BACKGROUND
bananas and plantains , which are staple foods due to their high protein content and nutrition value as well as the main income source in many developing countries, are among the most important crops in the world.
in fact, banana ranks as the fifth most important agricultural crop in world trade, making it the world’s leading fruit crop and a significant economic backbone to the export industry of many agriculture-based countries in asia, africa, and latin america  <cit> .
therefore, the global and local health of banana crops is of utmost importance to the world economy.
there are several devastating diseases that target the musa crop  <cit> .
one such disease, panama disease or fusarium wilt, is caused by the fungus fusarium oxysporum f. sp.
cubense   <cit>  and is widely regarded as one of the most destructive plant diseases in the world.
to date, foc has devastated banana production and continues to threaten crops  <cit> .
the disease was first reported in  <dig> in australia and later destroyed the export trade based on the variety ‘gros michel’ by the 1950s.
since the 1960s, the resistant ‘cavendish’  subgroup of cultivars has dominated banana exports, becoming the major commercial variety in the world.
however, an extremely virulent form of foc, called ‘tropical race 4’ , is capable of attacking the susceptible cavendish variety, causing large losses in banana production in recent years  <cit> .
foc infects the lateral or feeder roots of banana plants upon contact  <cit> .
foc infection causes wilt syndrome with the typical symptoms of necrosis and rotting of roots, rhizomes, and pseudostem vessels, which turn a reddish-brown/maroon color as the fungus grows through the tissues.
after the decay of infected plants, the pathogen can survive in soil in chlamydospore form over a long period of time to infect other plants.
foc spores can spread through water or soil, and by adhering to vehicles and footwear.
in the soil, fusarium is difficult to control by general chemical measures, such as fungicides or soil fumigants  <cit> .
therefore, resistance breeding is the preferred method of overcoming the fusarium wilt of banana plants.
however, because cavendish bananas have a triploid  genome, they do not produce seeds, which hinders conventional breeding strategies  <cit> .
genetic engineering methods can improve the disease resistance of banana plants to fusarium wilt  <cit> ; however, little is known about the actual transcriptional changes and their regulation during the pathogen-plant interaction.
understanding the underlying changes during this interaction would allow for the identification of signal transduction pathways affected by infection and the interaction mechanisms during infection, which can lead to improvement of disease resistance of the banana plants.
traditional genome-wide analysis of gene expression of organisms under different conditions or, in the case of pathogens, at different life cycle stages, has mainly been carried out by microarrays, suppression subtractive hybridization , and cdna-aflp methods  <cit> .
van den berg et al.
used ssh and microarrays to show that cell wall-strengthening genes may be important for banana resistance to fusarium wilt.
however, the approach that was used suffers a number of drawbacks, including the fact that the genes are far from complete with only  <dig> clones  <cit> .
recently, with the completion of banana genome sequence, a doubled haploid m. acuminata genotype  has been shown to be highly resistant to foc tr <dig> by phenotyping assays  <cit> , but further research on its mechanism has not been performed, especially with relation to transcription.
a resistant variety of the cavendish banana  was acquired by somaclonal variation, using the rna-seq and dge methods, and it was discovered that recognition of pamps  and defense-related transcripts are involved in banana resistance to foc tr <dig> infection  <cit> .
therefore, elucidation of the mechanism by which cavendish bananas respond to foc tr <dig> infection is imperative.
one such promising method developed in recent years is next-generation sequencing, by which an enormous amount of sequence data can be rapidly obtained within a short period of time due to its high-throughput and high-coverage nature  <cit> .
rna-seq technology, which is based on deep-sequencing, enables more precise quantification of genome-wide transcript levels than previous, microarray-based methods  <cit> .
in this technology, whole mrna or cdna is mechanically fragmented for deep-sequencing, the results of which can be then mapped on a reference genome or used in de novo assembly to obtain a genome-wide transcriptome.
another method of great value for expression analysis is digital gene-expression   <cit> .
dge uses 17– <dig> base pair  short fragments from the whole transcriptome as gene-specific tags and calculates the expression level of a gene from the frequency of its tag.
we previously used a green fluorescent protein -tagged strain of foc tr <dig> and characterized early events in infection and disease development of cavendish plantlets  <cit> .
the combination of dge and rna-seq allows us to easily perform transcriptome analysis without the need for an already-assembled reference genome.
despite the importance of the foc pathogen for global banana production, rna-seq and dge have not been used to investigate the main questions underlying the pathogen-banana interaction.
therefore, we aimed to investigate the changes in gene expression during foc tr <dig> infection of banana roots using rna-seq and dge analysis.
for this purpose, we generated over  <dig>  billion bases of high-quality dna sequence and demonstrated the suitability of short-read sequencing for assembly and annotation of genes expressed in a triploid-genome plant without previous whole-genome information.
we then identified  <dig>  distinct sequences.
furthermore, we compared the gene expression profiles during an infection time course using dge analysis.
the assembled and annotated gene expression profiles provide an invaluable resource for the identification of differentially expressed genes during foc tr <dig> infection of banana, which will enable us to screen for host susceptibility factors and to monitor shifts in foc tr <dig> virulence.
RESULTS
assembly of a high-quality banana root transcriptome
in the absence of a sequenced genome, de novo assembly of rna-seq data was the only viable option to study the banana transcriptome.
to obtain an overview of the expression profile of banana roots under foc tr <dig> stress, a cdna sample was prepared from the total rna of an equal mixture of roots not infected and infected with foc tr <dig> for  <dig>   <dig>  and  <dig> days to acquire the genes whose expression is specifically altered when the plant is infected by foc tr <dig>
deep-sequencing of this cdna sample produced  <dig> , <dig> sequence reads with a length of  <dig> bp each , which corresponded to approximately  <dig>  gigabase pairs  of raw data.
an overview of the sequencing and assembly is outlined in table  <dig>
the raw reads were first assembled into a draft using soap de novo-oases software  <cit> , and further assembly was achieved using cap <dig> sequence assembly software.
after assembling, reads were also mapped back to the assembled transcripts with a length ≥  <dig> bp.
if the coverage of two assembled reads was more than 80%, then the shorter one was eliminated.
the remaining sequences were then assembled into  <dig>  contigs .
the mean contig size was  <dig> bp with lengths ranging from  <dig> to  <dig>  bp, including  <dig> contigs larger than  <dig>  bp.
a total of  <dig>  contigs were confirmed using the banana est library .
the mean contig size in the final library was  <dig> bp with lengths ranging from  <dig> to  <dig>  bp, including  <dig> contigs larger than  <dig>  bp.
using paired-end joining and gap-filling, the contigs were further assembled into  <dig>  scaffolds with a mean size of  <dig>  bp, including  <dig>  scaffolds larger than  <dig>  bp.
the longest scaffold was  <dig>  bp .
to evaluate the quality of the dataset, we analyzed the gap-filling to assembled contigs length.
the total size of all contigs was  <dig> , <dig> bp with a total  <dig> , <dig> bp gap size.
the total size of the scaffolds was  <dig> , <dig> bp.
in order to evaluate our data, the assembled banana transcriptome was searched using blastn against plant cdnas  using a cut-off e-value of 10- <dig>  using this approach, approximately  <dig> % contigs  aligned successfully to plant cdna with a total gap size of  <dig> , <dig> bp, which was  <dig>  bp shorter than the banana gap size.
in addition,  <dig> % of the reads and  <dig> % of the paired-end joined sequences could be mapped onto the banana transcriptome.
importantly, 90% of the distinct gene sequences were unique.
our results indicated that the banana root transcriptome was of high quality.
transcripts with lengths ≥  <dig> bp were subsequently used for analysis.
functional annotation of the banana root transcriptome
we acquired  <dig>  distinct gene sequences,  <dig>  of which were longer than  <dig>  bp.
compared with the  <dig>  est and  <dig>  nucleotide sequences in ncbi database of banana, our data enriched the gene resources for banana.
to annotate, classify, and functionally map the  <dig>  distinct gene sequences, we used blastx to match the distinct gene sequences using a cut-off e-value of 10- <dig>  including the non-redundant protein database , gene ontology , and the kyoto encyclopedia of genes and genomes  database with a cut-off e-value of 10- <dig>  using this approach,  <dig>  distinct gene sequences  returned a valid blast result , confirming the high quality of our transcriptome assembly.
fourteen percent  of the distinct gene sequences could not be matched to known genes.
to annotate the distinct gene sequences that we identified in our transcriptome assembly, we initially searched against the plant proteins in the nr database.
as a result, we obtained  <dig>  significant blast hits , which confirmed that most genes could be annotated after assembly.
interestingly,  <dig> % of the  <dig> –  <dig>  bp query sequences,  <dig> % of the  <dig>  –  <dig>  bp, 85% of the  <dig>  –  <dig>  bp, and  <dig> % of the query sequences longer than  <dig>  bp were annotated successfully .
this result indicated that the longer sequences provided more accurate matches with the nr database, while the shorter sequences lacked sufficient gene information to match, despite being easier to annotate.
go classification
out of the  <dig>  annotated distinct gene sequences,  <dig>   were assigned  <dig>  go terms using blast2go.
forty-five go sub-categories were represented under three major categories .
the majority of the go annotations were in the cellular component category, assigned to  <dig>   distinct gene sequences, followed by the biological process category for  <dig>   distinct gene sequences, and the molecular function category for  <dig>   distinct gene sequences.
the major sub-categories are shown in figure 2: four major cellular component sub-categories were “cell” , “cell part” , “organelle” , and “organelle part” ; two major molecular functions sub-categories were “binding”  and “catalytic” ; and four major biological process sub-categories were “metabolic process” , “cellular process” , “metabolic process” , and “response stimulus” .
however, only  <dig> % of banana root distinct gene sequences were assigned with these go terms, possibly because the large number of uninformative gene descriptions of these plant protein hits.
kyoto encyclopedia of genes and genomes  pathway mapping
by mapping ec numbers to the reference canonical pathways,  <dig>   distinct gene sequences were assigned to  <dig> kegg pathways.
the pathways most represented by unique sequences were carbohydrate metabolism , amino acid metabolism , signal transduction , and cell growth and death .
taken together, these annotations provide a valuable resource for investigating the specific processes, structures, functions, and pathways involved in the response to the infection of foc tr <dig> in banana roots.
statistics of dge tags
using the dge method, which generates absolute rather than relative gene expression measurements and avoids many of the inherent limitations of microarray them with analysis, we analyzed the gene expression profile of banana roots after inoculating foc tr <dig>  total rna isolated from banana roots at  <dig>   <dig>   <dig>  and  <dig> days post-inoculation  were analyzed by illumina dge tag profiling to create transcriptome profiles of the four groups.
dge tags were derived from the 3’utr of transcripts and were 21-nucleotides long.
dge data provided a quantitative measure of transcript abundance in the rna population.
dge analysis also allowed for the identification of previously unannotated genes.
the majority of dge tags were expected to match only one location in the genome, with the remaining tags matching duplicate genes, alternate transcripts, antisense strands, or repeated sequences  <cit> .
we obtained a total of  <dig> , <dig>   <dig> , <dig>   <dig> , <dig>  and  <dig> , <dig> total tags and  <dig> ,  <dig> ,  <dig> , and  <dig>  distinct tags from the roots of the  <dig>   <dig>   <dig>  and  <dig> dpi time points, respectively.
heterogeneity and redundancy are two significant characteristics of mrna expression, and while the majority of mrnas are expressed at low levels, a small proportion is highly expressed.
therefore, the distribution of tag expression was used to evaluate the normality of the dge data.
as shown in figure  <dig>  the distribution of total tags and distinct tags over different tag abundance categories showed similar patterns for all four dge libraries.
mapping sequences to the reference transcriptome database
to identify the molecular events behind foc tr <dig> infection of banana roots, we mapped the tag sequences of the four dge libraries to our transcriptome reference database.
among the  <dig>  –  <dig>  distinct tags generated from the illumina sequencing of the four libraries,  <dig> –  <dig>  distinct tags were mapped to a gene in the reference database .
tags mapped to a unique sequence are the most critical subset in dge libraries, as they can explicitly identify a transcript.
up to  <dig> %  of the sequences in our transcriptome reference tag database could be unequivocally identified by a unique tag .
to determine whether our dge tags reached saturation, we compared the increase in the distinct tag number to the increase in total tag number.
when sequencing depths reached  <dig> million or more base pairs, the number of distinct tags discovered almost ceased to increase in all four libraries, which indicated that the sequencing was saturated .
the level of gene expression was then determined by calculating the number of unambiguous tags for each distinct gene sequence and then normalizing this to the number of transcripts per million tags .
additional file  <dig> provides a list of the top  <dig> most abundantly expressed genes in the  <dig> dpi library as an example.
comparing our results with those of van den berg , the expressions of two catalases, two pectin acetyl esterases and three pathogenesis-related proteins in our result in the  <dig> dpi library were consistent.
the result indicated that those genes responded to foc tr <dig> infection  <cit> .
gene expression profile changes in banana roots infected with foc tr4
to identify the signaling pathways involved in the banana response to foc tr <dig> infection, we identified tags that were differentially expressed between the  <dig> dpi and the later infection time points using an algorithm developed by audic et al.
<cit> .
a total of  <dig>  distinct gene sequences significantly changed between the  <dig> and  <dig> dpi libraries, where  <dig>  distinct gene sequences were upregulated and  <dig>  distinct gene sequences were downregulated after  <dig> days of foc tr <dig> infection.
between the  <dig> and  <dig> dpi libraries, a total of  <dig>  distinct gene sequences were detected with  <dig>  upregulated distinct gene sequences and  <dig>  downregulated gene sequences.
there were  <dig>  distinct gene sequences that were expressed at a different level in the  <dig> and  <dig> dpi libraries, with  <dig>  upregulated distinct gene sequences and  <dig>  downregulated distinct gene sequences after  <dig> days of infection .
gene ontology analysis was used for the above differential expression distinct gene sequences, and enrichment analysis was performed using a false discovery rate  adjusted p-value of ≤ <dig>  as the cutoff.
the downregulated distinct gene sequences did not enrich any go term, while the upregulated distinct gene sequences enriched  <dig>   <dig>  and  <dig> featured go terms at  <dig> dpi,  <dig> dpi and  <dig> dpi respectively .
in particular, response to stress  was enriched at  <dig> dpi and response to chemical stimulus  was enriched at  <dig> dpi, which suggested that banana root was subjected to stress and chemical stimulation because of foc tr <dig> infection at these two time points.
although down-regulated expression of distinct gene sequences did not enrich the go term, we did find that some distinct gene sequences had down-regulated expression, such as nsp-interacting kinase  <cit>  and sumo e <dig> protein ligase  <cit> .
these genes were included in the immune-related go term, which indicated that those distinct gene sequences did not respond to foc tr <dig> infection.
kegg pathway analysis of differentially expressed banana roots genes in response to infection by foc tr4
to understand the functions of differentially expressed distinct gene sequences, we mapped them to kegg terms to discover those genes involved in metabolic or signal transduction pathways that were significantly enriched.
additional file  <dig> shows enriched pathways at  <dig> dpi,  <dig> dpi and  <dig> dpi.
phenylalanine metabolism was enriched at  <dig> dpi,  <dig> dpi, and  <dig> dpi .
there are twenty-three peroxidases, twelve bacterial-induced peroxidase precursors, five 4-coumarate: coenzyme a ligases, three cinnamate 4-hydroxylases and three phenylalanine ammonia lyases enriched in this pathway.
meanwhile,  <dig> peroxidases, four 4-coumarate:coenzyme a ligases, two cinnamate 4-hydroxylases and one phenylalanine ammonia lyase were enriched in phenylpropanoid biosynthesis at  <dig> dpi.
it should be emphasized that peroxidases were enriched in both pathways.
the peroxidases enriched in those pathways may be involved in increased lignin biosynthesis  <cit> , and may acting as basal defense components: peroxidase is one source for the production of ros  <cit> .
that the peroxidases were upregulated suggests that banana roots responded to infection by foc tr <dig> by ros production.
similarly, enrichment of drug metabolism-cytochrome p <dig> was found at 2dpi.
there are  <dig> distinct gene sequences of glutathione s-transferases  or glutathione transferases in this pathway .
gsts, as a heterogeneous group of cell detoxifying enzymes, catalyse the conjugation of tripeptide glutathione  to electrophilic sites on a wide range of phytotoxic substrates  <cit> .
it is likely that even the susceptible cultivar activates some early mechanisms of defense against foc tr4; however, these are not sufficient to provide resistance against the pathogen.
at  <dig> dpi, alpha-linolenic acid metabolism was enriched, leading to jasmonic acid biosynthesis, which is one of the pathways associated with pathogen resistance and the genes in this pathway were significantly affected by foc tr <dig> infestation at all time points .
this is consistent with previous reports that biotic and abiotic stresses, such as pathogen infection, wounding and insect feeding, can trigger ja biosynthesis through direct activation of genes encoding the relevant biosynthetic enzymes  <cit> .
ethylene and sa biosynthetic and signaling related genes showed no significant differences between non-inoculation and inoculation in our results.
these results indicate that ja biological synthesis may be regulated by foc tr <dig> infection.
similarly, the expressions of ja biosynthetic and signaling related genes in a resistant variety were higher than in a susceptible variety  <cit> .
further study of these genes in this pathway could identify them as targets for testing whether a variety is resistant to foc tr <dig> infestation.
CONCLUSIONS
here, we present a rapid and low-cost method for triploid plant transcriptome assembly and dge analysis using illumina sequencing technology.
our findings provide a substantial contribution to the existing sequence resources for the banana and will certainly accelerate research regarding the devastating foc tr <dig> pathogen of this valuable staple food.
our expression analysis results provide promising leads for future functional studies for understanding how the foc tr <dig> pathogen infects and kills banana plants.

 bananas and plantains, which are staple foods due to their high protein content and nutrition value as well as the main income source in many developing countries, are among the most important crops in the world. in fact, banana ranks as the fifth most important agricultural crop in world trade, making it the world’s leading fruit crop and a significant economic backbone to the export industry of many agriculture-based countries in asia, africa, and latin america. therefore, the global and local health of banana crops is of utmost importance to the world economy. there are several devastating diseases that target the musa crop. one such disease, panama disease or fusarium wilt, is caused by the fungus fusarium oxysporum f. sp. cubense and is widely regarded as one of the most destructive plant diseases in the world. to date, foc has devastated banana production and continues to threaten crops. the disease was first reported in in australia and later destroyed the export trade based on the variety ‘gros michel’ by the 1950s. since the 1960s, the resistant ‘cavendish’ subgroup of cultivars has dominated banana exports, becoming the major commercial variety in the world. however, an extremely virulent form of foc, called ‘tropical race 4’, is capable of attacking the susceptible cavendish variety, causing large losses in banana production in recent years. foc infects the lateral or feeder roots of banana plants upon contact. foc infection causes wilt syndrome with the typical symptoms of necrosis and rotting of roots, rhizomes, and pseudostem vessels, which turn a reddish-brown/maroon color as the fungus grows through the tissues. after the decay of infected plants, the pathogen can survive in soil in chlamydospore form over a long period of time to infect other plants. foc spores can spread through water or soil, and by adhering to vehicles and footwear. in the soil, fusarium is difficult to control by general chemical measures, such as fungicides or soil fumigants. therefore, resistance breeding is the preferred method of overcoming the fusarium wilt of banana plants. however, because cavendish bananas have a triploid genome, they do not produce seeds, which hinders conventional breeding strategies. genetic engineering methods can improve the disease resistance of banana plants to fusarium wilt; however, little is known about the actual transcriptional changes and their regulation during the pathogen-plant interaction. understanding the underlying changes during this interaction would allow for the identification of signal transduction pathways affected by infection and the interaction mechanisms during infection, which can lead to improvement of disease resistance of the banana plants. traditional genome-wide analysis of gene expression of organisms under different conditions or, in the case of pathogens, at different life cycle stages, has mainly been carried out by microarrays, suppression subtractive hybridization, and cdna-aflp methods. van den berg et al. used ssh and microarrays to show that cell wall-strengthening genes may be important for banana resistance to fusarium wilt. however, the approach that was used suffers a number of drawbacks, including the fact that the genes are far from complete with only clones. recently, with the completion of banana genome sequence, a doubled haploid m. acuminata genotype has been shown to be highly resistant to foc tr by phenotyping assays, but further research on its mechanism has not been performed, especially with relation to transcription. a resistant variety of the cavendish banana was acquired by somaclonal variation, using the rna-seq and dge methods, and it was discovered that recognition of pamps and defense-related transcripts are involved in banana resistance to foc tr infection. therefore, elucidation of the mechanism by which cavendish bananas respond to foc tr infection is imperative. one such promising method developed in recent years is next-generation sequencing, by which an enormous amount of sequence data can be rapidly obtained within a short period of time due to its high-throughput and high-coverage nature. rna-seq technology, which is based on deep-sequencing, enables more precise quantification of genome-wide transcript levels than previous, microarray-based methods. in this technology, whole mrna or cdna is mechanically fragmented for deep-sequencing, the results of which can be then mapped on a reference genome or used in de novo assembly to obtain a genome-wide transcriptome. another method of great value for expression analysis is digital gene-expression. dge uses 17– base pair short fragments from the whole transcriptome as gene-specific tags and calculates the expression level of a gene from the frequency of its tag. we previously used a green fluorescent protein -tagged strain of foc tr and characterized early events in infection and disease development of cavendish plantlets. the combination of dge and rna-seq allows us to easily perform transcriptome analysis without the need for an already-assembled reference genome. despite the importance of the foc pathogen for global banana production, rna-seq and dge have not been used to investigate the main questions underlying the pathogen-banana interaction. therefore, we aimed to investigate the changes in gene expression during foc tr infection of banana roots using rna-seq and dge analysis. for this purpose, we generated over billion bases of high-quality dna sequence and demonstrated the suitability of short-read sequencing for assembly and annotation of genes expressed in a triploid-genome plant without previous whole-genome information. we then identified distinct sequences. furthermore, we compared the gene expression profiles during an infection time course using dge analysis. the assembled and annotated gene expression profiles provide an invaluable resource for the identification of differentially expressed genes during foc tr infection of banana, which will enable us to screen for host susceptibility factors and to monitor shifts in foc tr virulence.  assembly of a high-quality banana root transcriptome in the absence of a sequenced genome, de novo assembly of rna-seq data was the only viable option to study the banana transcriptome. to obtain an overview of the expression profile of banana roots under foc tr stress, a cdna sample was prepared from the total rna of an equal mixture of roots not infected and infected with foc tr for and days to acquire the genes whose expression is specifically altered when the plant is infected by foc tr deep-sequencing of this cdna sample produced, sequence reads with a length of bp each, which corresponded to approximately gigabase pairs of raw data. an overview of the sequencing and assembly is outlined in table the raw reads were first assembled into a draft using soap de novo-oases software, and further assembly was achieved using cap sequence assembly software. after assembling, reads were also mapped back to the assembled transcripts with a length ≥ bp. if the coverage of two assembled reads was more than 80%, then the shorter one was eliminated. the remaining sequences were then assembled into contigs. the mean contig size was bp with lengths ranging from to bp, including contigs larger than bp. a total of contigs were confirmed using the banana est library. the mean contig size in the final library was bp with lengths ranging from to bp, including contigs larger than bp. using paired-end joining and gap-filling, the contigs were further assembled into scaffolds with a mean size of bp, including scaffolds larger than bp. the longest scaffold was bp. to evaluate the quality of the dataset, we analyzed the gap-filling to assembled contigs length. the total size of all contigs was, bp with a total, bp gap size. the total size of the scaffolds was, bp. in order to evaluate our data, the assembled banana transcriptome was searched using blastn against plant cdnas using a cut-off e-value of 10- using this approach, approximately % contigs aligned successfully to plant cdna with a total gap size of, bp, which was bp shorter than the banana gap size. in addition, % of the reads and % of the paired-end joined sequences could be mapped onto the banana transcriptome. importantly, 90% of the distinct gene sequences were unique. our results indicated that the banana root transcriptome was of high quality. transcripts with lengths ≥ bp were subsequently used for analysis. functional annotation of the banana root transcriptome we acquired distinct gene sequences, of which were longer than bp. compared with the est and nucleotide sequences in ncbi database of banana, our data enriched the gene resources for banana. to annotate, classify, and functionally map the distinct gene sequences, we used blastx to match the distinct gene sequences using a cut-off e-value of 10- including the non-redundant protein database, gene ontology, and the kyoto encyclopedia of genes and genomes database with a cut-off e-value of 10- using this approach, distinct gene sequences returned a valid blast result, confirming the high quality of our transcriptome assembly. fourteen percent of the distinct gene sequences could not be matched to known genes. to annotate the distinct gene sequences that we identified in our transcriptome assembly, we initially searched against the plant proteins in the nr database. as a result, we obtained significant blast hits, which confirmed that most genes could be annotated after assembly. interestingly, % of the – bp query sequences, % of the – bp, 85% of the – bp, and % of the query sequences longer than bp were annotated successfully. this result indicated that the longer sequences provided more accurate matches with the nr database, while the shorter sequences lacked sufficient gene information to match, despite being easier to annotate. go classification out of the annotated distinct gene sequences, were assigned go terms using blast2go. forty-five go sub-categories were represented under three major categories. the majority of the go annotations were in the cellular component category, assigned to distinct gene sequences, followed by the biological process category for distinct gene sequences, and the molecular function category for distinct gene sequences. the major sub-categories are shown in figure 2: four major cellular component sub-categories were “cell”, “cell part”, “organelle”, and “organelle part”; two major molecular functions sub-categories were “binding” and “catalytic”; and four major biological process sub-categories were “metabolic process”, “cellular process”, “metabolic process”, and “response stimulus”. however, only % of banana root distinct gene sequences were assigned with these go terms, possibly because the large number of uninformative gene descriptions of these plant protein hits. kyoto encyclopedia of genes and genomes pathway mapping by mapping ec numbers to the reference canonical pathways, distinct gene sequences were assigned to kegg pathways. the pathways most represented by unique sequences were carbohydrate metabolism, amino acid metabolism, signal transduction, and cell growth and death. taken together, these annotations provide a valuable resource for investigating the specific processes, structures, functions, and pathways involved in the response to the infection of foc tr in banana roots. statistics of dge tags using the dge method, which generates absolute rather than relative gene expression measurements and avoids many of the inherent limitations of microarray them with analysis, we analyzed the gene expression profile of banana roots after inoculating foc tr total rna isolated from banana roots at and days post-inoculation were analyzed by illumina dge tag profiling to create transcriptome profiles of the four groups. dge tags were derived from the 3’utr of transcripts and were 21-nucleotides long. dge data provided a quantitative measure of transcript abundance in the rna population. dge analysis also allowed for the identification of previously unannotated genes. the majority of dge tags were expected to match only one location in the genome, with the remaining tags matching duplicate genes, alternate transcripts, antisense strands, or repeated sequences. we obtained a total of,,, and, total tags and,,, and distinct tags from the roots of the and dpi time points, respectively. heterogeneity and redundancy are two significant characteristics of mrna expression, and while the majority of mrnas are expressed at low levels, a small proportion is highly expressed. therefore, the distribution of tag expression was used to evaluate the normality of the dge data. as shown in figure the distribution of total tags and distinct tags over different tag abundance categories showed similar patterns for all four dge libraries. mapping sequences to the reference transcriptome database to identify the molecular events behind foc tr infection of banana roots, we mapped the tag sequences of the four dge libraries to our transcriptome reference database. among the – distinct tags generated from the illumina sequencing of the four libraries, – distinct tags were mapped to a gene in the reference database. tags mapped to a unique sequence are the most critical subset in dge libraries, as they can explicitly identify a transcript. up to % of the sequences in our transcriptome reference tag database could be unequivocally identified by a unique tag. to determine whether our dge tags reached saturation, we compared the increase in the distinct tag number to the increase in total tag number. when sequencing depths reached million or more base pairs, the number of distinct tags discovered almost ceased to increase in all four libraries, which indicated that the sequencing was saturated. the level of gene expression was then determined by calculating the number of unambiguous tags for each distinct gene sequence and then normalizing this to the number of transcripts per million tags. additional file provides a list of the top most abundantly expressed genes in the dpi library as an example. comparing our results with those of van den berg, the expressions of two catalases, two pectin acetyl esterases and three pathogenesis-related proteins in our result in the dpi library were consistent. the result indicated that those genes responded to foc tr infection. gene expression profile changes in banana roots infected with foc tr4 to identify the signaling pathways involved in the banana response to foc tr infection, we identified tags that were differentially expressed between the dpi and the later infection time points using an algorithm developed by audic et al.. a total of distinct gene sequences significantly changed between the and dpi libraries, where distinct gene sequences were upregulated and distinct gene sequences were downregulated after days of foc tr infection. between the and dpi libraries, a total of distinct gene sequences were detected with upregulated distinct gene sequences and downregulated gene sequences. there were distinct gene sequences that were expressed at a different level in the and dpi libraries, with upregulated distinct gene sequences and downregulated distinct gene sequences after days of infection. gene ontology analysis was used for the above differential expression distinct gene sequences, and enrichment analysis was performed using a false discovery rate adjusted p-value of ≤ as the cutoff. the downregulated distinct gene sequences did not enrich any go term, while the upregulated distinct gene sequences enriched and featured go terms at dpi, dpi and dpi respectively. in particular, response to stress was enriched at dpi and response to chemical stimulus was enriched at dpi, which suggested that banana root was subjected to stress and chemical stimulation because of foc tr infection at these two time points. although down-regulated expression of distinct gene sequences did not enrich the go term, we did find that some distinct gene sequences had down-regulated expression, such as nsp-interacting kinase and sumo e protein ligase. these genes were included in the immune-related go term, which indicated that those distinct gene sequences did not respond to foc tr infection. kegg pathway analysis of differentially expressed banana roots genes in response to infection by foc tr4 to understand the functions of differentially expressed distinct gene sequences, we mapped them to kegg terms to discover those genes involved in metabolic or signal transduction pathways that were significantly enriched. additional file shows enriched pathways at dpi, dpi and dpi. phenylalanine metabolism was enriched at dpi, dpi, and dpi. there are twenty-three peroxidases, twelve bacterial-induced peroxidase precursors, five 4-coumarate: coenzyme a ligases, three cinnamate 4-hydroxylases and three phenylalanine ammonia lyases enriched in this pathway. meanwhile, peroxidases, four 4-coumarate:coenzyme a ligases, two cinnamate 4-hydroxylases and one phenylalanine ammonia lyase were enriched in phenylpropanoid biosynthesis at dpi. it should be emphasized that peroxidases were enriched in both pathways. the peroxidases enriched in those pathways may be involved in increased lignin biosynthesis, and may acting as basal defense components: peroxidase is one source for the production of ros. that the peroxidases were upregulated suggests that banana roots responded to infection by foc tr by ros production. similarly, enrichment of drug metabolism-cytochrome p was found at 2dpi. there are distinct gene sequences of glutathione s-transferases or glutathione transferases in this pathway. gsts, as a heterogeneous group of cell detoxifying enzymes, catalyse the conjugation of tripeptide glutathione to electrophilic sites on a wide range of phytotoxic substrates. it is likely that even the susceptible cultivar activates some early mechanisms of defense against foc tr4; however, these are not sufficient to provide resistance against the pathogen. at dpi, alpha-linolenic acid metabolism was enriched, leading to jasmonic acid biosynthesis, which is one of the pathways associated with pathogen resistance and the genes in this pathway were significantly affected by foc tr infestation at all time points. this is consistent with previous reports that biotic and abiotic stresses, such as pathogen infection, wounding and insect feeding, can trigger ja biosynthesis through direct activation of genes encoding the relevant biosynthetic enzymes. ethylene and sa biosynthetic and signaling related genes showed no significant differences between non-inoculation and inoculation in our results. these results indicate that ja biological synthesis may be regulated by foc tr infection. similarly, the expressions of ja biosynthetic and signaling related genes in a resistant variety were higher than in a susceptible variety. further study of these genes in this pathway could identify them as targets for testing whether a variety is resistant to foc tr infestation.  here, we present a rapid and low-cost method for triploid plant transcriptome assembly and dge analysis using illumina sequencing technology. our findings provide a substantial contribution to the existing sequence resources for the banana and will certainly accelerate research regarding the devastating foc tr pathogen of this valuable staple food. our expression analysis results provide promising leads for future functional studies for understanding how the foc tr pathogen infects and kills banana plants. 
since there is very little known about the molecular mechanism of foc infections; therefore, we aimed to investigate the transcriptional changes induced by foc in banana roots. we generated a cdna library from total rna isolated from banana roots infected with foc tropical race at days and we generated over million high-quality reads from the cdna library using deep sequencing and assembled distinct gene sequences by de novo assembly and gap-filling. the expression of genes in the phenylalanine metabolism, phenylpropanoid biosynthesis and alpha-linolenic acid metabolism pathways was affected by foc tr infection. the assembled banana transcriptome provides an important resource for future investigations about the banana crop as well as the diseases that plague this valuable staple food. the combination of rna-seq and dge analysis provides a powerful method for analyzing the banana root transcriptome and investigating the transcriptional changes during the response of banana genes to foc tr infection. a total of unique sequences were annotated and were assigned to specific metabolic pathways using the kyoto encyclopedia of genes and genomes database. bananas and plantains are among the most important crops in the world due to their nutritional and export value. however, banana production has been devastated by fungal infestations caused by fusarium oxysporum f. sp. we used digital gene expression profiling to investigate the transcriptional changes in the banana root upon foc tr infection.
 bananas and plantains are among the most important crops in the world due to their nutritional and export value. however, banana production has been devastated by fungal infestations caused by fusarium oxysporum f. sp. cubense, which cannot be effectively prevented or controlled. since there is very little known about the molecular mechanism of foc infections; therefore, we aimed to investigate the transcriptional changes induced by foc in banana roots.  we generated a cdna library from total rna isolated from banana roots infected with foc tropical race at days and we generated over million high-quality reads from the cdna library using deep sequencing and assembled distinct gene sequences by de novo assembly and gap-filling. the average distinct gene sequence length was base pairs. a total of unique sequences were annotated and were assigned to specific metabolic pathways using the kyoto encyclopedia of genes and genomes database. we used digital gene expression profiling to investigate the transcriptional changes in the banana root upon foc tr infection. the expression of genes in the phenylalanine metabolism, phenylpropanoid biosynthesis and alpha-linolenic acid metabolism pathways was affected by foc tr infection.  the combination of rna-seq and dge analysis provides a powerful method for analyzing the banana root transcriptome and investigating the transcriptional changes during the response of banana genes to foc tr infection. the assembled banana transcriptome provides an important resource for future investigations about the banana crop as well as the diseases that plague this valuable staple food. 