analysis of the annotation results. It will become an indispensible tool for researchers studying chloroplast genomes. The software is freely accessible from http://www.herbalgenomics.org/cpgavas.

Ganoderma lucidum is a widely used medicinal macrofungus in traditional Chinese medicine that creates a diverse set of bioactive compounds. Here we report its 43.3-Mb genome, encoding 16,113 predicted genes, obtained using next-generation sequencing and optical mapping approaches. The sequence analysis reveals an impressive array of genes encoding cytochrome P450s (CYPs), transporters and regulatory proteins that cooperate in secondary metabolism. The genome also encodes one of the richest sets of wood degradation enzymes among all of the sequenced basidiomycetes. In all, 24 physical CYP gene clusters are identified. Moreover, 78 CYP genes are coexpressed with lanosterol synthase, and 16 of these show high similarity to fungal CYPs that specifically hydroxylate testosterone, suggesting their possible roles in triterpenoid biosynthesis. The elucidation of the G. lucidum genome makes this organism a potential model system for the study of secondary metabolic pathways and their regulation in medicinal fungi.

Salvia miltiorrhiza is an important medicinal plant with great economic and medicinal value. The complete chloroplast (cp) genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family, is reported here. The genome is 151,328 bp in length and exhibits a typical quadripartite structure of the large (LSC, 82,695 bp) and small (SSC, 17,555 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,539 bp). It contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs and four rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Four forward, three inverted and seven tandem repeats were detected in the Salvia miltiorrhiza cp genome. Simple sequence repeat (SSR) analysis among the 30 asterid cp genomes revealed that most SSRs are AT-rich, which contribute to the overall AT richness of these cp genomes. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the cp genomes. Entire cp genome comparison of Salvia miltiorrhiza and three other Lamiales cp genomes showed a high degree of sequence similarity and a relatively high divergence of intergenic spacers. Sequence divergence analysis discovered the ten most divergent and ten most conserved genes as well as their length variation, which will be helpful for phylogenetic studies in asterids. Our analysis also supports that both regional and functional constraints affect gene sequence evolution. Further, phylogenetic analysis demonstrated a sister relationship between Salvia miltiorrhiza and Sesamum indicum. The complete cp genome sequence of Salvia miltiorrhiza reported in this paper will facilitate population, phylogenetic and cp genetic engineering studies of this medicinal plant.

Danshen, Salvia miltiorrhiza Bunge, is one of the most widely used herbs in traditional Chinese medicine, wherein its rhizome/roots are particularly valued. The corresponding bioactive components include the tanshinone diterpenoids, the biosynthesis of which is a subject of considerable interest. Previous investigations of the S. miltiorrhiza transcriptome have relied on short-read next-generation sequencing (NGS) technology, and the vast majority of the resulting isotigs do not represent full-length cDNA sequences. Moreover, these efforts have been targeted at either whole plants or hairy root cultures. Here, we demonstrate that the tanshinone pigments are produced and accumulate in the root periderm, and apply a combination of NGS and single-molecule real-time (SMRT) sequencing to various root tissues, particularly including the periderm, to provide a more complete view of the S. miltiorrhiza transcriptome, with further insight into tanshinone biosynthesis as well. In addition, the use of SMRT long-read sequencing offered the ability to examine alternative splicing, which was found to occur in approximately 40% of the detected gene loci, including several involved in isoprenoid/terpenoid metabolism.

Salvia miltiorrhiza Bunge (Danshen) is a medicinal plant of the Lamiaceae family, and its dried roots have long been used in traditional Chinese medicine with hydrophilic phenolic acids and tanshinones as pharmaceutically active components (Zhang et al., 2014Zhang Y. Yan Y.P. Wu Y.C. Hua W.P. Chen C. Ge Q. Wang Z.Z. Pathway engineering for phenolic acid accumulations in Salvia miltiorrhiza by combinational genetic manipulation.Metab. Eng. 2014; 21: 71-80Crossref PubMed Scopus (60) Google Scholar, Xu et al., 2016Xu Z. Ji A. Zhang X. Song J. Chen S. Biosynthesis and regulation of active constituents in medicinal model plant Salvia miltiorrhiza.Chin. Herbal Med. 2016; 8: 3-11Crossref Google Scholar). The first step of tanshinone biosynthesis is bicyclization of the general diterpene precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) to copalyl diphosphate (CPP) by CPP synthases (CPSs), which is followed by a cyclization or rearrangement reaction catalyzed by kaurene synthase-like enzymes (KSL). The resulting intermediate is usually an olefin, which requires the insertion of oxygen by cytochrome P450 mono-oxygenases (CYPs) for the final production of diterpenoids (Zi et al., 2014Zi J. Mafu S. Peters R.J. To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism.Annu. Rev. Plant Biol. 2014; 65: 259-286Crossref PubMed Scopus (187) Google Scholar). While the CPS, KSL, and several early acting CYPs (CYP76AH1, CYP76AH3, and CYP76AK1) for tanshinone biosynthesis have been identified in S. miltiorrhiza (Gao et al., 2009Gao W. Hillwig M.L. Huang L. Cui G. Wang X. Kong J. Yang B. Peters R.J. A functional genomics approach to tanshinone biosynthesis provides stereochemical insights.Org. Lett. 2009; 11: 5170-5173Crossref PubMed Scopus (212) Google Scholar, Guo et al., 2013Guo J. Zhou Y.J. Hillwig M.L. Shen Y. Yang L. Wang Y. Zhang X. Liu W. Peters R.J. Chen X. et al.CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.Proc. Natl. Acad. Sci. USA. 2013; 110: 12108-12113Crossref PubMed Scopus (272) Google Scholar, Guo et al., 2016Guo J. Ma X. Cai Y. Ma Y. Zhan Z. Zhou Y.J. Liu W. Guan M. Yang J. Cui G. et al.Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.New Phytol. 2016; 210: 525-534Crossref PubMed Scopus (133) Google Scholar, Zi and Peters, 2013Zi J. Peters R.J. Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamiaceae.Org. Biomol. Chem. 2013; 11: 7650-7652Crossref PubMed Scopus (76) Google Scholar), the majority of the overall biosynthetic pathway, as well as the relevant regulatory factors associated with tanshinone production, remains elusive (Figure 1B ). Here we report the draft sequence and analysis of the S. miltiorrhiza genome by a hybrid assembly approach. First, genomic DNA was extracted from S. miltiorrhiza line 99-3, a strain cultivated by IMPLAD, and 158.2 Gb Illumina data were generated on a Hiseq 2000 platform (250-fold genome coverage; Supplemental Table 1) and assembled with Phusion2 (Mullikin and Ning, 2003Mullikin J.C. Ning Z. The phusion assembler.Genome Res. 2003; 13: 81-90Crossref PubMed Scopus (163) Google Scholar), which resulted in a draft assembly of 558 Mb, with contig N50 of 2.47 kb. Attempts with other assemblers, such as SOAPdenovo and Fermi, gave similar assembly metrics, suggesting intrinsic complexity of this plant genome. We then generated 8.19 Gb of data with the PacBio RS platform (3.74 kb read length on average) and 8.65 Gb Roche/454 data (Supplemental Table 1). Celera Assembler (v7.0) was used for PacBio reads assembly after base-error correction with Roche/454 data, and the resultant contigs were combined with 454 reads for re-assembly. Finally, Illumina reads were mapped onto these contigs to correct single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) in homozygotes, which were presumably introduced by sequencing chemistry bias. This led to a final genome assembly of 538 Mb, with contig and scaffold N50 of 12.38 kb and 51.02 kb, respectively (Supplemental Table 2). Compared with the estimated genome size of 615 Mb by flow cytometry analysis (Supplemental Figure 1), the relatively small size of the assembled genome might result from the high repeat content of this species, as multiple copies of repetitive elements are presumably collapsed together. By mapping the Illumina reads onto the draft assembly, 1 486 270 heterozygous SNPs (and 302 217 short indels) were identified, corresponding to 2.76 SNPs per kb (Supplemental Table 3). This heterozygosity value was comparable with that of Populus (2.6 polymorphisms per kb) and grape (3.6 SNPs per kb). Sequence annotation revealed that repetitive elements accounted for 54.44% of the genome (Supplemental Table 4), twice that of sesame, another species from the order Lamiales (Wang et al., 2014Wang L. Yu S. Tong C. Zhao Y. Liu Y. Song C. Zhang Y. Zhang X. Wang Y. Hua W. et al.Genome sequencing of the high oil crop sesame provides insight into oil biosynthesis.Genome Biol. 2014; 15: R39Crossref PubMed Scopus (188) Google Scholar). Long terminal repeats were the most abundant, spanning 18.03% of the genome, while 55.58% of the repeats (30.26% of the genome) were unclassified, implying lineage-specific repeat expansion. We predicted 30 478 protein-coding genes in the S. miltiorrhiza genome using ab initio and homology-based gene prediction methods (Supplemental Table 4), which were further validated by RNA-seq data (Xu et al., 2015Xu Z. Peters R.J. Weirather J. Luo H. Liao B. Zhang X. Zhu Y. Ji A. Zhang B. Hu S. et al.Full-length transcriptome sequences and splice variants obtained by a combination of sequencing platforms applied to different root tissues of Salvia miltiorrhiza and tanshinone biosynthesis.Plant J. 2015; 82: 951-961Crossref PubMed Scopus (255) Google Scholar). Most of these genes (91.2%) had homologs in the non-redundant (nr) database at GenBank (E value = 1e−5), and more than half (56.60%) of them could be assigned to KEGG pathways. Among them, 1620 genes encode transcription factors (TFs), including 171 APETALA2, 139 bHLH, 291 MYB, and 78 WRKY family TFs (Supplemental Table 5). Several of these TFs have been previously revealed to be involved in the biosynthesis of tanshinone and phenolic acid (Xu et al., 2016Xu Z. Ji A. Zhang X. Song J. Chen S. Biosynthesis and regulation of active constituents in medicinal model plant Salvia miltiorrhiza.Chin. Herbal Med. 2016; 8: 3-11Crossref Google Scholar). In addition, 82 terpene synthase genes (TPS; Supplemental Table 6) that are involved in the production of hemi-, mono-, sesqui-, or di-terpenes, along with 437 CYPs (Supplemental Table 7) that catalyze various oxidation reactions, were identified. Gene family evolution among eight plant species, including rice, Arabidopsis, grape, tomato, potato, bladderwort, sesame, and S. miltiorrhiza, was analyzed by CAFÉ (version 2.1). The result suggests that gene family contraction outnumbered expansion along each lineage (Figure 1A). Intriguingly, families undergoing significant expansion in S. miltiorrhiza (P < 0.01) were primarily involved in stilbenoid, diarylheptanoid or gingerol biosynthesis (Ko00945), and terpenoid biosynthesis (Ko00902) or steroid biosynthesis (Ko00100), which is consistent with the high production of tanshinones and phenolic acids by this medicinal plant. Phylogenomic analysis revealed that S. miltiorrhiza was most closely related to sesame, with an estimated divergence time of approximately 67 million years ago (Figure 1A). Physical clustering of TPSs and CYPs is frequently associated with consecutive enzymatic actions in terpenoid biosynthesis (Boutanaev et al., 2015Boutanaev A.M. Moses T. Zi J. Nelson D.R. Mugford S.T. Peters R.J. Osbourn A. Investigation of terpene diversification across multiple sequenced plant genomes.Proc. Natl. Acad. Sci. USA. 2015; 112: E81-E88Crossref PubMed Scopus (183) Google Scholar), and was investigated here. Four TPS/CYP pairs were found in the draft S. miltiorrhiza genome (Figure 1C–1F). Three of them have been previously characterized, with SmCPS1 and SmCPS2 involved in tanshinone biosynthesis in the roots and leaves, respectively, while SmCPS5 is required for gibberellin phytohormone metabolism (Cui et al., 2015Cui G. Duan L. Jin B. Qian J. Xue Z. Shen G. Snyder J.H. Song J. Chen S. Huang L. et al.Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza Bunge.Plant Physiol. 2015; 169: 1607-1618PubMed Google Scholar). Interestingly, both SmCPS1 and SmCPS2 are flanked by genes from the CYP76AH sub-family. Notably, this includes the previously characterized CYP76AH1 (Guo et al., 2013Guo J. Zhou Y.J. Hillwig M.L. Shen Y. Yang L. Wang Y. Zhang X. Liu W. Peters R.J. Chen X. et al.CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.Proc. Natl. Acad. Sci. USA. 2013; 110: 12108-12113Crossref PubMed Scopus (272) Google Scholar). More strikingly, one of CYP76AH sub-family members, CYP76AH3, was reported to be involved in tanshinone biosynthesis (Guo et al., 2016Guo J. Ma X. Cai Y. Ma Y. Zhan Z. Zhou Y.J. Liu W. Guan M. Yang J. Cui G. et al.Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.New Phytol. 2016; 210: 525-534Crossref PubMed Scopus (133) Google Scholar), further confirming the association of these biosynthetic gene clusters with tanshinone biosynthesis. Phylogenetic analysis suggests that the SmCPS1 and SmCPS2 clusters originated from a duplication event of an ancestral CPS/CYP76AH pair (Supplemental Figure 2). To further investigate the role of these clusters in tanshinone biosynthesis, the tissue-specific expression of the genes was analyzed using RNA-seq data. As previously reported (Cui et al., 2015Cui G. Duan L. Jin B. Qian J. Xue Z. Shen G. Snyder J.H. Song J. Chen S. Huang L. et al.Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza Bunge.Plant Physiol. 2015; 169: 1607-1618PubMed Google Scholar), SmCPS1 and SmCPS2 are most highly expressed in the roots and leaves/flowers, respectively. However, the expression patterns of the CYP76AH sub-family members do not simply follow that of the co-clustered CPSs. Instead, despite being clustered with the root-specific SmCPS1, CYP76AH12 is equally expressed in both the roots and leaves, although the linked CYP76AH13 is more specifically expressed in roots. In addition, despite being clustered with the more aerial tissue-specific SmCPS2, CYP76AH1 and CYP76AH3 are quite specifically expressed in roots, although the linked CYP76AH28P is more highly expressed in the leaves. All of these expression patterns were validated by qRT–PCR (Figure 1C–1F). Taken together, it seemed to imply that the decoupling of expression between CPSs and their flanking CYPs had occurred after the gene cluster duplication event. The SmCPS7/CYP cluster contains two members of the CYP71 family (Figure 1E), CYP71AT88 and CYP71BS4. Given that a number of CYPs from the CYP71 family are involved in (di)terpenoid biosynthesis (Zi et al., 2014Zi J. Mafu S. Peters R.J. To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism.Annu. Rev. Plant Biol. 2014; 65: 259-286Crossref PubMed Scopus (187) Google Scholar), this raises the possibility that this cluster might participate in a common diterpenoid biosynthetic pathway. For the SmCPS5/CYP cluster (Figure 1F), previous work had suggested that SmCPS5 is involved in gibberellin metabolism (Cui et al., 2015Cui G. Duan L. Jin B. Qian J. Xue Z. Shen G. Snyder J.H. Song J. Chen S. Huang L. et al.Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza Bunge.Plant Physiol. 2015; 169: 1607-1618PubMed Google Scholar), while CYP735A25v1 has no known function in such phytohormone metabolism. Thus, this particular pair of enzymes seems unlikely to operate together in a common pathway. We then compared the tissue-specific expression patterns of all 437 annotated CYP genes with that of SmCPS1. Thirty-two CYPs exhibited similar expression patterns to SmCPS1 across different organs examined (R2 > 0.85) (Supplemental Table 8). As expected, this includes CYP76AH1, whose role in tanshinone biosynthesis was firstly suggested on the basis of the similar co-expression analysis (Guo et al., 2013Guo J. Zhou Y.J. Hillwig M.L. Shen Y. Yang L. Wang Y. Zhang X. Liu W. Peters R.J. Chen X. et al.CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.Proc. Natl. Acad. Sci. USA. 2013; 110: 12108-12113Crossref PubMed Scopus (272) Google Scholar), as well as CYP76AH3 and CYP76AK1, which were recently demonstrated to play important roles in tanshinone biosynthesis using the same approach (Guo et al., 2016Guo J. Ma X. Cai Y. Ma Y. Zhan Z. Zhou Y.J. Liu W. Guan M. Yang J. Cui G. et al.Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.New Phytol. 2016; 210: 525-534Crossref PubMed Scopus (133) Google Scholar). Hence, the remaining co-regulated CYPs provide additional candidates for dissecting the tanshinone biosynthetic pathway. The traditional use of Danshen involves decoction with water, indicating an important role for the hydrophilic phenolic acids. These include rosmarinic acid (RA), salvianolic acid, and lithospermic acid B, whose biosynthesis involves both general phenylpropanoid metabolism and the more specific tyrosine-derived pathway. As reported previously, the genome contains 29 genes from nine families potentially involved in S. miltiorrhiza phenolic acid biosynthesis. Notably, most families had multiple genes with distinct expression patterns, implying diversified roles for these natural products. In addition, from the 80 laccases genes, five were potentially involved in the conversion of RA to salvianolic acid, based on their specific expression in the root phloem and xylem tissues. Thus, the genome sequence reported here provides important insights into the biosynthesis of these water-soluble natural products as well. Compared with the S. miltiorrhiza variety with purple flowers that was used for genome sequencing, the white-flowered landrace of S. miltiorrhiza is known for better medical quality. To evaluate the genetic differences between these varieties, a white-flowered plant was selected for sequencing and subsequent comparative analysis. The number of homozygous SNPs (1 719 024) was roughly twice than that of heterozygotes, corresponding to a fixed polymorphism level of 3.87 SNPs per kb. Overall, 49 521 non-synonymous SNPs were identified, among which 580 protein-coding genes were affected through the formation of premature stop codons. Nine KEGG pathways were significantly enriched with non-synonymous amino acid changes, including pathways for diterpenoid, flavonoid, and phenylpropanoid biosynthesis, as well as those for Toll-like receptor signaling and plant–pathogen interactions (Supplemental Figure 3). While the average sequencing depth for the white-flower plant was 42X, more than 10% of the genome had no coverage at all. For further investigation, 28.6 Mb genomic regions longer than 1 kb with no mapping coverage were analyzed. Interestingly, only 12.68% of these regions were composed of repetitive sequences, a much lower proportion than the genome average (54.44%). In total, these regions contained 107 genes, which appear to have been lost in the white-flower landrace, including 11 disease-resistance genes, four CYPs, and 13 TF encoding genes. At least some of this intergenomic diversity is hypothesized to contribute to the phenotypic differences between these two varieties, such as flower coloration, and tanshinone content, which require future investigations. In summary, we present a draft assembly of the S. miltiorrhiza genome using long reads from the PacBio RS platform to supplement short Illumina reads, which resulted in significant improvement of the assembly quality. This hybrid approach is proved to be effective for the highly repetitive and complex genome of S. miltiorrhiza, enabling the assembly of sufficiently large enough scaffolds for the identification of potential biosynthetic gene clusters. The four CPS/CYP gene clusters revealed here, along with other genes potentially encoding biosynthetic enzymes (e.g., in tashinone biosynthesis; Supplemental Table 9), provide a strong foundation for understanding the biochemical diversity and pharmaceutical qualities of S. miltiorrhiza. Moreover, access to the genome sequence is further expected to aid molecular breeding with this important traditional medicinal herb. This work was supported by the National Natural Science Foundation of China (81130069, 81573398, 31400278), the National Key Technology R&D Program (2012BAI29B01), the Key Project of Chinese National Programs for Fundamental Research and Development (2013CB127000), and the US National Institutes of Health (GM109773).

Ginseng, which contains ginsenosides as bioactive compounds, has been regarded as an important traditional medicine for several millennia. However, the genetic background of ginseng remains poorly understood, partly because of the plant's large and complex genome composition. We report the entire genome sequence of Panax ginseng using next-generation sequencing. The 3.5-Gb nucleotide sequence contains more than 60% repeats and encodes 42 006 predicted genes. Twenty-two transcriptome datasets and mass spectrometry images of ginseng roots were adopted to precisely quantify the functional genes. Thirty-one genes were identified to be involved in the mevalonic acid pathway. Eight of these genes were annotated as 3-hydroxy-3-methylglutaryl-CoA reductases, which displayed diverse structures and expression characteristics. A total of 225 UDP-glycosyltransferases (UGTs) were identified, and these UGTs accounted for one of the largest gene families of ginseng. Tandem repeats contributed to the duplication and divergence of UGTs. Molecular modeling of UGTs in the 71st, 74th, and 94th families revealed a regiospecific conserved motif located at the N-terminus. Molecular docking predicted that this motif captures ginsenoside precursors. The ginseng genome represents a valuable resource for understanding and improving the breeding, cultivation, and synthesis biology of this key herb.

To test whether the ITS2 region is an effective marker for use in authenticating of the family Fabaceae which contains many important medicinal plants. The ITS2 regions of 114 samples in Fabaceae were amplified. Sequence assembly was assembled by CodonCode Aligner V3.0. In combination with sequences from public database, the sequences were aligned by Clustal W, and genetic distances were computed using MEGA V4.0. The intra- vs. inter-specific variations were assessed by six metrics, wilcoxon two-sample tests and “barcoding gaps”. Species identification was accomplished using TaxonGAP V2.4, BLAST1 and the nearest distance method. ITS2 sequences had considerable variation at the genus and species level. The intra-specific divergence ranged from 0% to 14.4%, with an average of 1.7%, and the inter-specific divergence ranged from 0% to 63.0%, with an average of 8.6%. Twenty-four species found in the Chinese Pharmacopoeia, along with another 66 species including their adulterants, were successfully identified based on ITS2 sequences. In addition, ITS2 worked well, with over 80.0% of species and 100% of genera being correctly differentiated for the 1507 sequences derived from 1126 species belonging to 196 genera. Our findings support the notion that ITS2 can be used as an efficient and powerful marker and a potential barcode to distinguish various species in Fabaceae.

Five DNA regions, namely, rbcL, matK, ITS, ITS2, and psbA-trnH, have been recommended as primary DNA barcodes for plants. Studies evaluating these regions for species identification in the large plant taxon, which includes a large number of closely related species, have rarely been reported.The feasibility of using the five proposed DNA regions was tested for discriminating plant species within Asteraceae, the largest family of flowering plants. Among these markers, ITS2 was the most useful in terms of universality, sequence variation, and identification capability in the Asteraceae family. The species discriminating power of ITS2 was also explored in a large pool of 3,490 Asteraceae sequences that represent 2,315 species belonging to 494 different genera. The result shows that ITS2 correctly identified 76.4% and 97.4% of plant samples at the species and genus levels, respectively. In addition, ITS2 displayed a variable ability to discriminate related species within different genera.ITS2 is the best DNA barcode for the Asteraceae family. This approach significantly broadens the application of DNA barcoding to resolve classification problems in the family Asteraceae at the genera and species levels.

© The Willi Hennig Society 2010. ABSTRACT: The Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG) established the use of matK+rbcL as core barcodes and ITS2 as one of the supplementary loci for differentiating plants at the Third International Barcoding Conference. Here, we tested the applicability of four DNA regions (rbcL, matK, rpoC1 and ITS2) as the barcodes for identifying species within Rosaceae. Based on assessments of the success rates of PCR amplifications, the sequence quality, extent of specific genetic divergence, DNA barcoding gap and ability for species discrimination, our results suggest that ITS2 is the best of the four loci tested for barcoding Rosaceae. We further evaluated the effectiveness of ITS2 for identifying a wide range of species within Rosaceae. Of the 1410 plant samples collected from 893 species in 96 diverse genera, ITS2 successfully identified 78 and 100% of them at the species and genus levels, respectively. Therefore, our research indicated that the ITS2 region is a powerful, though not perfect, barcode for Rosaceae identification that also contributes valuable information for identifying closely related species in other plant taxonomic groups.

Panax ginseng C. A. Meyer is one of the most widely used medicinal plants. Complete genome information for this species remains unavailable due to its large genome size. At present, analysis of expressed sequence tags is still the most powerful tool for large-scale gene discovery. The global expressed sequence tags from P. ginseng tissues, especially those isolated from stems, leaves and flowers, are still limited, hindering in-depth study of P. ginseng.Two 454 pyrosequencing runs generated a total of 2,423,076 reads from P. ginseng roots, stems, leaves and flowers. The high-quality reads from each of the tissues were independently assembled into separate and shared contigs. In the separately assembled database, 45,849, 6,172, 4,041 and 3,273 unigenes were only found in the roots, stems, leaves and flowers database, respectively. In the jointly assembled database, 178,145 unigenes were observed, including 86,609 contigs and 91,536 singletons. Among the 178,145 unigenes, 105,522 were identified for the first time, of which 65.6% were identified in the stem, leaf or flower cDNA libraries of P. ginseng. After annotation, we discovered 223 unigenes involved in ginsenoside backbone biosynthesis. Additionally, a total of 326 potential cytochrome P450 and 129 potential UDP-glycosyltransferase sequences were predicted based on the annotation results, some of which may encode enzymes responsible for ginsenoside backbone modification. A BLAST search of the obtained high-quality reads identified 14 potential microRNAs in P. ginseng, which were estimated to target 100 protein-coding genes, including transcription factors, transporters and DNA binding proteins, among others. In addition, a total of 13,044 simple sequence repeats were identified from the 178,145 unigenes.This study provides global expressed sequence tags for P. ginseng, which will contribute significantly to further genome-wide research and analyses in this species. The novel unigenes identified here enlarge the available P. ginseng gene pool and will facilitate gene discovery. In addition, the identification of microRNAs and the prediction of targets from this study will provide information on gene transcriptional regulation in P. ginseng. Finally, the analysis of simple sequence repeats will provide genetic makers for molecular breeding and genetic applications in this species.

Summary A circular consensus sequencing ( CCS ) strategy involving single molecule, real‐time ( SMRT ) DNA sequencing technology was applied to de novo assembly and single nucleotide polymorphism ( SNP ) detection of chloroplast genomes. Chloroplast DNA was purified from enriched chloroplasts of pooled individuals to construct a shotgun library for each species. The sequencing reactions were performed on a PacBio RS platform. CCS sub‐reads were generated from polymerase reads that passed the native dumbbell‐shaped DNA templates multiple times. The complete chloroplast genome sequence was generated by mapping all reads to the draft sequence constructed in a step‐by‐step manner. The full‐chain, PCR ‐free approach eliminates the possible context‐specific biases in library construction and sequencing reaction. The chloroplast genome was easily and completely assembled using the data generated from one SMRT Cell without requiring a reference genome. Comparisons of the three assembled Fritillaria genomes to 34.1 kb of validation Sanger sequences revealed 100% concordance, and the detected intraspecies SNP s at a minimum variant frequency of 15% were all confirmed. This simple approach with potential for parallel sequencing yields high‐quality chloroplast genomes for sensitive SNP detection and comparative analyses. We recommend this approach for its powerful applicability for evolutionary genetics and genomics studies in plants based on the sequences of chloroplast genomes.

An ideal DNA barcoding region should be short enough to be amplified from degraded DNA. In this paper, we discuss the possibility of using a short nuclear DNA sequence as a barcode to identify a wide range of medicinal plant species. First, the PCR and sequencing success rates of ITS and ITS2 were evaluated based entirely on materials from dry medicinal product and herbarium voucher specimens, including some samples collected back to 90 years ago. The results showed that ITS2 could recover 91% while ITS could recover only 23% efficiency of PCR and sequencing by using one pair of primer. Second, 12861 ITS and ITS2 plant sequences were used to compare the identification efficiency of the two regions. Four identification criteria (BLAST, inter- and intradivergence Wilcoxon signed rank tests, and TaxonDNA) were evaluated. Our results supported the hypothesis that ITS2 can be used as a minibarcode to effectively identify species in a wide variety of specimens and medicinal materials.

Salvia miltiorrhiza is a Chinese herb with significant pharmacologic effects because of the bioactive compounds of tanshinones and phenolic acids. Methyl jasmonate ( MeJA ) has been used as an effective elicitor to enhance the production of these compounds. However, the molecular mechanism of MeJA ‐mediated tanshinone and salvianolic acid biosynthesis remains unclear. The transcriptional profiles of S. miltiorrhiza leaves at 12 h ( T12 ) after MeJA elicitation and mock‐treated leaves ( T0 ) were generated using the Illumina deep RNA sequencing ( RNA ‐seq) strategy to detect the changes in gene expression in response to MeJA . In total, 37 647 unique sequences were obtained from about 21 million reads, and 25 641 (71.53%) of these sequences were annotated based on the blast searches against the public databases. A total of 5287 unique sequences were expressed differentially between the samples of T0 and T12 , which covered almost all the known genes involved in tanshinone and phenolic acid biosynthesis in S. miltiorrhiza . Many of the transcription factors (e.g. MYB , bHLH and WRKY ) and genes involved in plant hormone biosynthesis and signal transduction were expressed differentially in response to the MeJA induction. Importantly, three and four candidate cytochrome P450s ( P450 s) that could be involved in the tanshinone and phenolic acid biosynthesis, respectively, were selected from the RNA ‐seq data based on co‐expressed pattern analysis with SmCPS1 / SmKSL1 and SmRAS , which are the key genes responsible for biosynthesis. This comprehensive investigation of MeJA ‐induced gene expression profiles can shed light on the molecular mechanisms of the MeJA ‐mediated bioactive compound biosynthesis and regulation in S. miltiorrhiza .

Salvia miltiorrhiza Bunge (Labiatae) is an emerging model plant for traditional medicine, and tanshinones are among the pharmacologically active constituents of this plant. Although extensive chemical and pharmaceutical studies of these compounds have been performed, studies on the basic helix-loop-helix (bHLH) transcription factors that regulate tanshinone biosynthesis are limited. In our study, 127 bHLH transcription factor genes were identified in the genome of S. miltiorrhiza, and phylogenetic analysis indicated that these SmbHLHs could be classified into 25 subfamilies. A total of 19 sequencing libraries were constructed for expression pattern analyses using RNA-Seq. Based on gene-specific expression patterns and up-regulated expression patterns in response to MeJA treatment, 7 bHLH genes were revealed as potentially involved in the regulation of tanshinone biosynthesis. Among them, the gene expression of SmbHLH37, SmbHLH74 and SmbHLH92 perfectly matches the accumulation pattern of tanshinone biosynthesis in S. miltiorrhiza. Our results provide a foundation for understanding the molecular basis and regulatory mechanisms of bHLH transcription factors in S. miltiorrhiza.

Camptotheca acuminata is a Nyssaceae plant, often called the "happy tree", which is indigenous in Southern China. C. acuminata produces the terpenoid indole alkaloid, camptothecin (CPT), which exhibits clinical effects in various cancer treatments. Despite its importance, little is known about the transcriptome of C. acuminata and the mechanism of CPT biosynthesis, as only few nucleotide sequences are included in the GenBank database.From a constructed cDNA library of young C. acuminata leaves, a total of 30,358 unigenes, with an average length of 403 bp, were obtained after assembly of 74,858 high quality reads using GS De Novo assembler software. Through functional annotation, a total of 21,213 unigenes were annotated at least once against the NCBI nucleotide (Nt), non-redundant protein (Nr), Uniprot/SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Arabidopsis thaliana proteome (TAIR) databases. Further analysis identified 521 ESTs representing 20 enzyme genes that are involved in the backbone of the CPT biosynthetic pathway in the library. Three putative genes in the upstream pathway, including genes for geraniol-10-hydroxylase (CaPG10H), secologanin synthase (CaPSCS), and strictosidine synthase (CaPSTR) were cloned and analyzed. The expression level of the three genes was also detected using qRT-PCR in C. acuminata. With respect to the branch pathway of CPT synthesis, six cytochrome P450s transcripts were selected as candidate transcripts by detection of transcript expression in different tissues using qRT-PCR. In addition, one glucosidase gene was identified that might participate in CPT biosynthesis. For CPT transport, three of 21 transcripts for multidrug resistance protein (MDR) transporters were also screened from the dataset by their annotation result and gene expression analysis.This study produced a large amount of transcriptome data from C. acuminata by 454 pyrosequencing. According to EST annotation, catalytic features prediction, and expression analysis, novel putative transcripts involved in CPT biosynthesis and transport were discovered in C. acuminata. This study will facilitate further identification of key enzymes and transporter genes in C. acuminata.

Ophiocordyceps sinensis is a highly valuable and popular medicinal fungus used as a tonic and roborant for thousands of years in traditional Asian medicine. However, unsustainable harvesting practices have endangered this species and very little is known about its developmental programming, its biochemistry and genetics. To begin to address this, the transcriptome of the medicinal O. sinensis fruiting body was analyzed by high-throughput. In this O. sinensis 454-EST dataset, four mating type genes and 121 genes that may be involved in fruiting body development, especially in signal transduction and transcription regulation, were discovered. Moreover, a model was developed for the synthesis of the primary medicinal compound, cordycepin, and the putative biosynthetic enzymes identified. This transcriptome dataset provides a significant new resource for gene discovery in O. sinensis and dissection of its valuable biosynthetic and developmental pathways.

Fungi have evolved powerful genomic and chemical defense systems to protect themselves against genetic destabilization and other organisms. However, the precise molecular basis involved in fungal defense remain largely unknown in Basidiomycetes. Here the complete genome sequence, as well as DNA methylation patterns and small RNA transcriptomes, was analyzed to provide a holistic overview of secondary metabolism and defense processes in the model medicinal fungus, Ganoderma sinense. We reported the 48.96 Mb genome sequence of G. sinense, consisting of 12 chromosomes and encoding 15,688 genes. More than thirty gene clusters involved in the biosynthesis of secondary metabolites, as well as a large array of genes responsible for their transport and regulation were highlighted. In addition, components of genome defense mechanisms, namely repeat-induced point mutation (RIP), DNA methylation and small RNA-mediated gene silencing, were revealed in G. sinense. Systematic bioinformatic investigation of the genome and methylome suggested that RIP and DNA methylation combinatorially maintain G. sinense genome stability by inactivating invasive genetic material and transposable elements. The elucidation of the G. sinense genome and epigenome provides an unparalleled opportunity to advance our understanding of secondary metabolism and fungal defense mechanisms.

DNA double-strand breaks (DSBs) are among the most lethal types of DNA damage and frequently cause genome instability. Sequencing-based methods for mapping DSBs have been developed but they allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. Here we propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induce spike-in DSBs by a site-specific endonuclease and use them to quantify detected DSBs (labeled, e.g., using i-BLESS). Utilizing qDSB-Seq, we determine numbers of DSBs induced by a radiomimetic drug and replication stress, and reveal two orders of magnitude differences in DSB frequencies. We also measure absolute frequencies of Top1-dependent DSBs at natural replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.

In this study, we tested the applicability of four DNA regions (rbcL, matK, ITS, and ITS2) as barcodes for identifying species within Euphorbiaceae. Based on assessments of the specific genetic divergence, the DNA barcoding gap, and the ability for species discrimination, the present results affirmed that ITS/ITS2 is a potential barcode for the Euphorbiaceae species. This study also provided a large-scale test to evaluate the effectiveness of ITS/ITS2 for differentiating species within Euphorbiaceae. Of the 1183 plant samples collected from 871 species in 66 diverse genera, ITS/ITS2 successfully identified > 90% and 100% of them at the species and genus levels, respectively. Therefore, our research indicates that use of the ITS/ITS2 region is a powerful technique for Euphorbiaceae identification.

Background Lonicera japonica Thunb. is a plant used in traditional Chinese medicine known for its anti-inflammatory, anti-oxidative, anti-carcinogenic, and antiviral pharmacological properties. The major active secondary metabolites of this plant are chlorogenic acid (CGA) and luteoloside. While the biosynthetic pathways of these metabolites are relatively well known, the genetic information available for this species, especially the biosynthetic pathways of its active ingredients, is limited. Methodology/Principal Findings We obtained one million reads (average length of 400 bp) in a whole sequence run using a Roche/454 GS FLX titanium platform. Altogether, 85.69% of the unigenes covering the entire life cycle of the plant were annotated and 325 unigenes were assigned to secondary metabolic pathways. Moreover, 2039 unigenes were predicted as transcription factors. Nearly all of the possible enzymes involved in the biosynthesis of CGA and luteoloside were discovered in L. japonica. Three hydroxycinnamoyl transferase genes, including two hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase genes and one hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) gene featuring high similarity to known genes from other species, were cloned. The HCT gene was discovered for the first time in L. japonica. In addition, 188 candidate cytochrome P450 unigenes and 245 glycosyltransferase unigenes were found in the expressed sequence tag (EST) dataset. Conclusion This study provides a high quality EST database for L. japonica by 454 pyrosequencing. Based on the EST annotation, a set of putative genes involved in CGA and luteoloside biosynthetic pathways were discovered. The database serves as an important source of public information on genetic markers, gene expression, genomics, and functional genomics in L. japonica.

Abstract Traditional herbal medicines adulterated and contaminated with plant materials from the Aristolochiaceae family, which contain aristolochic acids (AAs), cause aristolochic acid nephropathy. Approximately 256 traditional Chinese patent medicines, containing Aristolochiaceous materials, are still being sold in Chinese markets today. In order to protect consumers from health risks due to AAs, the hidden assassins, efficient methods to differentiate Aristolochiaceous herbs from their putative substitutes need to be established. In this study, 158 Aristolochiaceous samples representing 46 species and four genera as well as 131 non-Aristolochiaceous samples representing 33 species, 20 genera and 12 families were analyzed using DNA barcodes based on the ITS2 and psbA-trnH sequences. Aristolochiaceous materials and their non-Aristolochiaceous substitutes were successfully identified using BLAST1, the nearest distance method and the neighbor-joining (NJ) tree. In addition, based on sequence information of ITS2, we developed a Real-Time PCR assay which successfully identified herbal material from the Aristolochiaceae family. Using Ultra High Performance Liquid Chromatography-Mass Spectrometer (UHPLC-HR-MS), we demonstrated that most representatives from the Aristolochiaceae family contain toxic AAs. Therefore, integrated DNA barcodes, Real-Time PCR assays using TaqMan probes and UHPLC-HR-MS system provides an efficient and reliable authentication system to protect consumers from health risks due to the hidden assassins (AAs).

Although sperm is transcriptionally and translationally quiescent, complex populations of RNAs, including mRNAs and non-coding RNAs, exist in sperm. Previous microarray analysis of germ cell mutants identified hundreds of sperm genes in Caenorhabditis elegans. To take a more comprehensive view on C. elegans sperm genes, here, we isolate highly pure sperm cells and employ high-throughput technologies to obtain sperm transcriptome and proteome. First, sperm transcriptome consists of considerable amounts of non-coding RNAs, many of which have not been annotated and may play functional roles during spermatogenesis. Second, apart from kinases/phosphatases as previously reported, ion binding proteins are also enriched in sperm, underlying the crucial roles of intracellular ions in post-translational regulation in sperm. Third, while the majority of sperm genes/proteins have low abundance, a small number of sperm genes/proteins are hugely enriched in sperm, implying that sperm only rely on a small set of proteins for post-translational regulation. Lastly, by extensive RNAi screening of sperm enriched genes, we identified a few genes that control fertility. Our further analysis reveals a tight correlation between sperm transcriptome and sperm small RNAome, suggesting that the endogenous siRNAs strongly repress sperm genes. This leads to an idea that the inefficient RNAi screening of sperm genes, a phenomenon currently with unknown causes, might result from the competition between the endogenous RNAi pathway and the exogenous RNAi pathway. Together, the obtained sperm transcriptome and proteome serve as valuable resources to systematically study spermatogenesis in C. elegans.

Tanshinones and phenolic acids are the major bioactive constituents in the traditional medicinal crop Salvia miltiorrhiza ; however, transcription factors (TFs) are seldom investigated with regard to their regulation of the biosynthesis of these compounds. Here a complete overview of the APETALA2/ethylene‐responsive factor (AP2/ERF) transcription factor family in S. miltiorrhiza is provided, including phylogeny, gene structure, conserved motifs, and gene expression profiles of different organs (root, stem, leaf, flower) and root tissues (periderm, phloem, xylem). In total, 170 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (25 genes), DREB (61 genes), ethylene responsive factor (ERF; 79 genes), RAV (4 genes), and Soloist (1 gene). According to the distribution of bioactive constituents and the expression patterns of AP2/ERF genes in different organs and root tissues, the genes related to the biosynthesis of bioactive constituents were selected. On the basis of quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis, coexpression analysis, and the prediction of cis ‐regulatory elements in the promoters, we propose that two genes ( Sm128 and Sm152 ) regulate tanshinone biosynthesis and two genes ( Sm008 and Sm166 ) participate in controlling phenolic acid biosynthesis. The genes related to tanshinone biosynthesis belong to the ERF‐B3 subgroup. In contrast, the genes predicted to regulate phenolic acid biosynthesis belong to the ERF‐B1 and ERF‐B4 subgroups. These results provide a foundation for future functional characterization of AP2/ERF genes to enhance the biosynthesis of the bioactive compounds of S. miltiorrhiza .

Abstract Maintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

Medicinal pteridophytes are an important group used in traditional Chinese medicine; however, there is no simple and universal way to differentiate various species of this group by morphological traits. A novel technology termed "DNA barcoding" could discriminate species by a standard DNA sequence with universal primers and sufficient variation. To determine whether DNA barcoding would be effective for differentiating pteridophyte species, we first analyzed five DNA sequence markers (psbA-trnH intergenic region, rbcL, rpoB, rpoC1, and matK) using six chloroplast genomic sequences from GeneBank and found psbA-trnH intergenic region the best candidate for availability of universal primers. Next, we amplified the psbA-trnH region from 79 samples of medicinal pteridophyte plants. These samples represented 51 species from 24 families, including all the authentic pteridophyte species listed in the Chinese pharmacopoeia (2005 version) and some commonly used adulterants. We found that the sequence of the psbA-trnH intergenic region can be determined with both high polymerase chain reaction (PCR) amplification efficiency (94.1%) and high direct sequencing success rate (81.3%). Combined with GeneBank data (54 species cross 12 pteridophyte families), species discriminative power analysis showed that 90.2% of species could be separated/identified successfully by the TaxonGap method in conjunction with the Basic Local Alignment Search Tool 1 (BLAST1) method. The TaxonGap method results further showed that, for 37 out of 39 separable species with at least two samples each, between-species variation was higher than the relevant within-species variation. Thus, the psbA-trnH intergenic region is a suitable DNA marker for species identification in medicinal pteridophytes.

Abstract In 2009, the Consortium for the Barcode of Life (CBOL) recommended the combination of rbcL and matK as the plant barcode based on assessments of recoverability, sequencing quality, and levels of species discrimination. Subsequently, based on a study of more than 6600 samples belonging to 193 families from seven phyla, the internal transcribed spacer (ITS) 2 locus was proposed as a universal barcode sequence for all major plant taxa used in traditional herbal medicine. Neither of these two studies was based on a detailed analysis of a particular family. Here, Zingiberaceae plants, including many closely related species, were used to compare the genetic divergence and species identification efficiency of ITS2, rbcL , matK , psbK – psbI , trnH – psbA , and rpoB . The results indicate that ITS2 has the highest interspecific divergence and significant differences between inter‐ and intraspecific divergence, whereas matK and rbcL have much lower divergence values. Among 260 species belonging to 30 genera in Zingiberaceae, the discrimination ability of the ITS2 locus was 99.5% at the genus level and 73.1% at the species level. Thus, we propose that ITS2 is the preferred DNA barcode sequence for identifying Zingiberaceae plants.

Simple sequence repeats (SSRs) or microsatellites are one of the most popular sources of genetic markers and play a significant role in gene function and genome organization. We identified SSRs in the genome of Ganoderma lucidum and analyzed their frequency and distribution in different genomic regions. We also compared the SSRs in G. lucidum with six other Agaricomycetes genomes: Coprinopsis cinerea, Laccaria bicolor, Phanerochaete chrysosporium, Postia placenta, Schizophyllum commune and Serpula lacrymans. Based on our search criteria, the total number of SSRs found ranged from 1206 to 6104 and covered from 0.04% to 0.15% of the fungal genomes. The SSR abundance was not correlated with the genome size, and mono- to tri-nucleotide repeats outnumbered other SSR categories in all of the species examined. In G. lucidum, a repertoire of 2674 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. The highest SSR relative abundance was found in introns (108 SSRs/Mb), followed by intergenic regions (84 SSRs/Mb). A total of 684 SSRs were found in the protein-coding sequences (CDSs) of 588 gene models, with 81.4% of them being tri- or hexa-nucleotides. After scanning for InterPro domains, 280 of these genes were successfully annotated, and 215 of them could be assigned to Gene Ontology (GO) terms. SSRs were also identified in 28 bioactive compound synthesis-related gene models, including one 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), three polysaccharide biosynthesis genes and 24 cytochrome P450 monooxygenases (CYPs). Primers were designed for the identified SSR loci, providing the basis for the future development of SSR markers of this medicinal fungus.

Dioscorea is an important plant genus in terms of food supply and pharmaceutical applications. However, its classification and identification are controversial. DNA barcoding is a recent aid to taxonomic identification and uses a short standardized DNA region to discriminate plant species. In this study, the applicability of three candidate DNA barcodes (rbcL, matK, and psbA-trnH) to identify species within Dioscorea was tested.One-hundred and forty-eight individual plant samples of Dioscorea, encompassing 38 species, seven varieties and one subspecies, representing majority species distributed in China of this genus, were collected from its main distributing areas. Samples were assessed by PCR amplification, sequence quality, extent of specific genetic divergence, DNA barcoding gap, and the ability to discriminate between species. matK successfully identified 23.26% of all species, compared with 9.30% for rbcL and 11.63% for psbA-trnH. Therefore, matK is recommended as the best DNA barcoding candidate. We found that the combination of two or three loci achieved a higher success rate of species discrimination than one locus alone. However, experimental cost would be much higher if two or three loci, rather than a single locus, were assessed.We conclude that matK is a strong, although not perfect, candidate as a DNA barcode for Dioscorea identification. This assessment takes into account both its ability for species discrimination and the cost of experiments.

RNA editing is a widespread, post-transcriptional molecular phenomenon that diversifies hereditary information across various organisms. However, little is known about genome-scale RNA editing in fungi. In this study, we screened for fungal RNA editing sites at the genomic level in Ganoderma lucidum, a valuable medicinal fungus. On the basis of our pipeline that predicted the editing sites from genomic and transcriptomic data, a total of 8906 possible RNA-editing sites were identified within the G. lucidum genome, including the exon and intron sequences and the 5'-/3'-untranslated regions of 2991 genes and the intergenic regions. The major editing types included C-to-U, A-to-G, G-to-A, and U-to-C conversions. Four putative RNA-editing enzymes were identified, including three adenosine deaminases acting on transfer RNA and a deoxycytidylate deaminase. The genes containing RNA-editing sites were functionally classified by the Kyoto Encyclopedia of Genes and Genomes enrichment and gene ontology analysis. The key functional groupings enriched for RNA-editing sites included laccase genes involved in lignin degradation, key enzymes involved in triterpenoid biosynthesis, and transcription factors. A total of 97 putative editing sites were randomly selected and validated by using PCR and Sanger sequencing. We presented an accurate and large-scale identification of RNA-editing events in G. lucidum, providing global and quantitative cataloging of RNA editing in the fungal genome. This study will shed light on the role of transcriptional plasticity in the growth and development of G. lucidum, as well as its adaptation to the environment and the regulation of valuable secondary metabolite pathways.

Corrected by: Erratum for: Identification of Lonicera japonica and its Related Species Using the DNA Barcoding MethodPlanta Med 2011; 77(03): E10-E10DOI: 10.1055/s-0030-1250666

In this study, we tested the applicability of the core DNA barcode matK for identifying species within the Fabaceae family. Based on an evaluation of genetic variation, DNA barcoding gaps, and species discrimination power, matK is a useful barcode for Fabaceae species. Of 1355 plant samples collected from 1079 species belonging to 409 diverse genera, matK precisely identified approximately 80 % and 96 % of them at the species and genus levels, respectively. Therefore, our research indicates that the matK region is a valuable marker for plant species within Fabaceae.

Laccases are widely distributed in plant kingdom catalyzing the polymerization of lignin monolignols. Rosmarinic acid (RA) has a lignin monolignol-like structure and is converted into salvianolic acid B (SAB), which is a representatively effective hydrophilic compound of a well-known medicinal plant Salvia miltiorrhiza and also the final compound of phenolic acids metabolism pathway in the plant. But the roles of laccases in the biosynthesis of SAB are poorly understood. This work systematically characterizes S. miltiorrhiza laccase (SmLAC) gene family and identifies the SAB-specific candidates. Totally, 29 laccase candidates (SmLAC1-SmLAC29) are found to contain three signature Cu-oxidase domains. They present relatively low sequence identity and diverse intron-exon patterns. The phylogenetic clustering of laccases from S. miltiorrhiza and other ten plants indicates that the 29 SmLACs can be divided into seven groups, revealing potential distinct functions. Existence of diverse cis regulatory elements in the SmLACs promoters suggests putative interactions with transcription factors. Seven SmLACs are found to be potential targets of miR397. Putative glycosylation sites and phosphorylation sites are identified in SmLAC amino acid sequences. Moreover, the expression profile of SmLACs in different organs and tissues deciphers that 5 SmLACs (SmLAC7/8/20/27/28) are expressed preferentially in roots, adding the evidence that they may be involved in the phenylpropanoid metabolic pathway. Besides, silencing of SmLAC7, SmLAC20 and SmLAC28, and overexpression of SmLAC7 and SmLAC20 in the hairy roots of S. miltiorrhiza result in diversification of SAB, signifying that SmLAC7 and SmLAC20 take roles in SAB biosynthesis. The results of this study lay a foundation for further elucidation of laccase functions in S. miltiorrhiza, and add to the knowledge for SAB biosynthesis in S. miltiorrhiza.

Chinese caterpillar fungus (Ophiocordyceps sinensis) has been widely used as tonic in Asian medicine. Considering its curative effect and high cost, various counterfeit versions of O. sinensis have been introduced and are commercially available. These counterfeits have morphological characteristics that are difficult to distinguish based on morphology alone, thereby causing confusion and threatening its safe use. In this study, internal transcribed spacer (ITS) sequences as a DNA barcode were analyzed and assessed for rapid and accurate identification of 131 O. sinensis samples and 12 common counterfeits and closely related species. Results showed that sufficient ITS sequence differences, also known as 'barcode gaps', existed to distinguish between O. sinensis and counterfeit species. ITS sequence correctly identified 100% of the samples at the species and genus level using the Basic Local Alignment Search Tool 1 and the nearest distance method. Furthermore, O. sinensis, counterfeits, and closely related species can be successfully identified using tree-based methods including maximum parsimony, neighbor-joining, and maximum likelihood analysis. These results indicated that DNA barcoding could be used as a fast and accurate identification method to distinguish O. sinensis from counterfeits and closely related species to ensure its safe use.

The clinical significance of immune-related antigen

DNA barcoding is a technique in which species identification and discovery are performed by using short and standard fragments of DNA sequences. In this study, eleven species of Paris, including seven varieties, were sampled. Five chloroplast sequences, psbA-trnH, rpoB, rpoC1, rbcL, matK, and one nuclear marker, the second internal transcribed spacer (ITS2) of ribosomal DNA, were amplified and sequenced. The PCR amplification and sequencing efficiency, intra- and inter-specific divergence and barcoding gap were used to evaluate different loci, and the identification efficiency was assessed using BLAST1 and Nearest Distance methods. The ITS2 sequences in the studied samples of Paris were amplified and sequenced successfully using primers designed by our group, while matK showed low level in the amplification and psbA-trnH was difficult for sequencing because of over 800 bp and poly (A) structure. Analysis of the intra- and inter-specific divergence and barcoding gap showed ITS2 was superior to other loci. The ITS2 showed a much higher percentage of success (100%) in identification than other five loci, none of which indicated more than 50% except matK (52.9%). The 2-locus combination of rbcL+matK didn't improve ability of authentication. In addition, the rate of successful identification with ITS2 kept 100% when the samples were expanded to 67 samples of 29 species. In conclusion, ITS2 can be used to correctly identify medicinal plants of Paris, and it will be a potential DNA barcode for identifying medicinal plants of other taxa.

Species of genus Tripterygium (Celastraceae) have attracted much attention owing to their excellent effect on treating autoimmune and inflammatory diseases. However, due to high market demand causing overexploitation, natural populations of genus Tripterygium have rapidly declined. Tripterygium medicinal materials are mainly collected from the wild, making the quality of medicinal materials unstable. Additionally, identification of herbal materials from Tripterygium species and their adulterants is difficult based on morphological characters. Therefore, an accurate, convenient, and stability method is urgently needed. In this wok, we developed a DNA barcoding technique to distinguish T. wilfordii HOOK. f., T. hypoglaucum (LÉVL.) HUTCH, and T. regelii SPRAGUE et TAKEDA and their adulterants based on four uniform and standard DNA regions (internal transcribed spacer 2 (ITS2), matK, rbcL, and psbA–trnH). DNA was extracted from 26 locations of fresh leaves. Phylogenetic tree was constructed with Neighbor–Joining (NJ) method, while barcoding gap was analyzed to assess identification efficiency. Compared with the other DNA barcodes applied individually or in combination, ITS2+psbA–trnH was demonstrated as the optimal barcode. T. hypoglaucum and T. wilfordii can be considered as conspecific, while T. regelii was recognized as a separate species. Furthermore, identification of commercial Tripterygium samples was conducted using BLAST against GenBank and Species Identification System for Traditional Chinese Medicine. Our results indicated that DNA barcoding is a convenient, effective, and stability method to identify and distinguish Tripterygium and its adulterants, and could be applied as the quality control for Tripterygium medicinal preparations and monitoring of the medicinal herb trade in markets.

We developed a novel approach to sensitive detection of formamidopyrimidine-DNA glycosylase activity based on target-induced selfprimed rolling circle amplification and magnetic nanoprobes.

Ascaris suum (large roundworm of pigs) is a parasitic nematode that causes substantial losses to the meat industry. This nematode is suitable for biochemical studies because, unlike C. elegans, homogeneous tissue samples can be obtained by dissection. It has large sperm, produced in great numbers that permit biochemical studies of sperm motility. Widespread study of A. suum would be facilitated by more comprehensive genome resources and, to this end, we have produced a gonad transcriptome of A. suum.Two 454 pyrosequencing runs generated 572,982 and 588,651 reads for germline (TES) and somatic (VAS) tissues of the A. suum gonad, respectively. 86% of the high-quality (HQ) reads were assembled into 9,955 contigs and 69,791 HQ reads remained as singletons. 2.4 million bp of unique sequences were obtained with a coverage that reached 16.1-fold. 4,877 contigs and 14,339 singletons were annotated according to the C. elegans protein and the Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases. Comparison of TES and VAS transcriptomes demonstrated that genes participating in DNA replication, RNA transcription and ubiquitin-proteasome pathways are expressed at significantly higher levels in TES tissues than in VAS tissues. Comparison of the A. suum TES transcriptome with the C. elegans microarray dataset identified 165 A. suum germline-enriched genes (83% are spermatogenesis-enriched). Many of these genes encode serine/threonine kinases and phosphatases (KPs) as well as tyrosine KPs. Immunoblot analysis further suggested a critical role of phosphorylation in both testis development and spermatogenesis. A total of 2,681 A. suum genes were identified to have associated RNAi phenotypes in C. elegans, the majority of which display embryonic lethality, slow growth, larval arrest or sterility.Using deep sequencing technology, this study has produced a gonad transcriptome of A. suum. By comparison with C. elegans datasets, we identified sets of genes associated with spermatogenesis and gonad development in A. suum. The newly identified genes encoding KPs may help determine signaling pathways that operate during spermatogenesis. A large portion of A. suum gonadal genes have related RNAi phenotypes in C. elegans and, thus, might be RNAi targets for parasite control.

Quantitative real-time polymerase chain reaction (qRT-PCR) is widely used for the accurate analysis of gene expression. However, high homology among gene families might result in unsuitability of reference genes, which leads to the inaccuracy of qRT-PCR analysis. The release of the Ganoderma lucidum genome has triggered numerous studies to be done on the homology among gene families with the purpose of selecting reliable reference genes. Based on the G. lucdum genome and transcriptome database, 38 candidate reference genes including 28 novel genes were systematically selected and evaluated for qRT-PCR normalization. The result indicated that commonly used polyubiquitin (PUB), beta-actin (BAT), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were unsuitable reference genes because of the high sequence similarity and low primer specificity. According to the evaluation of RefFinder, cyclophilin 5 (CYP5) was ranked as the most stable reference gene for 27 tested samples under all experimental conditions and eighteen mycelial samples. Based on sequence analysis and expression analysis, our study suggested that gene characteristic, primer specificity of high homologous genes, allele-specificity expression of candidate genes and under-evaluation of reference genes influenced the accuracy and sensitivity of qRT-PCR analysis. This investigation not only revealed potential factors influencing the unsuitability of reference genes but also selected the superior reference genes from more candidate genes and testing samples than those used in the previous study. Furthermore, our study established a model for reference gene analysis by using the genomic sequence.

Fritillaria cirrhosa D. Don is an endangered species in the Liliaceae family, the bulb of which is the primary plant source for the Chinese traditional medicine “Chuan-beimu”, having activities that relieve coughs and eliminates phlegm. The major pharmacologically active constituents of F. cirrhosa are steroidal alkaloids. Two thousand one hundred and fifty-eight high-quality expressed sequence tags (ESTs) were generated from a cDNA library of F. cirrhosa bulbs and assembled to total 1343 unique transcripts. After removing ribosomal RNA sequences, 1330 putative protein coding sequences were obtained; among these, 765 (57.5%) had at least one significant match to the Swiss-Prot protein database via a BLASTX similarity search. The 1330 unique transcripts were further functionally classified for gene discovery purposes using the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases. More than ten transcripts that are likely involved in the biosynthesis of F. cirrhosa alkaloids and corresponding regulatory activities were discovered in this EST dataset, including HMGR, FPSs, CYP450s and aminotransferases. This report describes the first example of EST analysis from the Fritillaria genus and lays the foundation for further cloning and identification of candidate genes related to steroidal alkaloid biosynthesis in F. cirrhosa.   Key words: Fritillaria cirrhosa, expressed sequence tags, steroidal alkaloids.

This study aimed to provide guidance for the heterogenous gene expression, gene prediction and species evolution by analyzing codon usage bias of Catharanthus roseus.The codon composition and usage bias of 30 437 high-confidence coding sequences from C.roseus were analyzed and the proportion of rare codons of Escherichia coli and Saccharomyces cerevisiae in 25 genes involved in the biosynthesis of terpenoid indole alkaloids (TIAs) in C.roseus were calculated.The results showed that the average GC content of the genes was 42.47%; the average GC content of the third bases in codon was 35.89%.The relative synonymous codon usage (RSCU) of 28 codons were greater than 1 and 26 of them ended with A or T.The above 25 genes involved in TIA biosynthesis contained much more rare condons of E.coli than that of S.cerevisiae.It was concluded that C.roseus mainly prefered the codons ending with A or T and the rule of codon usage was more different to E.coli than S.cerevisiae.Thus, S.cerevisiae may be more suitable host for heterologous expression of these genes.

The dried succulent stems of Cistanche (Cistanche deserticola Y. C. Ma and Cistanche tubulosa Wight.) are one of the most widely used components of traditional Chinese medicines. However, it is often confused and substituted with the roots of Orobanche pycnostachya, Boschniakia rossica (Cham. & Schltdl.) Standl., Cistanche sinensis Beck, and Cistanche salsa (C. A. Mey.) Beck. In this study, we identified psbA-trnH regions from species and tested their suitable for the identification of the above mentioned taxa. The psbA-trnH sequences showed considerable variations between species and thus were revealed as a promising candidate for barcoding of Cistanche species. Additionally, the average genetic distance of psbA-trnH ranging from 0.077% to 0.743%. In contrast, the intra-specific variation among Cistanche species was found to be significantly different from those of other species, with percentages of variation studied ranged from 0% to 0.007%. The sequence difference between the psbA-trnH sequences of Cistanche species and Orobanche pycnostachya ranged from 0.979% to 1.149%. The distance between the Cistanche species and Boschniakia rossica ranged from 1.066% to 1.224%. Our results suggest that the psbA-trnH intergenic spacer region represent a barcode that can be used to identify Cistanche species and other morphologically undistinguishable species.

Magnoliaceae species have high ornamental and medicinal importance, but they are morphologically similar. DNA barcoding has been regarded as a rapid and effective approach for species identification. To determine the efficiency of expedient identification in Magnoliaceae species by DNA barcoding, in this study, we collected 83 samples belonging to 68 species in 10 genera of Magnoliaceae. Candidate DNA regions (i.e., psbA-trnH, matK, rbcL, ITS, ITS2, rpoB, and rpoC1) were amplified and sequenced for the evaluation of their PCR amplification, sequencing efficiency, intra- and inter-specific divergence and barcoding gap by sequence alignment and Kimura 2-Parameter (K2P) distance analysis, and the rate of correct identification was assessed by BLAST analysis. The results showed that psbA-trnH and matK exhibited high performance in efficiency of PCR amplification and the rate of successful sequencing, followed by rbcL. Associated with the analysis of 199 sequences for 96 species in 9 genera of Magnoliaceae retrieved from GenBank, it was discovered that psbA-trnH was highest in inter-specific divergence and rate of correct identification, indicating its efficiency in the identification of Magnoliaceae species. Besides, matK was also easy to amplify and had high rate of correct identification, suggesting its potential to distinguish Magnoliaceae species. This study indicates that DNA barcoding provides an effective technique for the expedient identification of morphologically similar species, and it is a powerful aid to the conventional methods for species identification.

Abstract DNA double-strand breaks (DSBs) can be detected by label-based sequencing or pulsed-field gel electrophoresis (PFGE). Sequencing yields population-average DSB frequencies genome-wide, while PFGE reveals percentages of broken chromosomes. We constructed a mathematical framework to combine advantages of both: high-resolution DSB locations and their population distribution. We also use sequencing read patterns to identify replication-induced DSBs and active replication origins. We describe changes in spatiotemporal replication program upon hydroxyurea-induced replication stress. We found that one-ended DSBs, resulting from collapsed replication forks, are population-representative, while majority of two-ended DSBs (79-100%) are not. To study replication fork collapse, we used strains lacking the checkpoint protein Mec1 and the endonuclease Mus81 and quantified that 19% and 13% of hydroxyurea-induced one-ended DSBs are Mec1-and Mus81-dependent, respectively. We also clarified that Mus81-induced one-ended DSBs are Mec1-dependent.

DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such as cancer. Therefore, there is a great need for a precise, sensitive, and universal method for DSB detection, to enable both the study of their mechanisms of formation and repair as well as to explore their therapeutic potential. We provide a detailed protocol for our recently developed ultrasensitive and genome-wide DSB detection method: immobilized direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (i-BLESS), which relies on the encapsulation of cells in agarose beads and labeling breaks directly and specifically with biotinylated linkers. i-BLESS labels DSBs with single-nucleotide resolution, allows detection of ultrarare breaks, takes 5 d to complete, and can be applied to samples from any organism, as long as a sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach to enable the measurement of absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Such normalization using qDSB-Seq is especially useful for the evaluation of spontaneous DSB levels and the estimation of DNA damage induced rather uniformly in the genome (e.g., by irradiation or radiomimetic chemotherapeutics). This protocol describes a genome-wide approach for ultrasensitive and quantitative detection of DNA double-strand breaks (DSBs) that relies on encapsulating cells in agarose beads and labeling breaks with biotinylated adapters.

ERF family transcription factor (TF) represented ethylene-responsive protein which harbored a conserved AP2 domain. After searching the plant transcription factor database, a total of 75 unigenes was found which contained AP2 domain from the transcriptome dataset of Panax quinquefolius L. One unique sequence of ERF transcript, named as PqERF1, was cloned with entire open reading frame of 933 base pairs (bp). Protein prediction result indicated that the gene was localized in nucleus and had a conserved AP2 domain. PqERF1 gene could be induced by methyl jasmonate (MeJA) which was consistent to the inducing profile of triterpene ginsenosides. InterproScan prediction indicated that PqERF1 was probably a pathogenesis-related gene. Sequence alignment and phylogenetic analysis demonstrated PqERF1 was with high identity and had relative close relationship to the NtERF4 (Nicotiana tabacum), PhERF12 (Petunia x hybrida) and DcERF1 (Daucus carota) which was related to plant defense, regulation of secondary metabolism and the flower senescence respectively. Therefore, the gene was likely involved in regulation of secondary metabolism, plant defense and physical processes which would provide gene resource for further study on secondary metabolite synthesis and molecular breeding of P. quinquefolius.

Cartilage is frequently used as scaffolds for repairing and reconstructing body surface organs. However, after successful plastic surgery, transplanted cartilage scaffolds often exhibit deformation and absorption over time. To enhance the shapping stability of cartilage scaffolds and improve patients’ satisfaction after reconstructions, we employed the ear folding models in New Zealand rabbit to confirm whether the 1064nm Nd:YAG laser could promote cartilage reshapping. There was an increase in collagen and aromatase (Cyp19) expression within the ear cartilage after laser treatment. Moreover, we have discovered that the Cyp19 inhibitor can suppress the shaping effect of the laser on cartilage, as well as the expression of collagen and Cyp19. The overall findings suggest that treatment with 1064nm Nd:YAG laser irradiation can enhance estrogen levels in local cartilage tissues by upregulating Cyp19 expression in chondrocytes through photobiomodulation, thereby promoting the proliferation and collagen secretion of chondrocytes to improve cartilage reshaping and stability.

Cartilage is frequently used as a scaffolds for repairing and reconstructing body surface organs. However, after successful plastic surgery, transplanted cartilage scaffolds often exhibit deformation and absorption over time. To enhance the shaping stability of cartilage scaffolds and improve patients' satisfaction after reconstructions, we employed the ear folding models in New Zealand rabbits to confirm whether the 1064nm neodymium-doped yttrium aluminum garnet (Nd:YAG) laser could promote cartilage reshaping. There was an increase in collagen and aromatase (Cyp19) expression within the ear cartilage after laser treatment. Moreover, we have found that the Cyp19 inhibitor can inhibit the laser's effect on cartilage shaping and reduce collagen and Cyp19 expression. The overall findings suggest that treatment with 1064nm Nd:YAG laser irradiation can enhance estrogen levels in local cartilage tissues by upregulating Cyp19 expression in chondrocytes through photobiomodulation, thereby promoting the proliferation and collagen secretion of chondrocytes to improve cartilage reshaping and stability.

We present CARAS, a web server that allows the automatic annotation of a chloroplast genome sequence, and the visualization and editing of the annotation results interactively and in real-time. CARAS accepts a complete chloroplast genome sequence as input. First, it accurately predicts protein-coding sequences and exon-intron structures by combining the results from two types of annotation approaches: ab initio prediction algorithms and similarity based methods. Second, tRNA genes and inverted repeats are identified using tRNAscan and vmatch. Using 220 chloroplast genome sequences as test, we show that CARAS outperforms a similar application DOGMA overall. Third, the annotation results are presented as a circular map that includes the name, location, orientation, and length of the various features. A Flex-based module is implemented, allowing the users to add, delete, and edit the features on-line. The results are stored on the server and can be retrieved from a given URL. Users also have options to download the annotation results in GFF3 format for further analyses in other third party software tools or in JPEG format for publication. Finally, CARAS can be used to create a Sequin file for GenBank submission of the annotated genome sequence. CARAS is freely available at http://caras.bicoup.com, and it will significantly facilitate research work involving chloroplast genomes.

Annals of the New York Academy of SciencesVolume 628, Issue 1 p. 151-152 Expression of Transforming Growth Factor-β, Tumor Necrosis Factor-α, and Leukemia Inhibitory Factor mRNAs in Rodent and Human Hematopoietic Cells K. F. WU, Corresponding Author K. F. WUa Address for correspondence: Dr. K. F. Wu, Institute of Hematology, Chinese Academy of Medical Sciences, No. 288 Nanjing Road, Tianjin, 300020, China.Search for more papers by this authorY. M. ZHU, Y. M. ZHU Institute of Hematology Chinese Academy of Medical Sciences Tianjin, 300020, ChinaSearch for more papers by this authorQ. RAO, Q. RAO Institute of Hematology Chinese Academy of Medical Sciences Tianjin, 300020, ChinaSearch for more papers by this authorJ. X. CHU, J. X. CHU Institute of Hematology Chinese Academy of Medical Sciences Tianjin, 300020, ChinaSearch for more papers by this authorJ. M. ZHAO, J. M. ZHAO Institute of Hematology Chinese Academy of Medical Sciences Tianjin, 300020, ChinaSearch for more papers by this author K. F. WU, Corresponding Author K. F. WUa Address for correspondence: Dr. K. F. Wu, Institute of Hematology, Chinese Academy of Medical Sciences, No. 288 Nanjing Road, Tianjin, 300020, China.Search for more papers by this authorY. M. ZHU, Y. M. ZHU Institute of Hematology Chinese Academy of Medical Sciences Tianjin, 300020, ChinaSearch for more papers by this authorQ. RAO, Q. RAO Institute of Hematology Chinese Academy of Medical Sciences Tianjin, 300020, ChinaSearch for more papers by this authorJ. X. CHU, J. X. CHU Institute of Hematology Chinese Academy of Medical Sciences Tianjin, 300020, ChinaSearch for more papers by this authorJ. M. ZHAO, J. M. ZHAO Institute of Hematology Chinese Academy of Medical Sciences Tianjin, 300020, ChinaSearch for more papers by this author First published: July 1991 https://doi.org/10.1111/j.1749-6632.1991.tb17233.xCitations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume628, Issue1Negative Regulators of Hematopoiesis: Studies on Their Nature, Action, and Potential Role in Cancer TherapyJuly 1991Pages 151-152 RelatedInformation

Emerging genome-wide methods for mapping DNA double-strand breaks (DSBs) by sequencing (e.g. BLESS) are limited to measuring relative frequencies of breaks between loci. Knowing the absolute DSB frequency per cell, however, is key to understanding their physiological relevance. Here, we propose quantitative DSB sequencing (qDSB-Seq), a method to infer the absolute DSB frequency genome-wide. qDSB-Seq relies on inducing spike-in DSBs by a site-specific endonuclease and estimating the efficiency of the endonuclease cleavage by sequencing or PCR. This spike-in frequency is used to quantify DSB sequencing data. We present validation of the qDSB-Seq method and results of its application. We quantify DSBs resulting from replication stress and the collapse of replication forks on natural fork barriers in the ribosomal DNA. The qDSB-Seq approach can be used with any DSB sequencing method and allows accurate comparisons of absolute DSB frequencies across samples and precise quantification of the impact of various DSB-causing agents.

Abstract DNA double-strand breaks (DSBs), are a major threat to genomic stability and may lead to cancer. Several technologies to accurately detect DSBs genome-wide have been developed recently, but still lacking publicly available tools for analysis of the resulting data. Here, we present a step-by-step iSeq package ( http://breakome.utmb.edu/software.html ), custom designed for analysis and interpretation of DSB-sequencing data. iSeq performs barcode trimming and read counting, and identifies DSB-enriched regions by statistical test and annotate them to the desired genomic features. Applying this package, users can identify and annotate DSB-enriched regions from base pair (eg. Cas9 cleavage sites) up to megabase (eg. DNA replication stress-induced) resolution, and if possible quantify DSB frequencies per cell genome-wide by combining with qDSB-Seq. iSeq can be used for any sequencing-based DSB detection techniques. The analysis for Steps 1-19 can be performed within ~4 hours.

Codon usage bias is an important characteristic of genetic information transfer in organisms. Analysis of codon usage bias of different species is important for understanding the rules on genetic information transfer. The previous method for analysis of codon usage bias is mainly based on genomic data. However, this method is greatly limited, because the genome sequences of higher organisms are still not available up to now. In this study, we found that we could obtain the same optimal codons of Ganoderma lucidum (Curtis: Fr.) P. Karst based on its whole genomic data or large-scale transcriptomic data from its liquid-cultured hyphae, primordium and fruiting body, separately. This result indicated the feasibility to understand the codon usage bias based on the large-scale transcriptomic data. By calculating the proportion of rare codons of Escherichia coli and Saccharomyces cerevisiae in 26 terpene synthases (TS) of G. lucidum, we found that the rare codons of S. cerevisiae have a higher proportion in TS genes, while the rare codons of E. coli have relatively lower, suggesting that the TS genes of G. lucidum are possibly more difficult to be expressed in S. cerevisiae than in E. coli. Chemical synthesis of TS genes according to the yeast optimal codons will be an effective way to solve the problem on the mismatch of gene codon bias between the foreign genes and the host strain.

Abstract Sequencing-based methods for mapping DNA double-strand breaks (DSBs) allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. We propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induced spike-in DSBs by a site-specific endonuclease and used them to quantify labeled DSBs (e.g. using i-BLESS). Utilizing qDSB-Seq, we determined numbers of DSBs induced by a radiomimetic drug and various forms of replication stress, and revealed several orders of magnitude differences in DSB frequencies. We also measured for the first time Top1-dependent absolute DSB frequencies at replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.

ABSTRACT Solving the structure of large, multi-subunit complexes is difficult despite recent advances in cryoEM, due to remaining challenges to express and purify complex subunits. Computational approaches that predict protein-protein interactions, including Direct Coupling Analysis (DCA), represent an attractive alternative to dissect interactions within protein complexes. However, due to high computational complexity and high false positive rate they are applicable only to small proteins. Here, we present a modified DCA to predict residues and domains involved in interactions of large proteins. To reduce false positive levels and increase accuracy of prediction, we use local Gaussian averaging and predicted secondary structure elements. As a proof-of-concept, we apply our method to two Integrator subunits, INTS9 and INTS11, which form a heterodimeric structure previously solved by crystallography. We accurately predict the domains of INTS9/11 interaction. We then apply this approach to predict the interaction domains of two complexes whose structure is currently unknown: 1) The heterodimer formed by the Cleavage and Polyadenylation Specificity Factor 100-kD (CPSF100) and 73-kD (CPSF73); 2) The heterotrimer formed by INTS4/9/11. Our predictions of interactions within these two complexes are supported by experimental data, demonstrating that our modified DCA is a useful method for predicting interactions and can easily be applied to other complexes.

Twenty six strains of nematophagous fungi were isolated from soil samples collected from Beijing and Yunnan Province. The fungi were identified to belong to 2 genera and 7 species, including Arthrobotrys oligospora, A. conoides, A. vermicola, dactyloides, Monacrosprium eudermatum, M. candidum, and M. thaumasum. A. oligospora was the most frequently-isolated species.

Annals of the New York Academy of SciencesVolume 628, Issue 1 p. 153-155 Suppression of Leukemic Growth by HFDI and LAI-615 K. F. WU, Corresponding Author K. F. WUa Address for correspondence: Dr. K. F. Wu, Institute of Hematology, Chinese Academy of Medical Sciences, No. 288 Nanjing Road, Tianjin, 300020, China.Search for more papers by this authorY. H. SONG, Y. H. SONG Institute of Hematology Chinese Academy of Medical Sciences Tianjin. 300020, ChinaSearch for more papers by this authorH. Z. LI, H. Z. LI Institute of Hematology Chinese Academy of Medical Sciences Tianjin. 300020, ChinaSearch for more papers by this authorQ. RAO, Q. RAO Institute of Hematology Chinese Academy of Medical Sciences Tianjin. 300020, ChinaSearch for more papers by this authorY. M. ZHU, Y. M. ZHU Institute of Hematology Chinese Academy of Medical Sciences Tianjin. 300020, ChinaSearch for more papers by this author K. F. WU, Corresponding Author K. F. WUa Address for correspondence: Dr. K. F. Wu, Institute of Hematology, Chinese Academy of Medical Sciences, No. 288 Nanjing Road, Tianjin, 300020, China.Search for more papers by this authorY. H. SONG, Y. H. SONG Institute of Hematology Chinese Academy of Medical Sciences Tianjin. 300020, ChinaSearch for more papers by this authorH. Z. LI, H. Z. LI Institute of Hematology Chinese Academy of Medical Sciences Tianjin. 300020, ChinaSearch for more papers by this authorQ. RAO, Q. RAO Institute of Hematology Chinese Academy of Medical Sciences Tianjin. 300020, ChinaSearch for more papers by this authorY. M. ZHU, Y. M. ZHU Institute of Hematology Chinese Academy of Medical Sciences Tianjin. 300020, ChinaSearch for more papers by this author First published: July 1991 https://doi.org/10.1111/j.1749-6632.1991.tb17234.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume628, Issue1Negative Regulators of Hematopoiesis: Studies on Their Nature, Action, and Potential Role in Cancer TherapyJuly 1991Pages 153-155 RelatedInformation