BACKGROUND
rubber is an indispensable commodity used in the manufacture of over  <dig>  products worldwide  <cit> .
approximately  <dig>  plant species synthesize rubber  <cit> , but hevea brasiliensis  muell.-arg., also known as pará rubber tree, is the primary commercial source for natural rubber  production.
this member of the euphorbiaceae family originated from the amazon basin, and it was not until the nineteenth century that it significantly began to be commercially exploited and its domestic cultivation was established outside of south america.
today, plantations are mainly found in the tropical regions of asia, africa, and latin america.
rubber trees start yielding latex after reaching 5– <dig> years of maturity and have a productive lifespan of 25– <dig> years.
according to the international rubber study group , global production of nr reached nearly  <dig> million tons in  <dig> with asia accounting for about 93% of the supply.
the demand for rubber  has steadily risen over the past century and is expected to continue to increase.
nr is a latex polymer with high elasticity, flexibility, resilience, impact resistance, and efficient heat dispersion  <cit> .
these properties make nr difficult to be replaced by synthetic rubber in many applications, such as medical gloves and heavy-duty tires for aircrafts and trucks.
nr consists of 94% cis- <dig> -polyisoprene and 6% proteins and fatty acids  <cit> .
cis- <dig> -polyisoprene biopolymers are made up of c <dig> monomeric isopentenyl diphosphate  units and are formed by sequential condensation on the surface of rubber particles.
the rubber chain elongation is catalyzed by cis-prenyltransferases , known as rubber polymerases  <cit> .
the molecular weight of the resulting polymer is an important determinant of rubber quality.
only a few plants produce large amounts of high quality nr , including h. brasiliensis and the potential alternative rubber crops parthenium argentatum  and taraxacum koksaghyz   <cit> .
in addition to nr, rubber trees are used as a source of timber, once their latex productivity is no longer economically viable.
rubberwood has become a major timber export of southeast asia  <cit> .
its natural light color and excellent physical properties make it suitable for flooring and household furniture.
owing to the value of this product, several superior latex-timber clones have been developed.
some of the issues concerning the rubber industry include pathogen attack and allergenicity.
fungal diseases, such as south american leaf blight  and leaf fall caused by colletotrichum, oidium, and corynespora, are major threats to rubber production  <cit> .
in the mid-1930s, salb collapsed the rubber industry in brazil.
asian plantations have not been hit by this disease yet, but an outbreak in the region could have devastating effects.
the allergenicity of nr is an issue which continues to be a global medical concern for those repeatedly exposed to latex-containing products .
these allergies are triggered by certain proteins present in hevea-derived nr.
in recent years, guayule has emerged as a source of hypoallergenic latex  <cit> .
difficulties with conventional breeding along with limited genome-based information have impeded efficient crop improvement of h. brasiliensis.
marker assisted selection can improve the efficiency of breeding by enabling the direct selection of targeted genotypes.
analysis of genetic linkage among markers and identification of the genetic locations of desirable phenotypes would further improve the selection accuracy.
a recent surge in high-throughput sequencing efforts  <cit>  has enhanced the genetic resources available for h. brasiliensis.
however, whole-genome information is still lacking.
while most of the studies have focused on transcriptome analysis, the non-coding regions of the genome are also essential for understanding the regulatory elements controlling gene expression, as well as for the development of a more comprehensive set of molecular markers.
here, we report the draft genome of h. brasiliensis, which provides a platform to help accelerate the future improvement of this economically important crop.
RESULTS
genome sequencing and annotation
we sequenced the genome of h. brasiliensis clone rrim  <dig>  a high yielding clone developed by the rubber research institute of malaysia .
the rubber tree genome is distributed over  <dig> pairs of chromosomes  <cit> , with the haploid genome estimated to be ~ <dig>  gb by feulgen microdensitometry  <cit> .
we used a whole-genome shotgun  approach to generate ~43× coverage of sequence data from the roche/ <dig>  illumina, and solid platforms .
newbler  <cit>  was chosen as the assembler for the final assembly since the majority of the sequencing data came from the roche/ <dig> platform with relatively longer read length, especially for single end reads  <cit> .
repeat motif identification on preliminary assemblies, removal of repeat-matching raw sequencing reads, and stringent assembling parameters were applied.
the final genome assembly, based on only  <dig>  gb data or ~13× coverage after filtering repeat-matching reads , resulted in scaffolds totaling  <dig>  mb with an n <dig> of  <dig>  bp .
we anchored  <dig> scaffolds and the associated  <dig>  genes onto the 18 h. brasiliensis linkage groups based on  <dig> microsatellite markers  <cit>  .
within the mapped scaffolds,  <dig> additional reported markers were also identified .
most of the markers were located in the intergenic regions.
we have exclusively used next-generation sequencing technologies for wgs assembly of the rubber tree genome, and only a few other plant genomes have taken a similar approach.
the strawberry genome was sequenced using a similar combination of roche/ <dig>  illumina, and solid reads  as in this study, but with a nearly 9× smaller genome  and considerably lower proportion of repetitive dna , much larger contigs/scaffolds could be achieved in that case  <cit> .
on the other hand, the largest scaffolds we assembled  were comparable with those of the cannabis genome , which was assembled from illumina and roche/ <dig> data  <cit> .
the major challenge of assembling the rubber tree genome was due to its highly repetitive content.
this was also a difficulty for the barley genome , as the wgs assembly based on illumina short reads resulted in relatively small contigs   <cit> .
however, when combined with a bac-based physical map and high-resolution genetic map, a highly structured chromosome-level framework was produced.
efforts such as incorporating a physical map or other methods to provide long-range linking information will be the next step for improving the rubber tree draft genome assembly.
using repeatmodeler and repeatmasker,  <dig> % of the assembly was identified as repetitive dna .
this is estimated to represent ~78% of the genome, similar to that of maize   <cit>  and barley   <cit> .
long terminal repeat  retrotransposons are the dominant class of transposable elements , of which the gypsy-type  and copia-type  are the most abundant .
less than 2% of the total repeat elements are dna transposons.
a major part of the repeat elements  could not be associated with any known families.
combining the evidences derived from several ab initio gene prediction programs along with transcriptome and protein alignments, evidencemodeler   <cit>  predicted  <dig>  gene models from the masked assembly .
the average gene, exon, and intron lengths are  <dig>  bp,  <dig> bp, and  <dig> bp, respectively .
of the  <dig>  expressed sequence tags  and assembled transcripts available for h. brasiliensis,  <dig> % are represented in the genome .
to provide additional support for gene model prediction and validation, we generated leaf transcriptome sequences , which were de novo assembled into  <dig>  contigs .
over 99% of these contigs and 81% of the roche/ <dig> transcriptome reads aligned to the genome assembly.
these results indicate that the draft assembly represents a large proportion of the gene space.
protein sequences from the final gene predictions were annotated through different databases, including the ncbi non-redundant database, swissprot  <cit> , interpro  <cit> , and kegg  <cit>  .
eukaryotic orthologous groups   <cit>  analysis revealed a significantly higher number of proteins in the ‘signal transduction mechanisms’ , ‘posttranslational modifications, protein turnover, chaperones’ , and ‘carbohydrate transport and metabolism’  categories .
in addition, leucine-rich repeats  are the most abundant pfam  <cit>  domain represented in the genome .
among the gene models,  <dig> % are predicted to have signal peptides, with the majority being plastidial and extracellular targeted .
other than protein-coding genes, we identified  <dig> trna genes including  <dig> suppressor  trnas,  <dig> pseudogenes, and  <dig> with undetermined function .
clustering of trna genes was noticed and interestingly, all sup trna genes were clustered into  <dig> scaffolds .
we also identified 5s ,  <dig> s , 18s , and 28s  rrna genes in the assembly.
phylogeny and lineage-specific genes
phylogenetic analysis using  <dig> single copy orthologous clusters from  <dig> sequenced plant genomes shows that h. brasiliensis shares the closest ancestry with manihot esculenta , consistent with the placement based on chloroplast genes  <cit> .
outside the euphorbiaceae, the closest sequenced genome is of populus.
trichocarpa.
in agreement with the angiosperm phylogeny derived from  <dig> nuclear genes  <cit> , our analysis reveals that malpighiales  shares a common ancestry with other members in malvidae.
we compared  <dig> representative plant genomes  and found that a core gene set of  <dig>  clusters are common to all groups, while  <dig>  are unique to the euphorbiaceae .
comparison of the four sequenced euphorbiaceae genomes  indicated that  <dig>  clusters comprising  <dig>  genes are hevea specific .
we were able to assign  <dig> gene ontology   <cit>  categories ,  <dig> interpro domains , and  <dig> pfam domains  to these hevea specific genes.
the most abundant interpro and pfam domains belong to lrr and protein kinases.
kog analyses revealed that majority of the genes are associated with signal transduction, cytoskeleton, and posttranslational modification .
rubber biosynthesis
rubber biosynthesis involves fixation of carbon in the leaf, loading and transportation of the assimilates, specialized metabolic processes driving the precursors for biosynthesis, and storage of polyisoprenes in the laticifer.
sucrose provides the carbon skeleton and energy supply for rubber biosynthesis, with laticifers serving as its strong sink  <cit> .
we reconstructed the entire metabolic pathway of rubber biosynthesis in h. brasiliensis .
the carbon assimilatory mechanism of rubber biosynthesis consists of  <dig> distinct sub-metabolic pathways , represented by  <dig> genes .
we validated the expression of these genes from leaf and/or latex cdna pools, detecting at least one isoform for the majority of the gene families .
we also made a comparison with the ests obtained from the rubber-producing bark tissue of guayule  <cit> .
we found that  <dig> of the h. brasiliensis genes were represented in the guayule ests, of which  <dig> showed more than 70% sequence identity with the best match .
it has been shown that sucrose transporters and their expression patterns are directly related to tapping and rubber production  <cit> .
sucrose and monosaccharides are imported into the laticifer cytosol via sucrose  and monosaccharide  transporters which are encoded by  <dig> and  <dig> genes, respectively.
β-fructofuranosidase and fructan β-fructosidase convert sucrose into monosaccharides, and the high number of genes  found in h. brasiliensis indicates the importance of this function for rubber biosynthesis.
excess sucrose is stored as fructan and starch which can later be used as a carbon source for rubber biosynthesis.
fructan metabolism consists of  <dig> enzymatic steps , while starch metabolism involves  <dig> reactions .
carbon is directed through glycolysis , alternative pentose phosphate pathway , and acetyl coa biosynthetic pathway  to produce intermediate substrates for the biosynthesis of rubber precursors.
isoprenoid precursors for rubber biosynthesis are provided by the cytosolic mevalonate dependent  pathway in the form of ipp  <cit> .
the plastidic mevalonate independent  pathway is also suggested to contribute ipp for rubber biosynthesis  <cit> .
recently, 13c-labelled studies on hevea seedlings suggested that the mep pathway contributes ipp for carotenoid biosynthesis, but not for rubber biosynthesis  <cit> .
however, expression analysis on mva and mep pathway genes suggest that the mep pathway can be an alternate provider of ipp in mature rubber trees or in clones which do not produce a large amount of carotenoid  <cit> .
in the h. brasiliensis genome, we identified  <dig> genes encoding enzymes for the mva pathway and  <dig> for the mep pathway.
for the initiation of rubber biosynthesis, a priming allylic diphosphate  is needed  <cit> .
the biosynthetic pathway leading to these compounds involves  <dig> enzymatic steps encoded by  <dig> genes in the assembly.
rubber polymerases, involved in the polymerization of isoprenoids, belong to the family of cpts  <cit> .
we identified eight cpts from the genome  which are divided into three groups according to evolutionary relationships .
we found that h. brasiliensis cpts in groups  <dig> and  <dig> are homologous to other plant cpts  which are responsible for the elongation of short-chain c5-isoprenes .
group  <dig>  comprising cpt 1– <dig>  is specific to h. brasiliensis and members belonging to this group were proven to catalyze the formation of long chain c5-isoprenes  <cit> .
only cpt  <dig>  has introns and it shares the least homology with others.
small rubber particle protein  and rubber elongation factor  are two other key proteins involved in rubber biosynthesis  <cit>  and are represented by  <dig> and  <dig> genes, respectively, in our assembly.
rubberwood
matured rubber trees that have reached the end of their latex-producing cycle are used as a source of timber for the manufacture of furniture and other products.
wood quality is associated with several lignocellulose biosynthesis genes  <cit> , and we identified  <dig> genes in h. brasiliensis .
there are  <dig> cellulose synthase -coding genes compared with  <dig> in arabidopsis <cit>  and  <dig> in populus <cit> .
genes associated with hemicellulose biosynthesis have been identified in h. brasiliensis and is similar to populus in having more α-l-fucosidases over α-l-fucosyltransferases  <cit> .
lignin, a heteropolymer of monolignols, determines the texture and hardness of the wood.
the complete set of genes involved in monolignol biosynthesis  showed the highest similarity with populus genes.
in comparison with the populus genome, caffeic acid o-methyl transferase  and cinnamyl alcohol dehydrogenase  showed noticeable difference in their numbers .
this is probably related to the hardness of populus wood  <cit>  compared with rubberwood.
the genes involved in transport, storage, and mobilization of monolignols and its final polymerization into lignin have also been identified.
disease resistance
rubber trees are highly susceptible to fungal diseases, so the identification of disease resistance genes is one of the major focuses of rubber tree research.
hypersensitive response  is the early defense response that causes necrosis and cell death to restrict the growth of the pathogen.
plant signaling molecules, salicylic and jasmonic acids, play a critical role in activating systemic acquired resistance  and induce certain pathogenesis-related  proteins  <cit> .
the nucleotide-binding site -coding r gene family is the largest group of disease resistance genes in plants  <cit> .
in h. brasiliensis we identified  <dig> members in this family, comparable to oryza sativa, which are divided into  <dig> sub-classes: toll-interleukin-like receptor -nbs, coiled-coil -nbs, nbs, tir-nbs-lrr, cc-nbs-lrr, and nbs-lrr .
the majority were those without lrr domains, in contrast with other plants where the lrr-containing classes are typically more abundant.
we also identified  <dig> pr and  <dig> early defense  associated genes in the assembly .
all these disease resistance genes were distributed in  <dig> scaffolds, and nbs-coding genes were often found to be in clusters .
in addition, we have reconstructed the sar and hr signaling pathways for h. brasiliensis .
the overall information can be potentially exploited for the biotic stress management of the plant.
latex allergens
allergy to natural rubber latex  is one of the major global medical concerns.
there are  <dig> internationally recognized nrl allergens, known as hevb  <dig> to hevb  <dig> .
these are encoded by  <dig> genes in h. brasiliensis .
most of the allergens are stress and defense-related proteins highly abundant in the latex  <cit> .
of the major allergens causing sensitization to nrl, hevb  <dig>  is encoded by  <dig> genes whereas hevb  <dig> is only a single copy gene.
hevb  <dig>  and hevb  <dig> , associated with rubber particle, are represented by  <dig> and  <dig> genes, respectively.
hevb  <dig>  with  <dig> genes and hevb  <dig>  with  <dig> genes are also known as glycoallergens.
there are  <dig>   <dig>  and  <dig> genes coding for the cross-reactivity proteins hevb  <dig> , hevb  <dig> , and hevb  <dig> , respectively.
defense-related allergens hevb  <dig>  and hevb  <dig>  are with  <dig> genes each.
domain analysis of hevb  <dig> shows the presence of an 18– <dig> amino acid long signal peptide which confers the systemic wounding response in plants  <cit> .
other than the aforementioned latex allergens,  <dig> types of non-latex allergens  were also identified in h. brasiliensis .
transcription factors
the h. brasiliensis genome contains ~ <dig> transcription factors distributed in  <dig> major families .
transcription factors account for  <dig> % of gene models in h. brasiliensis.
the bhlh, myb, c3h, g2-like, and wrky families are overrepresented.
bhlh, the largest transcription factor family in most plants, is represented by  <dig> members.
myb, a diverse family of transcription factors that co-interacts with the bhlh family to regulate secondary metabolism  <cit>  as well as biotic and abiotic stress, has  <dig> members.
the c3h family, involved in floral development, embryogenesis, wintering and leaf senescence  <cit> , is represented by  <dig> members followed by g2-like   <cit>  and wrky   <cit> .
mads-box genes encoding homeotic floral transcription factors are divided into  <dig> groups, mα, mβ, mγ, mδ , and mikcc <cit> , and are represented by  <dig> members.
there are  <dig> type ii mads-box  genes in h. brasiliensis while the number is 54– <dig> in arabidopsis, populus, and oryza.
in contrast, only  <dig> type i mads-box  genes are in h. brasiliensis compared to 29– <dig> present in the other  <dig> species.
only  <dig> %  mads-box genes in h. brasiliensis were clustered compared to 47% in the c. papaya genome  <cit> .
phytohormone biosynthesis and signaling
phytohormone biosynthetic and signaling-related genes are well represented in h. brasiliensis .
angiosperms dedicate a larger proportion of their genomes to auxin signaling, as evident by  <dig> gene families.
however, in h. brasiliensis a significant reduction in the number is observed for some of the auxin gene family members compared to other plants, especially for saur and iaa repressors.
ga-20-oxidase, a key regulatory enzyme in gibberellin biosynthesis, has  <dig> orthologs in h. brasiliensis compared to one in ricinus and oryza.
the ethylene-responsive element binding factor  proteins are overrepresented  in h. brasiliensis, compared to other plants.
the increased number of erf transcription factors may be involved in the ethylene-dependent processes specific to h. brasiliensis.
oxophytodienoic acid reductase, important in the jasmonic acid biosynthesis pathway, is encoded by  <dig> genes.
nitric oxide synthase, involved in the biosynthesis of nitric oxide as a defense mechanism, is a highly conserved single copy gene in arabidopsis, ricinus, populus, and oryza, whereas in h. brasiliensis there are  <dig> copies.
light signaling and circadian clock related genes
light signaling pathways and circadian clocks are interconnected and have profound effects on the plant’s physiology.
light is one of the most important environmental signals processed by the circadian clock to synchronize appropriate timing of physiological events  <cit> .
expansion in the number of genes involved in photoperception and circadian rhythm is observed in h. brasiliensis , compared to populus  and arabidopsis  .
these results indicate the intense involvement of environmental signals in regulating the physiology of the rubber tree.
f-box proteins
f-box proteins are part of the skp1p-cullin-f-box protein complex involved in the ubiquitin/26s proteasome pathway responsible for the selective degradation of proteins  <cit> .
they are characterized by a conserved f-box domain  at the n-terminus  <cit>  and are reported to be involved in the regulation of various developmental processes in plants such as leaf senescence, flowering, branching, phytochrome and phytohormone signaling, circadian rhythms, and self-incompatibility  <cit> .
in h. brasiliensis there are  <dig> f-box genes, compared to  <dig> in v. vinifera,  <dig> in c. papaya,  <dig> in p. trichocarpa,  <dig> in a. thaliana, and  <dig> in o. sativa <cit> .
this is interesting and contrary to the belief that the f-box gene family is expanded in herbaceous annuals compared to woody perennials  <cit> .
carotenoids
carotenoids have a pivotal role in light harvesting, photoprotection, photomorphogenesis, lipid peroxidation, and a vast array of plant developmental processes  <cit> .
carotenoids are found in nearly all types of plastids including the frey-wyssling particles of rubber latex, imparting yellowish color to the latex of some clones.
although the role of carotenoids in the latex is not well-defined, it could be a competing sink for ipp in the laticifers.
ipp from the mep pathway is proposed to be utilized for cis-polyisoprene synthesis in clones having low carotenoid content in the latex  <cit> .
it is observed that the genes for the carotenoid biosynthetic pathway in h. brasiliensis  underwent an expansion compared to the a. thaliana genome  .
phytoene synthase and phytoene desaturase, the enzymes catalyzing the initial committed steps of carotenoid biosynthesis, are highly expanded in hevea with  <dig> and  <dig> genes, compared to single copies in arabidopsis.
the overall observations indicate more efficient carotenoid biosynthetic machinery in hevea, possibly with diverse functions.
CONCLUSIONS
given the pivotal roles of nr production and sustainability, this draft genome sequence is an invaluable resource added to the spurge family.
it will facilitate and accelerate the genetic improvement of h. brasiliensis through molecular breeding and exploitation of genetic resources.
we observed the occurrence of a higher percentage  of repeat elements which could be attributed to the increased rate of non-homologous recombination and exon shuffling  <cit> , thereby reducing the consistency in the genetic purity of the progeny.
the high percentage of repeat elements together with a lack of chromosome level information is the major hurdle in assembling the whole h. brasiliensis genome.
the genome information together with the characterization of all available molecular markers linked to the desired genes will facilitate nr production by means of trait dependent molecular breeding.
alongside tree genome sequences available from populus, eucalyptus, and the herbaceous model arabidopsis, rubber research would specifically get assistance in the key areas of latex production, wood development, disease resistance, and allergenicity.

 rubber is an indispensable commodity used in the manufacture of over products worldwide. approximately plant species synthesize rubber, but hevea brasiliensis muell.-arg., also known as pará rubber tree, is the primary commercial source for natural rubber production. this member of the euphorbiaceae family originated from the amazon basin, and it was not until the nineteenth century that it significantly began to be commercially exploited and its domestic cultivation was established outside of south america. today, plantations are mainly found in the tropical regions of asia, africa, and latin america. rubber trees start yielding latex after reaching 5– years of maturity and have a productive lifespan of 25– years. according to the international rubber study group, global production of nr reached nearly million tons in with asia accounting for about 93% of the supply. the demand for rubber has steadily risen over the past century and is expected to continue to increase. nr is a latex polymer with high elasticity, flexibility, resilience, impact resistance, and efficient heat dispersion. these properties make nr difficult to be replaced by synthetic rubber in many applications, such as medical gloves and heavy-duty tires for aircrafts and trucks. nr consists of 94% cis- -polyisoprene and 6% proteins and fatty acids. cis- -polyisoprene biopolymers are made up of c monomeric isopentenyl diphosphate units and are formed by sequential condensation on the surface of rubber particles. the rubber chain elongation is catalyzed by cis-prenyltransferases, known as rubber polymerases. the molecular weight of the resulting polymer is an important determinant of rubber quality. only a few plants produce large amounts of high quality nr, including h. brasiliensis and the potential alternative rubber crops parthenium argentatum and taraxacum koksaghyz. in addition to nr, rubber trees are used as a source of timber, once their latex productivity is no longer economically viable. rubberwood has become a major timber export of southeast asia. its natural light color and excellent physical properties make it suitable for flooring and household furniture. owing to the value of this product, several superior latex-timber clones have been developed. some of the issues concerning the rubber industry include pathogen attack and allergenicity. fungal diseases, such as south american leaf blight and leaf fall caused by colletotrichum, oidium, and corynespora, are major threats to rubber production. in the mid-1930s, salb collapsed the rubber industry in brazil. asian plantations have not been hit by this disease yet, but an outbreak in the region could have devastating effects. the allergenicity of nr is an issue which continues to be a global medical concern for those repeatedly exposed to latex-containing products. these allergies are triggered by certain proteins present in hevea-derived nr. in recent years, guayule has emerged as a source of hypoallergenic latex. difficulties with conventional breeding along with limited genome-based information have impeded efficient crop improvement of h. brasiliensis. marker assisted selection can improve the efficiency of breeding by enabling the direct selection of targeted genotypes. analysis of genetic linkage among markers and identification of the genetic locations of desirable phenotypes would further improve the selection accuracy. a recent surge in high-throughput sequencing efforts has enhanced the genetic resources available for h. brasiliensis. however, whole-genome information is still lacking. while most of the studies have focused on transcriptome analysis, the non-coding regions of the genome are also essential for understanding the regulatory elements controlling gene expression, as well as for the development of a more comprehensive set of molecular markers. here, we report the draft genome of h. brasiliensis, which provides a platform to help accelerate the future improvement of this economically important crop.  genome sequencing and annotation we sequenced the genome of h. brasiliensis clone rrim a high yielding clone developed by the rubber research institute of malaysia. the rubber tree genome is distributed over pairs of chromosomes, with the haploid genome estimated to be ~ gb by feulgen microdensitometry. we used a whole-genome shotgun approach to generate ~43× coverage of sequence data from the roche/ illumina, and solid platforms. newbler was chosen as the assembler for the final assembly since the majority of the sequencing data came from the roche/ platform with relatively longer read length, especially for single end reads. repeat motif identification on preliminary assemblies, removal of repeat-matching raw sequencing reads, and stringent assembling parameters were applied. the final genome assembly, based on only gb data or ~13× coverage after filtering repeat-matching reads, resulted in scaffolds totaling mb with an n of bp. we anchored scaffolds and the associated genes onto the 18 h. brasiliensis linkage groups based on microsatellite markers. within the mapped scaffolds, additional reported markers were also identified. most of the markers were located in the intergenic regions. we have exclusively used next-generation sequencing technologies for wgs assembly of the rubber tree genome, and only a few other plant genomes have taken a similar approach. the strawberry genome was sequenced using a similar combination of roche/ illumina, and solid reads as in this study, but with a nearly 9× smaller genome and considerably lower proportion of repetitive dna, much larger contigs/scaffolds could be achieved in that case. on the other hand, the largest scaffolds we assembled were comparable with those of the cannabis genome, which was assembled from illumina and roche/ data. the major challenge of assembling the rubber tree genome was due to its highly repetitive content. this was also a difficulty for the barley genome, as the wgs assembly based on illumina short reads resulted in relatively small contigs. however, when combined with a bac-based physical map and high-resolution genetic map, a highly structured chromosome-level framework was produced. efforts such as incorporating a physical map or other methods to provide long-range linking information will be the next step for improving the rubber tree draft genome assembly. using repeatmodeler and repeatmasker, % of the assembly was identified as repetitive dna. this is estimated to represent ~78% of the genome, similar to that of maize and barley. long terminal repeat retrotransposons are the dominant class of transposable elements, of which the gypsy-type and copia-type are the most abundant. less than 2% of the total repeat elements are dna transposons. a major part of the repeat elements could not be associated with any known families. combining the evidences derived from several ab initio gene prediction programs along with transcriptome and protein alignments, evidencemodeler predicted gene models from the masked assembly. the average gene, exon, and intron lengths are bp, bp, and bp, respectively. of the expressed sequence tags and assembled transcripts available for h. brasiliensis, % are represented in the genome. to provide additional support for gene model prediction and validation, we generated leaf transcriptome sequences, which were de novo assembled into contigs. over 99% of these contigs and 81% of the roche/ transcriptome reads aligned to the genome assembly. these results indicate that the draft assembly represents a large proportion of the gene space. protein sequences from the final gene predictions were annotated through different databases, including the ncbi non-redundant database, swissprot, interpro, and kegg. eukaryotic orthologous groups analysis revealed a significantly higher number of proteins in the ‘signal transduction mechanisms’, ‘posttranslational modifications, protein turnover, chaperones’, and ‘carbohydrate transport and metabolism’ categories. in addition, leucine-rich repeats are the most abundant pfam domain represented in the genome. among the gene models, % are predicted to have signal peptides, with the majority being plastidial and extracellular targeted. other than protein-coding genes, we identified trna genes including suppressor trnas, pseudogenes, and with undetermined function. clustering of trna genes was noticed and interestingly, all sup trna genes were clustered into scaffolds. we also identified 5s, s, 18s, and 28s rrna genes in the assembly. phylogeny and lineage-specific genes phylogenetic analysis using single copy orthologous clusters from sequenced plant genomes shows that h. brasiliensis shares the closest ancestry with manihot esculenta, consistent with the placement based on chloroplast genes. outside the euphorbiaceae, the closest sequenced genome is of populus. trichocarpa. in agreement with the angiosperm phylogeny derived from nuclear genes, our analysis reveals that malpighiales shares a common ancestry with other members in malvidae. we compared representative plant genomes and found that a core gene set of clusters are common to all groups, while are unique to the euphorbiaceae. comparison of the four sequenced euphorbiaceae genomes indicated that clusters comprising genes are hevea specific. we were able to assign gene ontology categories, interpro domains, and pfam domains to these hevea specific genes. the most abundant interpro and pfam domains belong to lrr and protein kinases. kog analyses revealed that majority of the genes are associated with signal transduction, cytoskeleton, and posttranslational modification. rubber biosynthesis rubber biosynthesis involves fixation of carbon in the leaf, loading and transportation of the assimilates, specialized metabolic processes driving the precursors for biosynthesis, and storage of polyisoprenes in the laticifer. sucrose provides the carbon skeleton and energy supply for rubber biosynthesis, with laticifers serving as its strong sink. we reconstructed the entire metabolic pathway of rubber biosynthesis in h. brasiliensis. the carbon assimilatory mechanism of rubber biosynthesis consists of distinct sub-metabolic pathways, represented by genes. we validated the expression of these genes from leaf and/or latex cdna pools, detecting at least one isoform for the majority of the gene families. we also made a comparison with the ests obtained from the rubber-producing bark tissue of guayule. we found that of the h. brasiliensis genes were represented in the guayule ests, of which showed more than 70% sequence identity with the best match. it has been shown that sucrose transporters and their expression patterns are directly related to tapping and rubber production. sucrose and monosaccharides are imported into the laticifer cytosol via sucrose and monosaccharide transporters which are encoded by and genes, respectively. β-fructofuranosidase and fructan β-fructosidase convert sucrose into monosaccharides, and the high number of genes found in h. brasiliensis indicates the importance of this function for rubber biosynthesis. excess sucrose is stored as fructan and starch which can later be used as a carbon source for rubber biosynthesis. fructan metabolism consists of enzymatic steps, while starch metabolism involves reactions. carbon is directed through glycolysis, alternative pentose phosphate pathway, and acetyl coa biosynthetic pathway to produce intermediate substrates for the biosynthesis of rubber precursors. isoprenoid precursors for rubber biosynthesis are provided by the cytosolic mevalonate dependent pathway in the form of ipp. the plastidic mevalonate independent pathway is also suggested to contribute ipp for rubber biosynthesis. recently, 13c-labelled studies on hevea seedlings suggested that the mep pathway contributes ipp for carotenoid biosynthesis, but not for rubber biosynthesis. however, expression analysis on mva and mep pathway genes suggest that the mep pathway can be an alternate provider of ipp in mature rubber trees or in clones which do not produce a large amount of carotenoid. in the h. brasiliensis genome, we identified genes encoding enzymes for the mva pathway and for the mep pathway. for the initiation of rubber biosynthesis, a priming allylic diphosphate is needed. the biosynthetic pathway leading to these compounds involves enzymatic steps encoded by genes in the assembly. rubber polymerases, involved in the polymerization of isoprenoids, belong to the family of cpts. we identified eight cpts from the genome which are divided into three groups according to evolutionary relationships. we found that h. brasiliensis cpts in groups and are homologous to other plant cpts which are responsible for the elongation of short-chain c5-isoprenes. group comprising cpt 1– is specific to h. brasiliensis and members belonging to this group were proven to catalyze the formation of long chain c5-isoprenes. only cpt has introns and it shares the least homology with others. small rubber particle protein and rubber elongation factor are two other key proteins involved in rubber biosynthesis and are represented by and genes, respectively, in our assembly. rubberwood matured rubber trees that have reached the end of their latex-producing cycle are used as a source of timber for the manufacture of furniture and other products. wood quality is associated with several lignocellulose biosynthesis genes, and we identified genes in h. brasiliensis. there are cellulose synthase -coding genes compared with in arabidopsis and in populus. genes associated with hemicellulose biosynthesis have been identified in h. brasiliensis and is similar to populus in having more α-l-fucosidases over α-l-fucosyltransferases. lignin, a heteropolymer of monolignols, determines the texture and hardness of the wood. the complete set of genes involved in monolignol biosynthesis showed the highest similarity with populus genes. in comparison with the populus genome, caffeic acid o-methyl transferase and cinnamyl alcohol dehydrogenase showed noticeable difference in their numbers. this is probably related to the hardness of populus wood compared with rubberwood. the genes involved in transport, storage, and mobilization of monolignols and its final polymerization into lignin have also been identified. disease resistance rubber trees are highly susceptible to fungal diseases, so the identification of disease resistance genes is one of the major focuses of rubber tree research. hypersensitive response is the early defense response that causes necrosis and cell death to restrict the growth of the pathogen. plant signaling molecules, salicylic and jasmonic acids, play a critical role in activating systemic acquired resistance and induce certain pathogenesis-related proteins. the nucleotide-binding site -coding r gene family is the largest group of disease resistance genes in plants. in h. brasiliensis we identified members in this family, comparable to oryza sativa, which are divided into sub-classes: toll-interleukin-like receptor -nbs, coiled-coil -nbs, nbs, tir-nbs-lrr, cc-nbs-lrr, and nbs-lrr. the majority were those without lrr domains, in contrast with other plants where the lrr-containing classes are typically more abundant. we also identified pr and early defense associated genes in the assembly. all these disease resistance genes were distributed in scaffolds, and nbs-coding genes were often found to be in clusters. in addition, we have reconstructed the sar and hr signaling pathways for h. brasiliensis. the overall information can be potentially exploited for the biotic stress management of the plant. latex allergens allergy to natural rubber latex is one of the major global medical concerns. there are internationally recognized nrl allergens, known as hevb to hevb. these are encoded by genes in h. brasiliensis. most of the allergens are stress and defense-related proteins highly abundant in the latex. of the major allergens causing sensitization to nrl, hevb is encoded by genes whereas hevb is only a single copy gene. hevb and hevb, associated with rubber particle, are represented by and genes, respectively. hevb with genes and hevb with genes are also known as glycoallergens. there are and genes coding for the cross-reactivity proteins hevb, hevb, and hevb, respectively. defense-related allergens hevb and hevb are with genes each. domain analysis of hevb shows the presence of an 18– amino acid long signal peptide which confers the systemic wounding response in plants. other than the aforementioned latex allergens, types of non-latex allergens were also identified in h. brasiliensis. transcription factors the h. brasiliensis genome contains ~ transcription factors distributed in major families. transcription factors account for % of gene models in h. brasiliensis. the bhlh, myb, c3h, g2-like, and wrky families are overrepresented. bhlh, the largest transcription factor family in most plants, is represented by members. myb, a diverse family of transcription factors that co-interacts with the bhlh family to regulate secondary metabolism as well as biotic and abiotic stress, has members. the c3h family, involved in floral development, embryogenesis, wintering and leaf senescence, is represented by members followed by g2-like and wrky. mads-box genes encoding homeotic floral transcription factors are divided into groups, mα, mβ, mγ, mδ, and mikcc, and are represented by members. there are type ii mads-box genes in h. brasiliensis while the number is 54– in arabidopsis, populus, and oryza. in contrast, only type i mads-box genes are in h. brasiliensis compared to 29– present in the other species. only % mads-box genes in h. brasiliensis were clustered compared to 47% in the c. papaya genome. phytohormone biosynthesis and signaling phytohormone biosynthetic and signaling-related genes are well represented in h. brasiliensis. angiosperms dedicate a larger proportion of their genomes to auxin signaling, as evident by gene families. however, in h. brasiliensis a significant reduction in the number is observed for some of the auxin gene family members compared to other plants, especially for saur and iaa repressors. ga-20-oxidase, a key regulatory enzyme in gibberellin biosynthesis, has orthologs in h. brasiliensis compared to one in ricinus and oryza. the ethylene-responsive element binding factor proteins are overrepresented in h. brasiliensis, compared to other plants. the increased number of erf transcription factors may be involved in the ethylene-dependent processes specific to h. brasiliensis. oxophytodienoic acid reductase, important in the jasmonic acid biosynthesis pathway, is encoded by genes. nitric oxide synthase, involved in the biosynthesis of nitric oxide as a defense mechanism, is a highly conserved single copy gene in arabidopsis, ricinus, populus, and oryza, whereas in h. brasiliensis there are copies. light signaling and circadian clock related genes light signaling pathways and circadian clocks are interconnected and have profound effects on the plant’s physiology. light is one of the most important environmental signals processed by the circadian clock to synchronize appropriate timing of physiological events. expansion in the number of genes involved in photoperception and circadian rhythm is observed in h. brasiliensis, compared to populus and arabidopsis. these results indicate the intense involvement of environmental signals in regulating the physiology of the rubber tree. f-box proteins f-box proteins are part of the skp1p-cullin-f-box protein complex involved in the ubiquitin/26s proteasome pathway responsible for the selective degradation of proteins. they are characterized by a conserved f-box domain at the n-terminus and are reported to be involved in the regulation of various developmental processes in plants such as leaf senescence, flowering, branching, phytochrome and phytohormone signaling, circadian rhythms, and self-incompatibility. in h. brasiliensis there are f-box genes, compared to in v. vinifera, in c. papaya, in p. trichocarpa, in a. thaliana, and in o. sativa. this is interesting and contrary to the belief that the f-box gene family is expanded in herbaceous annuals compared to woody perennials. carotenoids carotenoids have a pivotal role in light harvesting, photoprotection, photomorphogenesis, lipid peroxidation, and a vast array of plant developmental processes. carotenoids are found in nearly all types of plastids including the frey-wyssling particles of rubber latex, imparting yellowish color to the latex of some clones. although the role of carotenoids in the latex is not well-defined, it could be a competing sink for ipp in the laticifers. ipp from the mep pathway is proposed to be utilized for cis-polyisoprene synthesis in clones having low carotenoid content in the latex. it is observed that the genes for the carotenoid biosynthetic pathway in h. brasiliensis underwent an expansion compared to the a. thaliana genome. phytoene synthase and phytoene desaturase, the enzymes catalyzing the initial committed steps of carotenoid biosynthesis, are highly expanded in hevea with and genes, compared to single copies in arabidopsis. the overall observations indicate more efficient carotenoid biosynthetic machinery in hevea, possibly with diverse functions.  given the pivotal roles of nr production and sustainability, this draft genome sequence is an invaluable resource added to the spurge family. it will facilitate and accelerate the genetic improvement of h. brasiliensis through molecular breeding and exploitation of genetic resources. we observed the occurrence of a higher percentage of repeat elements which could be attributed to the increased rate of non-homologous recombination and exon shuffling, thereby reducing the consistency in the genetic purity of the progeny. the high percentage of repeat elements together with a lack of chromosome level information is the major hurdle in assembling the whole h. brasiliensis genome. the genome information together with the characterization of all available molecular markers linked to the desired genes will facilitate nr production by means of trait dependent molecular breeding. alongside tree genome sequences available from populus, eucalyptus, and the herbaceous model arabidopsis, rubber research would specifically get assistance in the key areas of latex production, wood development, disease resistance, and allergenicity. 
hevea brasiliensis, a member of the euphorbiaceae family, is the major commercial source of natural rubber. most of the key genes associated with rubber biosynthesis, rubberwood formation, disease resistance, and allergenicity have been identified. nr is a latex polymer with high elasticity, flexibility, and resilience that has played a critical role in the world economy since here, we report the draft genome sequence of h. brasiliensis. the knowledge gained from this genome sequence will aid in the future development of high-yielding clones to keep up with the ever increasing need for natural rubber. overall, ~78% of the genome was identified as repetitive dna.
 hevea brasiliensis, a member of the euphorbiaceae family, is the major commercial source of natural rubber. nr is a latex polymer with high elasticity, flexibility, and resilience that has played a critical role in the world economy since  here, we report the draft genome sequence of h. brasiliensis. the assembly spans ~ gb of the estimated gb haploid genome. overall, ~78% of the genome was identified as repetitive dna. gene prediction shows gene models, of which % are unique to hevea. most of the key genes associated with rubber biosynthesis, rubberwood formation, disease resistance, and allergenicity have been identified.  the knowledge gained from this genome sequence will aid in the future development of high-yielding clones to keep up with the ever increasing need for natural rubber. hevea brasiliensiseuphorbiaceaenatural rubbergenome 