BACKGROUND
aquilaria agallocha is one of the largest producers of agarwood, a valuable product derived from aquilaria and gyrinops trees.
agarwood-producing tree species have become endangered due to the deforestation of tropical forests and the international trade of agarwood has come under strict control with a listing in appendix ii of the convention on international trade in endangered species of wild fauna and flora.
the use of agarwood is prevalent in many cultures, particularly in the middle east and asia where it has been used for over a thousand years.
in particular, the use of agarwood is prevalent in religious ceremonies, herbal medicine, and as fragrances for perfumes and aromatherapy.
the main compounds related to the medicinal properties of agarwood are terpenes and phenylethyl chromone derivatives
which can be highly variable in content and composition among different agarwood-producing tree species.
previous studies have focused on sesquiterpenes, the most abundant terpenes compound in agarwood
<cit> .
however, little is known about triterpenoids in agarwood.
terpenoid content is induced under biotic stress as an immune response to resist various pathogens and its derivatives have been shown to exhibit anti-microorganism and anti-tumour functions
<cit> .
a specific compound of interest, cucurbitacin, is produced to combat fungal and bacterial pathogens
<cit> .
cucurbitacins have previously been isolated in chinese medicinal herbs and have been shown to have pharmacological effects
<cit> .
in particular, cucurbitacin i is known to repress cancer cell motility by perturbing actin dynamics and has also been known to exhibit cytotoxicity against mda-mb- <dig> human breast cancer cells from animal models and indirectly interrupt actin dynamics
<cit> .
the study of anti-tumour compounds and related pathways is thus an important field in agarwood research.
in this study, we identified cucurbitacin e and i from in vitro a. agallocha agarwood and present a draft genome for a. agallocha.
in vitro materials were used to perform this study due to the lengthy growth period of resinous material in this species as well as to avoid contamination from microorganisms.
importantly, this process is applicable to plant factories for large-scale production in the future.
the dna and rna sequence data were obtained using illumina hiseq  <dig> sequencing technology, from which we performed de novo genome assembly and gene annotation.
we inferred a putative pathway for cucurbitacin e and i from the genomic and transcriptomic data in order to better understand these important medicinal compounds in agarwood.
the transcripts discovered to be related to the cucurbitacin pathway, were validated through qrt-pcr.
to the best of our knowledge, this is the first draft genome for any species of aquilaria as well as the first study to identify cucurbitacin e and i in agarwood from in vitro materials.
RESULTS
agarwood contains high cucurbitacin content
the strain of a. agallocha used in this study was originally derived from myanmar and domesticated in taiwan, after import.
in vitro materials from this strain were analyzed using lc-esi-ms where the presence of cucurbitacin i and e were detected .
after identification, in vitro callus, shoot and plant materials were analyzed for cucurbitacin e and i content  where it was seen to be most abundant in in vitro plant.
to the best of our knowledge, the cucurbitacin i content from in vitro a. agallocha produced agarwood is significantly higher than in any other chinese medicinal herb studied previously, with an average concentration of  <dig>  μg/g observed in this study as compared to previously reported concentrations of  <dig>  μg/g and 25 μg/g from studies by afifi et al.
and wu et al.
respectively
<cit> .figure  <dig>
agarwood endogenous cucurbitacin e and i content.
the endogenous cucurbitacin content was measured from different stages of agarwood plant.
data is represented as mean ± standard deviation .
the endogenous cucurbitacin content of 2 month old agarwood in vitro shoot material was measured after treatment with various concentrations of methyl jasmonate .
data is represented as mean ± standard deviation .
as mentioned, agarwood produces terpenoids under biotic stress
<cit> .
to investigate whether pathogens induce cucurbitacin content, the in vitro material was treated with methyl jasmonate , an important signal in pathogen related pathways.
various concentrations of mj were applied to mimic different levels of biotic stress and the change in cucurbitacin content in response to different levels of biotic stress was measured.
it was seen that cucurbitacin content increased as mj concentration increased, up to 218 μg/g of cucurbitacin i at  <dig>  mm mj and  <dig>  μg/g of cucurbitacin e at  <dig>  mm mj .
these results reveal that cucurbitacin pathway related genes were induced by mj, indicating that agarwood may produce cucurbitacins and triterpenoids under biotic stress.
with consideration for both cucurbitacin e and i peak values, the 0 mm and  <dig>  mm mj treatment samples from in vitro shoot were chosen for rna-seq analysis.
assembly of a draft genome for a. agallocha
although transcriptome assembly has become commonplace, the majority of transcripts obtained from transcriptome assembly are partial transcripts.
genome assembly may potentially allow for more complete transcript information to be obtained when compared to a de novo transcriptome procedure, thus easing primer design.
as well, the sequence information of 5′ utr regions is more easily obtainable from a genome assembly than from transcriptome studies.
these factors allow for deeper studies of gene expression mechanisms.
the size of the haploid a. agallocha genome was estimated to be approximately 736 mb by flow cytometry .
five dna libraries were constructed for a total of  <dig>  gb, which represents approximately 196x sequencing coverage .
the dna libraries contained one pair-end library with a fragment length of 300 bp and four mate-pair libraries of various fragment lengths.
for the a. agallocha transcriptome, two rna libraries were constructed to represent mj treatment conditions  where polya + rna from in vitro shoot was sequenced for a total of  <dig>  gb.table  <dig>
sequencing read libraries for agarwood
the dna libraries were utilized in a de novo assembly procedure where the resulting draft genome  contained  <dig>  scaffolds with an n <dig> of  <dig>  kb, a  <dig>  mb longest sequence, and a total size of  <dig>  mb, approximately 98% genome coverage .table  <dig>
summary of dna pair-end libraries and
de novo
genome assembly
gene annotation
the tigr plant repeat database
<cit>  and repbase  were combined with ab initio repeat prediction to perform repeat masking.
this resulted in  <dig> % of the draft genome classified as repeat sequences, with  <dig> % classified as long tandem repeat  elements.
transposable elements are generally non-coding dna sequences that can change its location within a genome and can play an important role in development and evolution
<cit> .
thus, annotation of transposable elements was performed in this study using the tigr plant repeat database .table  <dig>
repeated sequences annotation of transposable elements
via
tigr database
rna-seq data was aligned to the repeat-masked genome  to provide extrinsic support for gene prediction.
ab initio gene prediction combined with protein alignment resulted in annotations for  <dig>  protein-coding genes, among which  <dig>  genes encoded for multiple isoforms, representing  <dig>  transcripts in total.
on average, the predicted gene-models consisted of transcript lengths of  <dig> .72 bp, coding lengths of  <dig> .27 bp, and  <dig>  exons per gene.
a total of  <dig> % of the predicted gene-models had matches in the ncbi non-redundant protein database, uniprot enzyme database, or matched a protein functional domain in pfam.
the transcriptome sequence data was able to be mapped to the draft genome at a mapping rate of  <dig> %.
as well, a total of  <dig> % of gene-models were supported by  <dig>  of  <dig>  transcript sequences constructed from rna-seq using the draft genome as a reference.
a de novo assembled transcriptome was also aligned to the draft genome, where  <dig> % of predicted exons were supported by  <dig>  of  <dig>  exons able to be mapped to the draft genome.
functional classification for the set of annotated transcripts was performed using gene ontology  .figure  <dig>
functional classification of annotated proteins.
the gene count for various protein classes as determined by gene ontology for the set of annotated proteins in a. agallocha agarwood.
we compared our gene set to those from a recent study by xu et al.
<cit> , where a de novo transcriptome assembly for aquilaria sinensis was performed.
in their study,  <dig>  transcripts were annotated with an average assembled sequence length of  <dig>  bp as compared to our results of  <dig>  annotated transcripts with an average length coding length of  <dig> .27 bp.
as well,  <dig>  transcripts in our gene set contained both start and stop codons, which is another indicator for completeness.
for gene discovery, assembling a draft genome clearly aids in the number of genes discovered as well as in the completeness of the assembled sequence.
differential expression
under biotic stress and wounded conditions, mj is an important signal in a plant’s defence system and will induce secondary metabolites.
thus, the set of annotated transcripts were analyzed for differential expression between the two treatment conditions, 0 mm and  <dig>  mm mj, in order to observe the effect of mj on gene expression.
the short reads from rna-seq data were aligned to the set of annotated transcripts and the gene expression for each treatment condition was quantified, resulting in  <dig>  differentially expressed genes  with at least a two-fold change in expression, of which  <dig>  genes were up-regulated and  <dig>  genes were down-regulated.
functional classification was performed for the set of differentially expressed genes, using go .
after treatment with  <dig>  mm mj, the activity of transcripts in the categories of metabolic processes and catalytic activity was observed to have increased, which is consistent with observations in previous studies
<cit> .figure  <dig>
functional classification of differentially expressed transcripts.
functional classifications of the set of differentially expressed transcripts through gene ontology, separated into up and down modes of regulation.
it can be seen that metabolic processes and catalytic activity were enriched with up-regulated genes in the presence of biotic stress.
putative pathway for cucurbitacin e and i
cucurbitacins belong to the class of cytotoxic triterpenoids and are synthesized from mep/doxp and mevalonate pathways
<cit> .
although the importance of medicinal compounds in a. agallocha agarwood is known, knowledge of its molecular mechanism is lacking and a transgenic line for this species is not able to be created.
thus, an assay of the cucurbitacin bio-synthetic pathway is currently not available.
to investigate the cucurbitacin pathway in a. agallocha, transcripts were annotated using arabidopsis thaliana proteins as well as uniprot enzymes.
the annotated transcripts were then used to infer a putative cucurbitacin pathway in a. agallocha by referring to the mevalonate pathway in a. thaliana from kegg
<cit>  as well as many differentially expressed cytochrome p450s  and s-adenosyl-l-methionine-dependent methyltransferases  as putative downstream genes .figure  <dig>
cucurbitacin e and i pathways in
a. agallocha.
a schematic illustration showing cucurbitacin e and i bio-synthesis, including isoprenoid precursors via mep and mevalonate pathways.
the voz transcription factor’s cis-element was observed to exist in hmgr.
we identified genes which encode for enzymes in the cucurbitacin e and i pathway , including three important gene categories: hmgr
<cit> , dxps
<cit> , and sqs
<cit> ; as well as genes which encode for synthases in cucurbitacin metabolism.
these gene expression results are consistent with the knowledge that cucurbitacins are synthesized to resist pathogens
<cit> .
we also investigated the vascular plant one-zinc-finger transcription factor , described as follows.table  <dig>
enzymes identified in the cucurbitacin e and i pathway and their change in gene expression
vozs are transcription factors that are highly conserved in land plant evolution
<cit> .
it has been shown to bind to the cis-element gcgtnx7acgc, which belongs to the nac subgroup viii-2
<cit> .
vozs have been observed to be both positive and negative transcription factors of biotic and abiotic stress-response pathways, respectively, in a. thaliana.
although the voz1voz2- <dig> mutant did not notably change in endogenous aba content, the voz1voz <dig> double mutant was inable to combat pathogens  due to low gene expression from defense-response genes
<cit> .
this indicates that vozs are positive regulators in the sa and mj signaling pathways in land plants.
therefore, we speculated that voz plays a postive role in the cucurbitacin pathway.
a transcript from our gene set homologous to voz in a. thaliana and zea mays was able to be identified with identities of  <dig> % and  <dig> % respectively.
we identified hmgr as containing the voz cis-element in its promoter region.
in can be seen from table
<dig> that voz expression was slightly up-regulated and hmgr was significantly up-regulated after mj treatment.
this provides evidence that voz transcription factors are a positive regulator that play a role, directly or indirectly, in the cucurbitacin pathway and biotic stress-response related genes.
the putative pathway also includes cyp450s and sam-mtases .
cyp450s are one of the largest gene families in plants and catalyzes most oxidation steps in secondary metabolism such as in the biosynthesis of defense compounds, pigment, and antioxidants
<cit> .
putatively, cyp450s may catalyze the conversion of cucurbitadienol.
sam-mtases may also act on cucurbitadienol by catalyzing methylation, as it is known that many compounds with anti-microorganism functions have cucurbitadienol backbones activated by methylation
<cit> .
we annotated  <dig> cytochrome p450s and 66 s-adenosyl-l-methionine-dependent methyltransferases  in the a. agallocha genome, of which,  <dig> cyp450s and  <dig> sam-mtases showed significant up-regulation.
these genes can be considered candidate genes that are possibly involved in the cucurbitacin pathway.
as well, we identified a small number of sam-mtases that contained the voz cis-element, though their expression was not observed to be significantly up-regulated.
the transcripts related to the cucurbitacin pathway and the voz transcription factor were validated using qrt-pcr .
our results are consistent with the expectation of cucurbitacin pathway genes being up-regulated in response to biotic stress.
there is some disparity between qrt-pcr and rna-seq values.
however, this is likely due to differences in platform.
the qrt-pcr primer sequences were designed for the 3′ ends of transcripts, which is highly stable, as compared to quantifying expression using the whole transcript with rna-seq, which is typically not uniformly sequenced.figure  <dig>
qrt-pcr validation.
the qrt-pcr analysis results showing relative change in expression for each gene involved in the cucurbitacin pathway, as well as the voz transcription factor and the internal control achistone, after treatment with mj.
CONCLUSIONS
cucurbitacin, a compound of interest with medicinal value which exhibits anti-microorganism and anti-tumour properties was identified and observed to be abundant in in vitro a. agallocha agarwood.
to better understand the genes and pathways related to cucurbitacin, a draft genome was assembled, from which, many genes and protein functions were able to be annotated and analyzed.
the expression change for cucurbitacins was shown to be consistent with known responses of a. agallocha to biotic stress.
as well, the dna and rna data was utilized to infer a putative pathway for cucurbitacin e and i in a. agallocha and a set of homologous genes in a. thaliana related to cucurbitacin bio-synthesis was presented.
the voz transcription factor, a positive regulator of biotic stress-response pathways was identified as well as a putative regulation target among the genes related to the cucurbitacin pathway, hmgr, in which the voz cis-element was present in its promoter.
the draft genome from this study can provide a resource for the investigation of secondary metabolite pathways not only in aquilaria trees, but also in other non-model medicinal plants.
the confirmation that cucurbitacins can be obtained from in vitro materials with a relatively high yield has positive implications with regards to plant factories to save on costs and time, as well as limiting the effects of research on this endangered species in the wild.

 aquilaria agallocha is one of the largest producers of agarwood, a valuable product derived from aquilaria and gyrinops trees. agarwood-producing tree species have become endangered due to the deforestation of tropical forests and the international trade of agarwood has come under strict control with a listing in appendix ii of the convention on international trade in endangered species of wild fauna and flora. the use of agarwood is prevalent in many cultures, particularly in the middle east and asia where it has been used for over a thousand years. in particular, the use of agarwood is prevalent in religious ceremonies, herbal medicine, and as fragrances for perfumes and aromatherapy. the main compounds related to the medicinal properties of agarwood are terpenes and phenylethyl chromone derivatives which can be highly variable in content and composition among different agarwood-producing tree species. previous studies have focused on sesquiterpenes, the most abundant terpenes compound in agarwood. however, little is known about triterpenoids in agarwood. terpenoid content is induced under biotic stress as an immune response to resist various pathogens and its derivatives have been shown to exhibit anti-microorganism and anti-tumour functions. a specific compound of interest, cucurbitacin, is produced to combat fungal and bacterial pathogens. cucurbitacins have previously been isolated in chinese medicinal herbs and have been shown to have pharmacological effects. in particular, cucurbitacin i is known to repress cancer cell motility by perturbing actin dynamics and has also been known to exhibit cytotoxicity against mda-mb- human breast cancer cells from animal models and indirectly interrupt actin dynamics. the study of anti-tumour compounds and related pathways is thus an important field in agarwood research. in this study, we identified cucurbitacin e and i from in vitro a. agallocha agarwood and present a draft genome for a. agallocha. in vitro materials were used to perform this study due to the lengthy growth period of resinous material in this species as well as to avoid contamination from microorganisms. importantly, this process is applicable to plant factories for large-scale production in the future. the dna and rna sequence data were obtained using illumina hiseq sequencing technology, from which we performed de novo genome assembly and gene annotation. we inferred a putative pathway for cucurbitacin e and i from the genomic and transcriptomic data in order to better understand these important medicinal compounds in agarwood. the transcripts discovered to be related to the cucurbitacin pathway, were validated through qrt-pcr. to the best of our knowledge, this is the first draft genome for any species of aquilaria as well as the first study to identify cucurbitacin e and i in agarwood from in vitro materials.  agarwood contains high cucurbitacin content the strain of a. agallocha used in this study was originally derived from myanmar and domesticated in taiwan, after import. in vitro materials from this strain were analyzed using lc-esi-ms where the presence of cucurbitacin i and e were detected. after identification, in vitro callus, shoot and plant materials were analyzed for cucurbitacin e and i content where it was seen to be most abundant in in vitro plant. to the best of our knowledge, the cucurbitacin i content from in vitro a. agallocha produced agarwood is significantly higher than in any other chinese medicinal herb studied previously, with an average concentration of  μg/g observed in this study as compared to previously reported concentrations of  μg/g and 25 μg/g from studies by afifi et al. and wu et al. respectively.figure agarwood endogenous cucurbitacin e and i content. the endogenous cucurbitacin content was measured from different stages of agarwood plant. data is represented as mean ± standard deviation. the endogenous cucurbitacin content of 2 month old agarwood in vitro shoot material was measured after treatment with various concentrations of methyl jasmonate. data is represented as mean ± standard deviation. as mentioned, agarwood produces terpenoids under biotic stress. to investigate whether pathogens induce cucurbitacin content, the in vitro material was treated with methyl jasmonate, an important signal in pathogen related pathways. various concentrations of mj were applied to mimic different levels of biotic stress and the change in cucurbitacin content in response to different levels of biotic stress was measured. it was seen that cucurbitacin content increased as mj concentration increased, up to 218 μg/g of cucurbitacin i at  mm mj and  μg/g of cucurbitacin e at  mm mj. these results reveal that cucurbitacin pathway related genes were induced by mj, indicating that agarwood may produce cucurbitacins and triterpenoids under biotic stress. with consideration for both cucurbitacin e and i peak values, the 0 mm and  mm mj treatment samples from in vitro shoot were chosen for rna-seq analysis. assembly of a draft genome for a. agallocha although transcriptome assembly has become commonplace, the majority of transcripts obtained from transcriptome assembly are partial transcripts. genome assembly may potentially allow for more complete transcript information to be obtained when compared to a de novo transcriptome procedure, thus easing primer design. as well, the sequence information of 5′ utr regions is more easily obtainable from a genome assembly than from transcriptome studies. these factors allow for deeper studies of gene expression mechanisms. the size of the haploid a. agallocha genome was estimated to be approximately 736 mb by flow cytometry. five dna libraries were constructed for a total of gb, which represents approximately 196x sequencing coverage. the dna libraries contained one pair-end library with a fragment length of 300 bp and four mate-pair libraries of various fragment lengths. for the a. agallocha transcriptome, two rna libraries were constructed to represent mj treatment conditions where polya + rna from in vitro shoot was sequenced for a total of gb.table sequencing read libraries for agarwood the dna libraries were utilized in a de novo assembly procedure where the resulting draft genome contained scaffolds with an n of  kb, a  mb longest sequence, and a total size of  mb, approximately 98% genome coverage.table summary of dna pair-end libraries and de novo genome assembly gene annotation the tigr plant repeat database and repbase were combined with ab initio repeat prediction to perform repeat masking. this resulted in % of the draft genome classified as repeat sequences, with % classified as long tandem repeat elements. transposable elements are generally non-coding dna sequences that can change its location within a genome and can play an important role in development and evolution. thus, annotation of transposable elements was performed in this study using the tigr plant repeat database.table repeated sequences annotation of transposable elements via tigr database rna-seq data was aligned to the repeat-masked genome to provide extrinsic support for gene prediction. ab initio gene prediction combined with protein alignment resulted in annotations for protein-coding genes, among which genes encoded for multiple isoforms, representing transcripts in total. on average, the predicted gene-models consisted of transcript lengths of.72 bp, coding lengths of.27 bp, and exons per gene. a total of % of the predicted gene-models had matches in the ncbi non-redundant protein database, uniprot enzyme database, or matched a protein functional domain in pfam. the transcriptome sequence data was able to be mapped to the draft genome at a mapping rate of %. as well, a total of % of gene-models were supported by of transcript sequences constructed from rna-seq using the draft genome as a reference. a de novo assembled transcriptome was also aligned to the draft genome, where % of predicted exons were supported by of exons able to be mapped to the draft genome. functional classification for the set of annotated transcripts was performed using gene ontology.figure functional classification of annotated proteins. the gene count for various protein classes as determined by gene ontology for the set of annotated proteins in a. agallocha agarwood. we compared our gene set to those from a recent study by xu et al., where a de novo transcriptome assembly for aquilaria sinensis was performed. in their study, transcripts were annotated with an average assembled sequence length of  bp as compared to our results of annotated transcripts with an average length coding length of.27 bp. as well, transcripts in our gene set contained both start and stop codons, which is another indicator for completeness. for gene discovery, assembling a draft genome clearly aids in the number of genes discovered as well as in the completeness of the assembled sequence. differential expression under biotic stress and wounded conditions, mj is an important signal in a plant’s defence system and will induce secondary metabolites. thus, the set of annotated transcripts were analyzed for differential expression between the two treatment conditions, 0 mm and  mm mj, in order to observe the effect of mj on gene expression. the short reads from rna-seq data were aligned to the set of annotated transcripts and the gene expression for each treatment condition was quantified, resulting in differentially expressed genes with at least a two-fold change in expression, of which genes were up-regulated and genes were down-regulated. functional classification was performed for the set of differentially expressed genes, using go. after treatment with  mm mj, the activity of transcripts in the categories of metabolic processes and catalytic activity was observed to have increased, which is consistent with observations in previous studies.figure functional classification of differentially expressed transcripts. functional classifications of the set of differentially expressed transcripts through gene ontology, separated into up and down modes of regulation. it can be seen that metabolic processes and catalytic activity were enriched with up-regulated genes in the presence of biotic stress. putative pathway for cucurbitacin e and i cucurbitacins belong to the class of cytotoxic triterpenoids and are synthesized from mep/doxp and mevalonate pathways. although the importance of medicinal compounds in a. agallocha agarwood is known, knowledge of its molecular mechanism is lacking and a transgenic line for this species is not able to be created. thus, an assay of the cucurbitacin bio-synthetic pathway is currently not available. to investigate the cucurbitacin pathway in a. agallocha, transcripts were annotated using arabidopsis thaliana proteins as well as uniprot enzymes. the annotated transcripts were then used to infer a putative cucurbitacin pathway in a. agallocha by referring to the mevalonate pathway in a. thaliana from kegg as well as many differentially expressed cytochrome p450s and s-adenosyl-l-methionine-dependent methyltransferases as putative downstream genes.figure cucurbitacin e and i pathways in a. agallocha. a schematic illustration showing cucurbitacin e and i bio-synthesis, including isoprenoid precursors via mep and mevalonate pathways. the voz transcription factor’s cis-element was observed to exist in hmgr. we identified genes which encode for enzymes in the cucurbitacin e and i pathway, including three important gene categories: hmgr, dxps, and sqs; as well as genes which encode for synthases in cucurbitacin metabolism. these gene expression results are consistent with the knowledge that cucurbitacins are synthesized to resist pathogens. we also investigated the vascular plant one-zinc-finger transcription factor, described as follows.table enzymes identified in the cucurbitacin e and i pathway and their change in gene expression vozs are transcription factors that are highly conserved in land plant evolution. it has been shown to bind to the cis-element gcgtnx7acgc, which belongs to the nac subgroup viii-2. vozs have been observed to be both positive and negative transcription factors of biotic and abiotic stress-response pathways, respectively, in a. thaliana. although the voz1voz2- mutant did not notably change in endogenous aba content, the voz1voz double mutant was inable to combat pathogens due to low gene expression from defense-response genes. this indicates that vozs are positive regulators in the sa and mj signaling pathways in land plants. therefore, we speculated that voz plays a postive role in the cucurbitacin pathway. a transcript from our gene set homologous to voz in a. thaliana and zea mays was able to be identified with identities of % and % respectively. we identified hmgr as containing the voz cis-element in its promoter region. in can be seen from table  that voz expression was slightly up-regulated and hmgr was significantly up-regulated after mj treatment. this provides evidence that voz transcription factors are a positive regulator that play a role, directly or indirectly, in the cucurbitacin pathway and biotic stress-response related genes. the putative pathway also includes cyp450s and sam-mtases. cyp450s are one of the largest gene families in plants and catalyzes most oxidation steps in secondary metabolism such as in the biosynthesis of defense compounds, pigment, and antioxidants. putatively, cyp450s may catalyze the conversion of cucurbitadienol. sam-mtases may also act on cucurbitadienol by catalyzing methylation, as it is known that many compounds with anti-microorganism functions have cucurbitadienol backbones activated by methylation. we annotated cytochrome p450s and 66 s-adenosyl-l-methionine-dependent methyltransferases in the a. agallocha genome, of which, cyp450s and sam-mtases showed significant up-regulation. these genes can be considered candidate genes that are possibly involved in the cucurbitacin pathway. as well, we identified a small number of sam-mtases that contained the voz cis-element, though their expression was not observed to be significantly up-regulated. the transcripts related to the cucurbitacin pathway and the voz transcription factor were validated using qrt-pcr. our results are consistent with the expectation of cucurbitacin pathway genes being up-regulated in response to biotic stress. there is some disparity between qrt-pcr and rna-seq values. however, this is likely due to differences in platform. the qrt-pcr primer sequences were designed for the 3′ ends of transcripts, which is highly stable, as compared to quantifying expression using the whole transcript with rna-seq, which is typically not uniformly sequenced.figure qrt-pcr validation. the qrt-pcr analysis results showing relative change in expression for each gene involved in the cucurbitacin pathway, as well as the voz transcription factor and the internal control achistone, after treatment with mj.  cucurbitacin, a compound of interest with medicinal value which exhibits anti-microorganism and anti-tumour properties was identified and observed to be abundant in in vitro a. agallocha agarwood. to better understand the genes and pathways related to cucurbitacin, a draft genome was assembled, from which, many genes and protein functions were able to be annotated and analyzed. the expression change for cucurbitacins was shown to be consistent with known responses of a. agallocha to biotic stress. as well, the dna and rna data was utilized to infer a putative pathway for cucurbitacin e and i in a. agallocha and a set of homologous genes in a. thaliana related to cucurbitacin bio-synthesis was presented. the voz transcription factor, a positive regulator of biotic stress-response pathways was identified as well as a putative regulation target among the genes related to the cucurbitacin pathway, hmgr, in which the voz cis-element was present in its promoter. the draft genome from this study can provide a resource for the investigation of secondary metabolite pathways not only in aquilaria trees, but also in other non-model medicinal plants. the confirmation that cucurbitacins can be obtained from in vitro materials with a relatively high yield has positive implications with regards to plant factories to save on costs and time, as well as limiting the effects of research on this endangered species in the wild. 
in this study, we present a draft genome and a putative pathway for cucurbitacin e and i, compounds with known medicinal value, from in vitro aquilaria agallocha agarwood. dna and rna data are utilized to annotate many genes and protein functions in the draft genome. this study is the first attempt to identify cucurbitacin e and i from in vitro agarwood and the first draft genome for any species of aquilaria. the expression changes for cucurbitacin e and i are shown to be consistent with known responses of a. agallocha to biotic stress and a set of homologous genes in arabidopsis thaliana related to cucurbitacin bio-synthesis is presented and validated through qrt-pcr. agarwood is derived from aquilaria trees, the trade of which has come under strict control with a listing in appendix ii of the convention on international trade in endangered species of wild fauna and flora. the results of this study will aid in future investigations of secondary metabolite pathways in aquilaria and other non-model medicinal plants. many secondary metabolites of agarwood are known to have medicinal value to humans, including compounds that have been shown to elicit sedative effects and exhibit anti-cancer properties. however, little is known about the genome, transcriptome, and the biosynthetic pathways responsible for producing such secondary metabolites in agarwood.
 agarwood is derived from aquilaria trees, the trade of which has come under strict control with a listing in appendix ii of the convention on international trade in endangered species of wild fauna and flora. many secondary metabolites of agarwood are known to have medicinal value to humans, including compounds that have been shown to elicit sedative effects and exhibit anti-cancer properties. however, little is known about the genome, transcriptome, and the biosynthetic pathways responsible for producing such secondary metabolites in agarwood.  in this study, we present a draft genome and a putative pathway for cucurbitacin e and i, compounds with known medicinal value, from in vitro aquilaria agallocha agarwood. dna and rna data are utilized to annotate many genes and protein functions in the draft genome. the expression changes for cucurbitacin e and i are shown to be consistent with known responses of a. agallocha to biotic stress and a set of homologous genes in arabidopsis thaliana related to cucurbitacin bio-synthesis is presented and validated through qrt-pcr.  this study is the first attempt to identify cucurbitacin e and i from in vitro agarwood and the first draft genome for any species of aquilaria. the results of this study will aid in future investigations of secondary metabolite pathways in aquilaria and other non-model medicinal plants. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords agarwoodcucurbitacinaquilariagenomeissue-copyright-statement© the author 2014 