[Functional characterization of ent-kaurane-type diterpenoid synthases from Stellera chamaejasme].

Sequential catalysis by ent-copalyl diphosphate(CPS) and ent-kaurene synthase(KS) is a critical step for plants to initiate the biosynthesis of gibberellin with geranylgeranyl pyrophosphate(GGPP) as the substrate. This study mined the transcriptome data of Stellera chamaejasme and cloned two key diterpene synthase genes, SchCPS and SchKS, involved in the gibberellin pathway. The two genes had the complete open reading frames of 2 595 bp and 1 701 bp, encoding two hydrophilic proteins composed of 864 and 566 amino acid residues and with the relative molecular mass of 97.9 kDa and 64.6 kDa and the theoretical isoelectric points of 5.61 and 6.12, respectively. Sequence comparison and phylogenetic tree showed that SchCPS contained LHS, PNV, and DxDD motifs conserved in the CPS family and was categorized in the TPS-c subfamily, while SchKS contained DDxxD, NSE/DTE and PIx motifs conserved in the KS family and was categorized in the TPS-e subfamily. Functional validation showed that SchCPS catalyzed the protonation and cyclization of GGPP to ent-CPP, while SchKS acted on ent-CPP dephosphorylation and re-cyclization to ent-kaurene. In this study, the full-length sequences of SchCPS and SchKS were cloned and functionally verified for the first time, which not only enriched the existing CPS and KS gene libraries but also laid a foundation for the cloning and biosynthesis pathway analysis of more genes involved in the synthesis of active components in S. chamaejasme.

Molecular evolution and characterization of type III polyketide synthase gene family in Aquilaria sinensis.

2-(2-Phenylethyl) chromone (PEC) and its derivatives are markers of agarwood formation and are also related to agarwood quality. However, the biosynthetic and regulatory mechanisms of PECs still remain mysterious. Several studies suggested that type III polyketide synthases (PKSs) contribute to PEC biosynthesis in Aquilaria sinensis. Furthermore, systematic studies on the evolution of PKSs in A. sinensis have rarely been reported. Herein, we comprehensively analyzed PKS genes from 12 plant genomes and characterized the AsPKSs in detail. A unique branch contained only AsPKS members was identified through evolutionary analysis, including AsPKS01 that was previously indicated to participate in PEC biosynthesis. AsPKS07 and AsPKS08, two tandem-duplicated genes of AsPKS01 and lacking orthologous genes in evolutionary models, were selected for their transient expression in the leaves of Nicotiana benthamiana. Subsequently, PECs were detected in the extracts of N. benthamiana leaves, suggesting that AsPKS07 and AsPKS08 promote PEC biosynthesis. The interaction between the promoters of AsPKS07, AsPKS08 and five basic leucine zippers (bZIPs) from the S subfamily indicated that their transcripts could be regulated by these transcription factors (TFs) and might further contribute to PECs biosynthesis in A. sinensis. Our findings provide valuable insights into the molecular evolution of the PKS gene family in A. sinensis and serve as a foundation for advancing PEC production through the bioengineering of gene clusters. Ultimately, this contribution is expected to shed light on the mechanism underlying agarwood formation.

High-throughput sequencing-based analysis of the composition and diversity of the endophyte community in roots of Stellera chamaejasme.

Stellera chamaejasme (S. chamaejasme) is an important medicinal plant with heat-clearing, detoxifying, swelling and anti-inflammatory effects. At the same time, it is also one of the iconic plants of natural grassland degradation in northwest China, playing a key role in the invasion process. Plant endophytes live in healthy plant tissues and can synthesize substances needed for plant growth, induce disease resistance in host plants, and enhance plant resistance to environmental stress. Therefore, studying the root endophytes of S. chamaejasme is of great significance for mining beneficial microbial resources and biological prevention and control of S. chamaejasme. This study used Illumina MiSeq high-throughput sequencing technology to analyze the composition and diversity of endophytes in the roots of S. chamaejasme in different alpine grasslands (BGC, NMC and XGYZ) in Tibet. Research results show that the main phylum of endophytic fungi in the roots of S. chamaejasme in different regions is Ascomycota, and the main phyla of endophytic bacteria are Actinobacteria, Proteobacteria and Firmicutes (Bacteroidota). Overall, the endophyte diversity of the NMC samples was significantly higher than that of the other two sample sites. Principal coordinate analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) results showed significant differences in the composition of endophytic bacterial and fungal communities among BGC, NMC and XGYZ samples. Co-occurrence network analysis of endophytes showed that there were positive correlations between fungi and some negative correlations between bacteria, and the co-occurrence network of bacteria was more complex than that of fungi. In short, this study provides a vital reference for further exploring and utilizing the endophyte resources of S. chamaejasme and an in-depth understanding of the ecological functions of S. chamaejasme endophytes.

Identification and characterization of two O-methyltransferases involved in biosynthesis of methylated 2-(2-phenethyl)chromones in agarwood.

The 2-(2-phenethyl)chromones (PECs) are the signature constituents responsible for the fragrance and pharmacological properties of agarwood. O-Methyltransferases (OMTs) are necessary for the biosynthesis of methylated PECs, but there is little known about OMTs in Aquilaria sinensis. In this study, we identified 29 OMT genes from the A. sinensis genome. Expression analysis showed they were differentially expressed in different tissues and responded to drill wounding. Comprehensive analysis of the gene expression and methylated PEC content revealed that AsOMT2, AsOMT8, AsOMT11, AsOMT16, and AsOMT28 could potentially be involved in methylated PECs biosynthesis. In vitro enzyme assays and functional analysis in Nicotiana benthamiana demonstrated that AsOMT11 and AsOMT16 could methylate 6-hydroxy-2-(2-phenylethyl)chromone to form 6-methoxy-2-(2-phenylethyl)chromone. A transient overexpression experiment in the variety 'Qi-Nan' revealed that AsOMT11 and AsOMT16 could significantly promote the accumulation of three major methylated PECs. Our results provide candidate genes for the mass production of methylated PECs using synthetic biology.

Comprehensive Analysis of NAC Transcription Factors Reveals Their Evolution in Malvales and Functional Characterization of AsNAC019 and AsNAC098 in Aquilaria sinensis.

NAC is a class of plant-specific transcription factors that are widely involved in the growth, development and (a)biotic stress response of plants. However, their molecular evolution has not been extensively studied in Malvales, especially in Aquilaria sinensis, a commercial and horticultural crop that produces an aromatic resin named agarwood. In this study, 1502 members of the NAC gene family were identified from the genomes of nine species from Malvales and three model plants. The macroevolutionary analysis revealed that whole genome duplication (WGD) and dispersed duplication (DSD) have shaped the current architectural structure of NAC gene families in Malvales plants. Then, 111 NAC genes were systemically characterized in A. sinensis. The phylogenetic analysis suggests that NAC genes in A. sinensis can be classified into 16 known clusters and four new subfamilies, with each subfamily presenting similar gene structures and conserved motifs. RNA-seq analysis showed that AsNACs presents a broad transcriptional response to the agarwood inducer. The expression patterns of 15 AsNACs in A. sinensis after injury treatment indicated that AsNAC019 and AsNAC098 were positively correlated with the expression patterns of four polyketide synthase (PKS) genes. Additionally, AsNAC019 and AsNAC098 were also found to bind with the AsPKS07 promoter and activate its transcription. This comprehensive analysis provides valuable insights into the molecular evolution of the NAC gene family in Malvales plants and highlights the potential mechanisms of AsNACs for regulating secondary metabolite biosynthesis in A. sinensis, especially for the biosynthesis of 2-(2-phenyl) chromones in agarwood.

[Characterization and phylogenetic analysis of complete chloroplast genome of cultivated Qinan agarwood].

"Tangjie" leaves of cultivated Qinan agarwood were used to obtain the complete chloroplast genome using high-throughput sequencing technology. Combined with 12 chloroplast genomes of Aquilaria species downloaded from NCBI, bioinformatics method was employed to determine the chloroplast genome characteristics and phylogenetic relationships. The results showed that the chloroplast genome sequence length of cultivated Qinan agarwood "Tangjie" leaves was 174 909 bp with a GC content of 36.7%. A total of 136 genes were annotated, including 90 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Sequence repeat analysis detected 80 simple sequence repeats(SSRs) and 124 long sequence repeats, with most SSRs composed of A and T bases. Codon preference analysis revealed that AUU was the most frequently used codon, and codons with A and U endings were preferred. Comparative analysis of Aquilaria chloroplast genomes showed relative conservation of the IR region boundaries and identified five highly variable regions: trnD-trnY, trnT-trnL, trnF-ndhJ, petA-cemA, and rpl32, which could serve as potential DNA barcodes specific to the Aquilaria genus. Selection pressure analysis indicated positive selection in the rbcL, rps11, and rpl32 genes. Phylogenetic analysis revealed that cultivated Qinan agarwood "Tangjie" and Aquilaria agallocha clustered together(100% support), supporting the Chinese origin of Qinan agarwood from Aquilaria agallocha. The chloroplast genome data obtained in this study provide a foundation for studying the genetic diversity of cultivated Qinan agarwood and molecular identification of the Aquilaria genus.

Exploration of the B3 transcription factor superfamily in Aquilaria sinensis reveal their involvement in seed recalcitrance and agarwood formation.

The endangered tree species of the Aquilaria genus produce agarwood, a high value material produced only after wounding; however, conservation of Aquilaria seeds is difficult. The B3 transcription factor family has diverse important functions in plant development, especially in seed development, although their functions in other areas, such as stress responses, remain to be revealed. Here germination tests proved that the seeds of A. sinensis were recalcitrant seeds. To provide insights into the B3 superfamily, the members were identified and characterized by bioinformatic approaches and classified by phylogenetic analysis and domain structure. In total, 71 members were identified and classified into four subfamilies. Each subfamily not only had similar domains, but also had conserved motifs in their B3 domains. For the seed-related LAV subfamily, the B3 domain of AsLAV3 was identical to that of AsVALs but lacked a typical zf-CW domain such as VALs. AsLAV5 lacks a typical PHD-L domain present in Arabidopsis VALs. qRT-PCR expression analysis showed that the LEC2 ortholog AsLAV4 was not expressed in seeds. RAVs and REMs induced after wound treatment were also identified. These findings provide insights into the functions of B3 genes and seed recalcitrance of A. sinensis and indicate the role of B3 genes in wound response and agarwood formation.This is the first work to investigate the B3 family in A. sinensis and to provide insights of the molecular mechanism of seed recalcitrance.This will be a valuable guidance for studies of B3 genes in stress responses, secondary metabolite biosynthesis, and seed development.

Identification and characterization of novel sesquiterpene synthases TPS9 and TPS12 from Aquilaria sinensis.

Sesquiterpenes are characteristic components and important quality criterions for agarwood. Although sesquiterpenes are well-known to be biosynthesized by sesquiterpene synthases (TPSs), to date, only a few TPS genes involved in agarwood formation have been reported. Here, two new TPS genes, namely, TPS9 and TPS12, were isolated from Aquilaria sinensis (Lour.) Gilg, and their functions were examined in Escherichia coli BL21(DE3), with farnesyl pyrophosphate (FPP) and geranyl pyrophosphate (GPP) as the substrate of the corresponding enzyme activities. They were both identified as a multiproduct enzymes. After incubation with FPP, TPS9 liberated ß-farnesene and cis-sesquisabinene hydrate as main products, with cedrol and another unidentified sesquiterpene as minor products. TPS12 catalyzes the formation of ß-farnesene, nerolidol, γ-eudesmol, and hinesol. After incubation with GPP, TPS9 generated citronellol and geraniol as main products, with seven minor products. TPS12 converted GPP into four monoterpenes, with citral as the main product, and three minor products. Both TPS9 and TPS12 showed much higher expression in the two major tissues emitting floral volatiles: flowers and agarwood. Further, RT-PCR analysis showed TPS9 and TPS12 are typical genes mainly expressed during later stages of stress response, which is better known than that of chromone derivatives. This study will advance our understanding of agarwood formation and provide a solid theoretical foundation for clarifying its mechanism in A. sinensis.

Systematic investigation of the R2R3-MYB gene family in Aquilaria sinensis reveals a transcriptional repressor AsMYB054 involved in 2-(2-phenylethyl)chromone biosynthesis.

Trees in the genus Aquilaria produce agarwood, a valuable resin used in medicine, perfumes, and incense. 2-(2-Phenethyl)chromones (PECs) are characteristic components of agarwood; however, molecular mechanisms underlying PEC biosynthesis and regulation remain largely unknown. The R2R3-MYB transcription factors play important regulatory roles in the biosynthesis of various secondary metabolites. In this study, 101 R2R3-MYB genes in Aquilaria sinensis were systematically identified and analyzed at the genome-wide level. Transcriptomic analysis revealed that 19 R2R3-MYB genes were significantly regulated by an agarwood inducer, and showed significant correlations with PEC accumulation. Expression and evolutionary analyses revealed that AsMYB054, a subgroup 4 R2R3-MYB, was negatively correlated with PEC accumulation. AsMYB054 was located in the nucleus and functioned as a transcriptional repressor. Moreover, AsMYB054 could bind to the promoters of the PEC biosynthesis related genes AsPKS02 and AsPKS09, and inhibit their transcriptional activity. These findings suggested that AsMYB054 functions as a negative regulator of PEC biosynthesis via the inhibition of AsPKS02 and AsPKS09 in A. sinensis. Our results provide a comprehensive understanding of the R2R3-MYB subfamily in A. sinensis and lay a foundation for further functional analyses of R2R3-MYB genes in PEC biosynthesis.

Revealing the Roles of the JAZ Family in Defense Signaling and the Agarwood Formation Process in Aquilaria sinensis.

Jasmonate ZIM-domain family proteins (JAZs) are repressors in the signaling cascades triggered by jasmonates (JAs). It has been proposed that JAs play essential roles in the sesquiterpene induction and agarwood formation processes in Aquilaria sinensis. However, the specific roles of JAZs in A. sinensis remain elusive. This study employed various methods, including phylogenetic analysis, real-time quantitative PCR, transcriptomic sequencing, yeast two-hybrid assay, and pull-down assay, to characterize A. sinensis JAZ family members and explore their correlations with WRKY transcription factors. The bioinformatic analysis revealed twelve putative AsJAZ proteins in five groups and sixty-four putative AsWRKY transcription factors in three groups. The AsJAZ and AsWRKY genes exhibited various tissue-specific or hormone-induced expression patterns. Some AsJAZ and AsWRKY genes were highly expressed in agarwood or significantly induced by methyl jasmonate in suspension cells. Potential relationships were proposed between AsJAZ4 and several AsWRKY transcription factors. The interaction between AsJAZ4 and AsWRKY75n was confirmed by yeast two-hybrid and pull-down assays. This study characterized the JAZ family members in A. sinensis and proposed a model of the function of the AsJAZ4/WRKY75n complex. This will advance our understanding of the roles of the AsJAZ proteins and their regulatory pathways.

Aquilariperoxide A, a Sesquiterpene Dimer from Agarwood of Aquilaria sinensis with Dual Antitumor and Antimalarial Effects.

Aquilariperoxide A (1), an unprecedented sesquiterpene dimer characterized by a dioxepane ring connecting two sesquiterpene units via a C-C bond, was isolated from agarwood of Aquilaria sinensis-containing resins. The structure was elucidated by spectroscopic and computational methods. A bioassay revealed that 1 significantly inhibits cell proliferation and migration in human cancer cells. The mechanism of 1 against cancer cells was briefly discussed by analysis of RNA sequence data and epithelial-mesenchymal transition. Besides, the antimalarial activity of 1 was also evaluated.

RNA-Sequencing Reveals the Involvement of Sesquiterpene Biosynthesis Genes and Transcription Factors during an Early Response to Mechanical Wounding of Aquilaria sinensis.

Plants respond to wounding by reprogramming the expression of genes involved in secondary metabolism. Aquilaria trees produce many bioactive secondary metabolites in response to wounding, but the regulatory mechanism of agarwood formation in the early response to mechanical wounding has remained unclear. To gain insights into the process of transcriptome changes and to determine the regulatory networks of Aquilaria sinensis to an early response (15 days) to mechanical wounding, we collected A. sinensis samples from the untreated (Asc1) and treated (Asf1) xylem tissues and performed RNA sequencing (RNA-seq). This generated 49,102,523 (Asc1) and 45,180,981 (Asf1) clean reads, which corresponded to 18,927 (Asc1) and 19,258 (Asf1) genes, respectively. A total of 1596 differentially expressed genes (DEGs) were detected in Asf1 vs. Asc1 (|log2 (fold change)| ≥ 1, Padj ≤ 0.05), of which 1088 were up-regulated and 508 genes were down-regulated. GO and KEGG enrichment analysis of DEGs showed that flavonoid biosynthesis, phenylpropanoid biosynthesis, and sesquiterpenoid and triterpenoid biosynthesis pathways might play important roles in wound-induced agarwood formation. Based on the transcription factor (TF)-gene regulatory network analysis, we inferred that the bHLH TF family could regulate all DEGs encoding for farnesyl diphosphate synthase, sesquiterpene synthase, and 1-deoxy-D-xylulose-5-phosphate synthase (DXS), which contribute to the biosynthesis and accumulation of agarwood sesquiterpenes. This study provides insight into the molecular mechanism regulating agarwood formation in A. sinensis, and will be helpful in selecting candidate genes for improving the yield and quality of agarwood.

Genome-wide investigation of Cytochrome P450 superfamily of Aquilaria agallocha: Association with terpenoids and phenylpropanoids biosynthesis.

Agarwood is a dark resinous wood, produced when Aquilaria tree responds to wounding and microbial infection resulting in the accumulation of fragrant metabolites. Sesquiterpenoids and 2-(2-phenylethyl) chromones are the major phytochemicals in agarwood and Cytochrome P450s (CYPs) are one of the important enzymes in the biosynthesis of these fragrant chemicals. Thus, understanding the repertoire of CYP superfamily in Aquilaria can not only give insights into the fundamentals of agarwood formation, but can also provide a tool for the overproduction of the aroma chemicals. Therefore, current study was designed to investigate CYPs of an agarwood producing plant, Aquilaria agallocha. We identified 136 CYP genes from A. agallocha genome (AaCYPs) and classified them into 8 clans and 38 families. The promoter regions had stress and hormone-related cis-regulatory elements which indicate their participation in the stress response. Duplication and synteny analysis revealed segmental and tandem duplicated and evolutionary related CYP members in other plants. Potential members involved in the biosynthesis of sesquiterpenoids and phenylpropanoids were identified and found to be upregulated in methyl jasmonate-induced callus and infected Aquilaria trees by real-time quantitative PCR analyses. This study highlights the possible involvement of AaCYPs in agarwood resin development and their complex regulation during stress exposure.

Characterization and phylogenetic analysis of complete chloroplast genome of cultivated Qinan agarwood / 中国中药杂志

"Tangjie" leaves of cultivated Qinan agarwood were used to obtain the complete chloroplast genome using high-throughput sequencing technology. Combined with 12 chloroplast genomes of Aquilaria species downloaded from NCBI, bioinformatics method was employed to determine the chloroplast genome characteristics and phylogenetic relationships. The results showed that the chloroplast genome sequence length of cultivated Qinan agarwood "Tangjie" leaves was 174 909 bp with a GC content of 36.7%. A total of 136 genes were annotated, including 90 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Sequence repeat analysis detected 80 simple sequence repeats(SSRs) and 124 long sequence repeats, with most SSRs composed of A and T bases. Codon preference analysis revealed that AUU was the most frequently used codon, and codons with A and U endings were preferred. Comparative analysis of Aquilaria chloroplast genomes showed relative conservation of the IR region boundaries and identified five highly variable regions: trnD-trnY, trnT-trnL, trnF-ndhJ, petA-cemA, and rpl32, which could serve as potential DNA barcodes specific to the Aquilaria genus. Selection pressure analysis indicated positive selection in the rbcL, rps11, and rpl32 genes. Phylogenetic analysis revealed that cultivated Qinan agarwood "Tangjie" and Aquilaria agallocha clustered together(100% support), supporting the Chinese origin of Qinan agarwood from Aquilaria agallocha. The chloroplast genome data obtained in this study provide a foundation for studying the genetic diversity of cultivated Qinan agarwood and molecular identification of the Aquilaria genus.

Comparative transcriptome analysis reveals different defence responses during the early stage of wounding stress in Chi-Nan germplasm and ordinary Aquilaria sinensis.

BACKGROUND: Agarwood is a valuable Chinese medicinal herb and spice that is produced from wounded Aquilaria spp., is widely used in Southeast Asia and is highly traded on the market. The lack of highly responsive Aquilaria lines has seriously restricted agarwood yield and the development of its industry. In this article, a comparative transcriptome analysis was carried out between ordinary A. sinensis and Chi-Nan germplasm, which is a kind of A. sinensis tree with high agarwood-producing capacity in response to wounding stress, to elucidate the molecular mechanism underlying wounding stress in different A. sinensis germplasm resources and to help identify and breed high agarwood-producing strains. RESULTS: A total of 2427 and 1153 differentially expressed genes (DEGs) were detected in wounded ordinary A. sinensis and Chi-Nan germplasm compared with the control groups, respectively. KEGG enrichment analysis revealed that genes participating in starch metabolism, secondary metabolism and plant hormone signal transduction might play major roles in the early regulation of wound stress. 86 DEGs related to oxygen metabolism, JA pathway and sesquiterpene biosynthesis were identified. The majority of the expression of these genes was differentially induced between two germplasm resources under wounding stress. 13 candidate genes related to defence and sesquiterpene biosynthesis were obtained by WGCNA. Furthermore, the expression pattern of genes were verified by qRT-PCR. The candidate genes expression levels were higher in Chi-Nan germplasm than that in ordinary A. sinensis during early stage of wounding stress, which may play important roles in regulating high agarwood-producing capacity in Chi-Nan germplasm. CONCLUSIONS: Compared with A. sinensis, Chi-Nan germplasm invoked different biological processes in response to wounding stress. The genes related to defence signals and sesquiterepene biosynthesis pathway were induced to expression differentially between two germplasm resources. A total of 13 candidate genes were identified, which may correlate with high agarwood-producting capacity in Chi-Nan germplasm during the early stage of wounding stress. These genes will contribute to the development of functional molecular markers and the rapid breeding highly of responsive Aquilaria lines.

Genome-wide investigation and expression analysis of the AP2/ERF family for selection of agarwood-related genes in Aquilaria sinensis (Lour.) Gilg.

Aquilaria sinensis is an important non-timber tree species for producing high-value agarwood, which is widely used as a traditional medicine and incense. Agarwood is the product of Aquilaria trees in response to injury and fungal infection. The APETALA2/ethylene responsive factor (AP2/ERF) transcription factors (TFs) play important roles in plant stress responses and metabolite biosynthesis. In this study, 119 AsAP2/ERF genes were identified from the A. sinensis genome and divided into ERF, AP2, RAV, and Soloist subfamilies. Their conserved motif, gene structure, chromosomal localization, and subcellular localization were characterized. A stress/defense-related ERF-associated amphiphilic repression (EAR) motif and an EDLL motif were identified. Moreover, 11 genes that were highly expressed in the agarwood layer in response to whole-tree agarwood induction technique (Agar-Wit) treatment were chosen, and their expression levels in response to methyl jasmonate (MeJA), salicylic acid (SA), or salt treatment were further analyzed using the quantitative real time PCR (qRT-PCR). Among the 11 genes, eight belonged to subgroup B-3. All 11 genes were significantly upregulated under salt treatment, while eight genes were significantly induced by both MeJA and SA. In addition, the gene clusters containing these upregulated genes on chromosomes were observed. The results obtained from this research not only provide useful information for understanding the functions of AP2/ERF genes in A. sinensis but also identify candidate genes and gene clusters to dissect their regulatory roles in agarwood formation for future research.

Genetic relationship and source species identification of 58 Qi-Nan germplasms of Aquilaria species in China that easily form agarwood.

Recently, Qi-Nan germplasm, the germplasm of Aquilaria species that easily forms agarwood, has been widely cultivated in Guangdong and Hainan Provinces in China. Since the morphological characteristics of Qi-Nan germplasm are similar to those of Aquilaria species and germplasm is bred by grafting, it is difficult to determine the source species of this germplasm by traditional taxonomic characteristics. In this study, we performed a DNA barcoding analysis of 58 major Qi-Nan germplasms as well as Aquilaria sinensis, A. yunnanensis, A. crassna, A. malaccensis and A. hirta with 5 primers (nuclear gene internal transcribed spacer 2 (ITS2) and the chloroplast genes matK, trnH-psbA, rbcL and trnL-trnF). This field survey in the Qi-Nan germplasm plantations in Guangdong and Hainan Provinces aimed to accurately identify the source species of Qi-Nan germplasm. According to the results, ITS2 and matK showed the most variability and the highest divergence at all genetic distances. This ITS2+matK combination, screened for with TaxonDNA analysis, showed the highest success rate in species identification of the Qi-Nan germplasm. Clustering in the phylogenetic trees constructed with Bayesian inference and maximum likelihood indicated that the Qi-Nan germplasm was most closely related to A. sinensis and more distantly related to A. yunnanensis, A. crassna, A. malaccensis and A. hirta. Therefore, this study determined that the source species of the Qi-Nan germplasm is A. sinensis.

Genome-wide analysis of basic helix-loop-helix (bHLH) transcription factors in Aquilaria sinensis.

The basic helix-loop-helix (bHLH) transcription factors are involved in several biological processes both in plant development and stress responses. Agarwood, a major active and economical product, is only induced and accumulated when the roots, stems, or branches are wounded in Aquilaria sinensis. Although genome-wide comprehensive analyses of the bHLH family have been identified in many plants, no systematic study of the genes in this family has been conducted in A. sinensis. In this study, 105 bHLH genes were identified in A. sinensis through genome-wide analysis and named according to their chromosomal locations. Based on a phylogenetic tree, AsbHLH family proteins were classified into 18 subfamilies. Most of them were distributed on eight chromosomes, with the exception of two genes. Based on the tissue-specific expression characteristics and expression patterns in response to methyl jasmonate (MeJA) treatment, seven AsbHLH genes were likely involved in wound-induced agarwood formation. The results provide comprehensive information on AsbHLHs that can be used to elucidate the molecular functions and physiological roles of these proteins in A. sinensis.

DNA databases of a CITES listed species Aquilaria malaccensis (Thymelaeaceae) as the tracking tools for forensic identification and chain of custody certification.

Aquilaria malaccensis (Thymelaeaceae) is the main source of high-grade agarwood in Southeast Asia. Aggressive collections and trade activities over the past decades have put great pressure on the natural stands and raised concerns over the long-term survival potential of A. malaccensis. Tracking and authentication of agarwood require method with a high degree of accuracy. Therefore, this study aimed to develop DNA databases of A. malaccensis as the tracking tools at species, population and individual levels for forensic identification and chain of custody certification. Using two cpDNA (rbcL and matK) and an rDNA (ITS2) markers, species identification database of Aquilaria was developed to distinguish A. malaccensis from A. hirta, A. microcarpa, A. beccariana, A. crassna, A. sinensis and A. rostrata. In addition, based on 35 populations of A. malaccensis throughout Peninsular Malaysia, cpDNA haplotype and STR allele frequency databases were developed for population and individual identification. A haplotype distribution map based on 29 haplotypes derived from seven cpDNA showed that the A. malaccensis in Peninsular Malaysia can be associated to Kedah-Perak and Kelantan-Johor regions. Similarly, genetic relatedness and Bayesian clustering analyses based on 10 STR markers also divided the 35 populations into two main genetic clusters, corresponding to Kedah-Perak and Kelantan-Johor regions. The STR allele frequency databases were established and characterized according to these two regions. To determine the performance of the STR allele frequency databases for population identification, independent self-assignment tests showed that the percentage of individuals correctly assigned into the origin population was 93.88% in Kedah-Perak and 90.29% in Kelantan-Johor. For the STR allele frequency databases to be used for individual identification, conservativeness tests showed that the θ should be adjusted to 0.250 and 0.200 in the Kedah-Perak and Kelantan-Johor databases, respectively. To ensure consistency in allele calling for the dinucleotide repeat loci across different electrophoretic platforms or laboratories, allelic ladders have been developed for the 10 STR loci. Two case studies are presented of how these databases were used to track A. malaccensis to the origin population and stump. These databases are ready to be used to provide admissible forensic evidence for legal proceedings against the illegal harvesters of agarwood and for agarwood certification to meet the consumer country regulations.

A tetraploidization event shaped the Aquilaria sinensis genome and contributed to the ability of sesquiterpenes synthesis.

BACKGROUND: Agarwood, generated from the Aquilaria sinensis, has high economic and medicinal value. Although its genome has been sequenced, the ploidy of A. sinensis paleopolyploid remains unclear. Moreover, the expression changes of genes associated with agarwood formation were not analyzed either. RESULTS: In the present work, we reanalyzed the genome of A. sinensis and found that it experienced a recent tetraploidization event ~ 63-71 million years ago (Mya). The results also demonstrated that the A. sinensis genome had suffered extensive gene deletion or relocation after the tetraploidization event, and exhibited accelerated evolutionary rates. At the same time, an alignment of homologous genes related to different events of polyploidization and speciation were generated as well, which provides an important comparative genomics resource for Thymelaeaceae and related families. Interestingly, the expression changes of genes related to sesquiterpene synthesis in wounded stems of A. sinensis were also observed. Further analysis demonstrated that polyploidization promotes the functional differentiation of the key genes in the sesquiterpene synthesis pathway. CONCLUSIONS: By reanalyzing its genome, we found that the tetraploidization event shaped the A. sinensis genome and contributed to the ability of sesquiterpenes synthesis. We hope that these results will facilitate our understanding of the evolution of A. sinensis and the function of genes involved in agarwood formation.