BACKGROUND
longan , a tropical/subtropical fruit tree in the family sapindaceae, is of great economic importance in southeast asia.
because the status of embryo development determines seed size, fruit quality, percentage of fruit set, and yield in longan, efforts to improve fruit quality and yield have included studies on regulation of longan embryo development using cytological, molecular, and proteomics approaches  <cit> .
such research has been hampered, however, by the extremely high genetic heterozygosity of longan and early embryo sampling difficulties  <cit> .
because plant somatic embryogenesis  shows close similarities on morphological and molecular levels to normal zygotic embryogeny  <cit> , the longan se system has been used as a system for investigating regulation of in vitro and in vivo embryogenesis in longan  <cit> .
studies focusing on molecular biology and proteomics of the longan se system have been conducted using differential display reverse transcription pcr , homology cloning, quantitative real-time pcr , two-dimensional electrophoresis, and protein bio-mass spectrometry , resulting in the isolation and identification of hundreds of related genes and proteins  <cit> .
but little genomic or proteomic information of the above-mentioned studies is available for the longan embryo.
as of july  <dig>  only  <dig> nucleotide sequences and  <dig> expressed sequence tags  had been deposited in the ncbi genbank database.
although many key longan genes and proteins have been cloned and identified, molecular resouces of longan are still limited because genomic and transcriptomic information is lacking.
consequently, an accelerated effort to acquire transcriptomes of longan embryogenesis is needed.
a few transcriptomic studies of embryogenesis have been conducted in rice  <cit> , poplar  <cit> , arabidopsis <cit> , gossypium hirsutum <cit> , solanum tuberosum <cit> , elaeis guineensis <cit> , brassica napus <cit> , soybean  <cit> , and maize  <cit> ; these studies were mainly focused on calli or embryogenic calli, and involved techniques such as illumina sequencing, massively parallel signature sequencing , est analysis, microarray analysis, and suppression subtraction hybridization .
no research has been performed on the longan transcriptome.
to assist in the identification, quantification, and classification of genes expressed in longan embryogenic callus , we generated a global transcriptome from longan ec using high-throughput illumina rna sequencing, and analyzed functions, classification, and metabolic pathways of the resulting unigenes using bioinformatics.
we then comparatively analyzed expression patterns to reveal  <dig> selected unigenes participating in longan se.
the resulting assembled and annotated transcriptome should serve as a highly useful resource for the identification of genes involved in longan se.
RESULTS
illumina sequencing, de novo assembly, and sequence analysis of the d. longan transcriptome
to obtain a global overview of the longan ec transcriptome, we constructed a cdna library from a longan ec rna sample.
using an illumina hiseq  <dig> sequencing system,  <dig> , <dig> clean reads  were obtained after removing low-quality reads and adaptor sequences.
q <dig>  n, and gc percentages were  <dig> %,  <dig> %, and  <dig> %, respectively .
using the soapdenovo assembly program, all high-quality reads were assembled into  <dig>  contigs longer than  <dig> bp, with a median length of  <dig> bp and an n <dig> of  <dig> bp.
the size distribution of these contigs is shown in additional file  <dig>  the length of  <dig>  contigs  ranged from  <dig> to  <dig> bp;  <dig>  contigs  were longer than  <dig> bp, and the remaining were mainly between 200– <dig> bp in length.
using a paired-end sequencing strategy, contigs from the same transcript can be identified and the distances between these contigs evaluated.
<dig>  scaffolds, with a median length of  <dig> bp and an n <dig> of  <dig> bp, were generated .
length distributions of the resulting scaffolds were as follows: 100– <dig> bp , 500– <dig> bp , 1000– <dig> bp , and > <dig> bp  .
then, the ratio of gap length to length of scaffold was analyzed;  <dig>   scaffolds had no gap at all,  <dig>   had gap lengths less than 10% and only  <dig>   exhibited gap lengths ranging from 20–40% of the total length.
finally, paired-end reads were used again for gap filling of scaffolds to generate unigenes with the smallest number of ns.
<dig>  unigenes, with an average length of  <dig> bp and an n <dig> of  <dig> bp, were constructed from the scaffolds .
unigenes with lengths ranging from 100– <dig> bp, 500– <dig> bp, and 1000– <dig> bp accounted for  <dig> % ,  <dig> % , and  <dig> %  of the total, respectively; in addition,  <dig>   unigenes were ≥  <dig> bp long .
of the  <dig>  unigenes,  <dig> %  had no gap and  <dig> %  had gap lengths less than 10% of the total length.
protein coding region  prediction of the d. longan transcriptome
to determine the function of longan embryogenic unigenes, blastx alignment  between unigenes and nr, swiss-prot, kegg and cog protein databases was carried out, and the results were used to predict unigene transcriptional orientations and coding regions.
a total of  <dig>  unigenes  were identified in the longan ec library, with  <dig>  unigenes remaining unidentified.
for validation and annotation of gene names, cds, and predicted proteins, all assembled unigenes were first searched against nr and swiss-prot databases using blastx.
in total,  <dig> %  of the putative protein unigenes showed significant similarity to known plant proteins in the databases.
the distribution of unigenes with homologous matches was 200– <dig> bp , 600– <dig> bp , 1100– <dig> bp , and > <dig> bp .
furthermore,  <dig>   unigene cdss had no gaps at all and  <dig>  exhibited gap lengths less than 10% of the total length.
the coding region unigenes were translated into amino sequences using a standard codon table.
there were  <dig>   unigenes coding for polypeptides approximately  <dig> aa long and  <dig>   with polypeptide lengths ranging from  <dig> to  <dig> aa.
in addition, there were a few unigenes with polypeptide lengths greater than  <dig> aa; these included unigenes coding for zinc finger family protein , vacuolar protein sorting-associated protein 13c , auxin transport protein , wd <dig> g-beta repeats , phosphatidylinositol-4-phosphate 5-kinase family protein , and caax amino terminal protease family protein .
the remaining  <dig>  unigenes with no homologs in the above databases were scanned again using estscan.
<dig>  putative protein unigenes were identified,  <dig>   with no gaps.
putative protein unigenes with lengths ranging from 200– <dig> bp accounted for  <dig> %  of these; other approximate lengths represented were  <dig> bp ,  <dig> bp , and  <dig> bp.
of the putative protein unigenes identified using estscan,  <dig> %  translated to polypeptide sequences about  <dig> aa long.
in total,  <dig> %  of putative protein coding unigenes were annotated by homology analysis using nr and swiss- prot databases or estscan predictions.
with respect to plant growth and developmental functions, analysis of unigenes longer than  <dig> bp  showed that at least  <dig> unigenes of embryogenesis-related genes were expressed in longan ec.
among them, pentatricopeptide repeat-containing protein genes  were the most dominant group, followed by emb  family genes , and then mee  family genes .
surprisingly, in addition to the embryogenesis-related genes mentioned above, many reproductive growth-related genes were also expressed in ec, including genes related to flowering, meiosis, floral organ development, female and male gametophyte development, embryo sac development, ovule development, endosperm development, pollen tube growth, inflorescence meristem growth, floral organ number control, petal loss, and tapetum formation.
furthermore, some vegetative growth-related genes, such as those related to apical meristem growth, root growth, and mycorrhizal formation, were also expressed in ec .
go functional annotation and classification of the d. longan transcriptome
to functionally categorize d.longan expressed genes, gene ontology  terms were assigned to assembled unigenes.
based on blastx hits against the nr database, blast2go  <cit>  and wego  <cit>  were used to obtain go annotations and classifications according to molecular function, biological process, and cellular component ontologies.
based on nr annotations,  <dig>  unigenes were assigned to the three main go categories and  <dig> sub-categories, which included cellular process, metabolic process, death, development process, cell, organelle, antioxidant activity, catalytic activity, binding, enzyme regulator activity, transcription regulator activity, and translation regulator activity .
of the three main go categories, cellular component was the most dominant category , followed by biological process  and molecular function  .
the biological process category was divided into  <dig> sub-categories.
among them, metabolic processes  were the most highly represented, followed by cellular processes  and biological regulation .
only a few unigenes were assigned into sub-categories such as development process , death , growth , and immune system process  .
the cellular component category included  <dig>  unigenes in  <dig> sub-categories, including cell , organelle , macromolecular complex , envelope , extracellular region , and membrane-enclosed lumen  .
with respect to molecular function,  <dig>  unigenes could be sub-categorized into  <dig> functional groups.
these included  <dig>   unigenes assigned to binding, followed by catalytic activity  and transporter activity .
in addition, a few unigenes were associated with transcription regulator activity , structural molecule activity , molecular transducer activity , translation regulator activity , antioxidant activity , enzyme regulator activity , nutrient reservoir activity , and metallochaperone activity  .
cog functional annotation and classification of the d. longan transcriptome
the clusters of orthologous groups  database is based on a set of coding proteins with complete genomes and information about systematic evolutionary relationships of bacteria, algae, and eukaryotes.
all longan unigenes were searched against the cog database to predict and classify by possible function.
overall,  <dig>   unigenes were assigned to  <dig> cog categories .
of the  <dig> cog categories, the cluster for general function prediction associated with basic physiological and metabolic functions represented the largest group , followed by replication, recombination and repair , transcription , post-translational modification, protein turnover, chaperones , signal transduction mechanisms , carbohydrate transport and metabolism , translation, ribosomal structure and biogenesis , and amino acid transport and metabolism .
a few unigenes were assigned to cell motility , nuclear structure , and extracellular structures .
in addition,  <dig>  of longan unigenes were assigned into the function unknown cluster .
kegg functional classification of the d. longan transcriptome
the kyoto encyclopedia of genes and genomes  database can be used to analyze gene products of metabolic processes and related cellular processes and to further research the genetics of biologically complex behaviors.
to identify biological pathways in d.longan, unigenes were compared against the kegg database using blastx; as a result,  <dig>  unigenes were assigned to  <dig> kegg pathways .
among the  <dig> kegg pathways, the pathways most represented by unigenes were metabolic pathways , primarily starch and sucrose metabolism , purine metabolism , pyrimidine metabolism , ubiquitin mediated proteolysis , glycolysis/gluconeogenesis , cysteine and methionine metabolism , and pyruvate metabolism .
in contrast, only a few unigenes were assigned to thiamine metabolism , riboflavin metabolism , biotin metabolism , vitamin b <dig> metabolism , c5-branched dibasic acid metabolism , and caffeine metabolism .
in addition,  <dig>  of longan unigenes mapped to the plant-pathogen interaction pathway , illustrating that many disease-resistance genes are expressed in longan ec.
furthermore,  <dig>   unigenes were classified into biosynthesis of secondary metabolites pathways, including stilbenoid, diarylheptanoid and gingerol biosynthesis , flavonoid biosynthesis , zeatin biosynthesis , carotenoid biosynthesis , biosynthesis of unsaturated fatty acids , ubiquinone and other terpenoid-quinone biosynthesis , terpenoid backbone biosynthesis , steroid biosynthesis , diterpenoid biosynthesis , glucosinolate biosynthesis , flavone and flavonol biosynthesis , tropane, piperidine and pyridine alkaloid biosynthesis , brassinosteroid biosynthesis , folate biosynthesis  , and anthocyanin biosynthesis .
in addition to the pathways mentioned above, many longan unigenes were associated with genetic information processing involving spliceosome  , ribosome , rna degradation , nucleotide excision repair , rna polymerase , dna replication , base excision repair , homologous recombination , mismatch repair , protein processing in endoplasmic reticulum , and protein export .
finally, longan unigenes were also involved in carbon fixation in photosynthetic organisms , photosynthesis  , and photosynthesis- antenna proteins .
taken together, the annotated longan unigenes provided valuable information for investigating specific processes, functions, and pathways involved in longan ec development, and allowed identification of novel genes in non-model organisms.
gene validation and expression analysis during longan se using quantitative real-time pcr
to experimentally confirm that unigenes obtained from sequencing and computational analysis were indeed expressed,  <dig> unigenes longer than  <dig> bp, including  <dig> embryogenesis-related genes ,  <dig> reproductive growth-related genes , and  <dig> vegetative growth-related genes , were selected for qpcr analysis across the six sequential developmental stages of longan se: friable-embryogenic callus , incomplete compact pro-embryogenic cultures , globular embryos , heart-shaped embryos , torpedo-shaped embryos , and cotyledonary embryos  .
based on the analyzed qpcr data, all selected unigenes were expressed at varying levels in different embryogenic tissues .
mrh <dig>  npgr <dig>  ppr <dig>  ref <dig>  npg <dig>  swp <dig>  and vln <dig> were expressed throughout the different tissue culture developmental stages, although no significantly expression profiles were observed.
expression levels of emb <dig>  emb <dig>  frp, spk <dig>  vln <dig>  aah, and swp <dig> were low in te, and high in he and ce, while bel1-like exhibited the highest expression in te.
ghmp <dig>  ppr <dig>  and npgr <dig> were highly expressed in icpec, ghmp <dig>  emb <dig>  and swa <dig> showed strong expression in he, moderate expression in ec, and weak expression in icpec.
eda <dig> and swa <dig> were highly expressed in ec.
these results confirm that differential expressions of these unigenes have potential roles during longan se.
discussion
feasibility of illumina paired-end sequencing and assembly for non-model species with unsequenced genomes such as longan
understanding the dynamics of plant transcriptomes is helpful for studying the complexity of transcriptional regulation and its impact on phenotype  <cit> .
transcriptome sequencing is one of the most important tools for gene discovery and expression pattern identification, but traditional est sequencing based on the sanger method is time-consuming and costly.
because of their high throughput, accuracy, and low cost, next-generation sequencing technologies, such as illumina/solexa,  <dig>  and mpss, have been used successfully for plant genomic and transcriptomic analyses in many organisms  <cit> .
in this study, approximately  <dig> million clean reads  were obtained from longan ec using illumina hiseq  <dig> sequencing and assembled into  <dig>  unigenes, more than that reported for plants such as s.indicum <cit> , t.chinensis <cit> , c.sinensis <cit> , g. hirsutum <cit>  and i.batatas <cit>  using the same technology.
compared with previous studies, these sequences produced shorter unigenes  than those assembled from sesamum , taxus , i.batatas , poncirus trifoliata  and jatropha curcas   <cit> , but longer than those generated from c.sinensis <cit> , fagopyrum   <cit> , and maize   <cit> .
more importantly,  <dig>   of the assembled unigenes were longer than  <dig> bp.
these results demonstrate that illumina sequencing technology can be an effective tool for gene discovery in non- model organisms.
moreover,  <dig> %  of the putative protein unigenes showed significant similarity to known plant proteins in databases, a higher percentage than that reported for s.indicum   <cit> , i.batatas   <cit> , and epimedium sagittatum   <cit> .
on the other hand, average unigene length in our study was shorter than that obtained for most plant species, and there were many unassembled reads; difficulties with the de novo transcriptome assembly may be attributed to various factors, such as short sequence fragments, assembly options, genes expressed at low levels, repetitive sequences, alternative splicing, and lack of a reference genome  <cit> .
quantity and variety of genes expressed in longan ec
plant callus transcriptomes have been obtained for a number of species, including rice  <cit> , populus <cit> , a.thaliana <cit> , g.hirsutum <cit> , s.tuberosum <cit>  and e.guineensis <cit> .
in this study,  <dig>  unigenes from longan ec were assembled, more than the number reported from calli of rice in one study   <cit> , g. hirsutum   <cit>  and s.tuberosum   <cit> , but less than from rice in another study   <cit> , populus   <cit> , and a.thaliana   <cit> .
the above studies failed to obtain global expression profiles of callus genes, either because there was insufficient transcriptome information or because no further analysis was conducted.
studies have shown that plant se is morphologically and molecularly similar to zygotic embryogenesis  <cit> .
just as a plant zygotic embryo, the most complex plant organ, epitomizes the entire plant, so too a plant somatic embryo can be considered to represent an entire plant.
in our study, we uncovered  <dig>  unigenes, the majority of which reflected expression of genes required for plant in vitro embryogenesis, such as pentatricopeptide repeat proteins  and embryo defective  family genes.
a previous study has shown that mutations of different pprs have distinct impacts on embryo morphogenesis  <cit> .
in our study,  <dig> ppr unigenes longer than  <dig> bp were identified, and two highly abundent ppr unigenes were further confirmed and found to be expressed throughout longan se.
ppr1_unigene  <dig> was highly expressed in icpec and he, while ppr2_unigene  <dig> mrna was abundant in ec and icpec.
these results suggest the involvement of these genes during early developmental stages of longan se.
in arabidopsis,  <dig> emb genes have been confirmed to be required for normal embryo development  <cit> , with emb <dig> displaying aberrant cell organization and undergoing morphological arrest before the globular-heart transition  <cit> .
in our study, three emb unigenes longer than  <dig> bp—emb1_unigene <dig>  emb2_unigene <dig>  and emb3_unigene1123—were verified and strongly expressed in he and ce.
in addition, emb1_unigene <dig> was also highly expressed in ge and moderately detectable in ec and te, while emb2_unigene <dig> and emb3_unigene <dig> exhibited moderate expression in ge and te.
high expression levels of these selected embs in longan he and ce indicate their possible roles in the development of longan se.
surprisingly, however, there were also many unigenes expressed in ec associated with reproductive growth characteristics , and vegetative growth .
in our study,  <dig> reproductive growth-related genes longer than  <dig> bp were confirmed by qpcr during the developmental stages of longan se.
for example, the fertility-related frp , was strongly expressed in he and ce, and also weakly in te.
genes related to pollen growth—npgr , npgr , and vln —displayed ubiquitous but weak expression during longan se.
npgr2_unigene <dig> was highly expressed in icpec, and vln2_unigene <dig> was accumulated in he and ce, but barely detectable in te.
swa1/ <dig>  essential for gametogenesis in arabidopsis <cit> , were also differentially expressed during longan se; while they were both highly expressed in ec, while swa1_unigene <dig> was also expressed in he.
the ghmp kinase enzyme family, a primary determinant of sexual fate in caenorhabditis elegans <cit> , was also detected in our study.
ghmp1_unigene <dig> expression was high in he, and ghmp2_unigene <dig> transcripts accumulated in icpec.
bel1-like, is required for cytokinin and auxin signaling during ovule development in arabidopsis <cit> , was expressed highly in te and moderately in he and ce, but was barely detectable in icpec; this suggests it may play a major role in longan se during late embryonic stages.
eda <dig>  related to embryo sac development, was strongly expressed in ec, te, and ce.
these results all demonstrate that these  <dig> reproductive growth-related genes also play roles during longan se development.
five vegetative growth-related genes were also chosen for qpcr analysis across the six sequential developmental stages of longan se.
these genes included mrh <dig> , which is likely involved in polarized growth of root hairs in arabidopsis <cit> , ref <dig> , which plays divergent roles in the regulation of arabidopsis flowering  <cit> , and swp , which plays an important role in defining the duration of cell proliferation  <cit> .
all of these genes were expressed at varied levels in different embryogenic tissues, suggesting their wide involvement in various developmental stages during longan se.
in particular, swp1_unigene <dig> was highly expressed during late stages of longan se, but was barely detectable in ge.
in addition, spk <dig> , required for normal cell shape control and tissue development  <cit> , and aah , related to nitrogen fixation, were both strongly expressed in he and ce.
the number and variety of expressed genes in ec suggests that ec practically reflects the entire se profile, and even that of the entire plant.
in addition, ec might be considered as a “gene pool” for isolating various plant target genes, such as genes related to flowering, pollen development, root and shoot growth, and plant-pathogen interactions.
this ec dataset provides new candidates with possible roles in longan somatic embryogenesis.
in arabidopsis, the largest number of unannotated signatures was found in callus:  <dig>  , compared with  <dig> in inflorescences,  <dig> in leaves,  <dig>  in roots, and  <dig> in siliques  <cit> .
in our study, we also found many unigenes  with unknown function, demonstrating how little is known about the biology of undifferentiated plant cells.
thus, the ec transcriptome can be used to more effectively discover new genes in longan.
using solexa sequencing, 27% of identified unigenes in populus euphratica callus were found to be differentially expressed in response to salt stress; these genes were mainly involved in transport, transcription, cellular communication, and metabolism  <cit> .
during arabidopsis callus development,  <dig> genes were found to be up-regulated and  <dig> to be down-regulated.
the most highly up-regulated genes encoded an unknown protein  and acireductone dioxygenase , and the most highly down-regulated genes included a dr <dig> protease inhibitor , two peroxidase genes , two pearli  <dig> genes , and two that encoded subtilases   <cit> .
in rice callus cells,  <dig>  expressed genes were identified using the microarray suite   <dig>  detection algorithm  <cit> .
these studies demonstrate that a large number of genes involved in various biological and metabolic pathways are expressed in plant ec.
in our study,  <dig>   unigenes were assigned to  <dig> kegg pathways, including metabolic pathways, plant-pathogen interactions, biosynthesis of secondary metabolites, and photosynthesis and genetic information processing-related pathways.
these results lay a foundation for further identification of longan ec-related genes.
CONCLUSIONS
in summary, our study generated the first large-scale transcriptome dataset of longan ec.
in addition, the types and quantities of genes expressed in longan, as well as their functions, classification, and metabolic pathways, were revealed for the first time.
twenty-three unigenes related to embryogenesis and reproductive and vegetative growth were differentially expressed in various embryogenic cultures, indicating their possible roles in longan se.
this transcriptome dataset provides new insights into molecular processes in d. longan.

 longan, a tropical/subtropical fruit tree in the family sapindaceae, is of great economic importance in southeast asia. because the status of embryo development determines seed size, fruit quality, percentage of fruit set, and yield in longan, efforts to improve fruit quality and yield have included studies on regulation of longan embryo development using cytological, molecular, and proteomics approaches. such research has been hampered, however, by the extremely high genetic heterozygosity of longan and early embryo sampling difficulties. because plant somatic embryogenesis shows close similarities on morphological and molecular levels to normal zygotic embryogeny, the longan se system has been used as a system for investigating regulation of in vitro and in vivo embryogenesis in longan. studies focusing on molecular biology and proteomics of the longan se system have been conducted using differential display reverse transcription pcr, homology cloning, quantitative real-time pcr, two-dimensional electrophoresis, and protein bio-mass spectrometry, resulting in the isolation and identification of hundreds of related genes and proteins. but little genomic or proteomic information of the above-mentioned studies is available for the longan embryo. as of july only nucleotide sequences and expressed sequence tags had been deposited in the ncbi genbank database. although many key longan genes and proteins have been cloned and identified, molecular resouces of longan are still limited because genomic and transcriptomic information is lacking. consequently, an accelerated effort to acquire transcriptomes of longan embryogenesis is needed. a few transcriptomic studies of embryogenesis have been conducted in rice, poplar, arabidopsis, gossypium hirsutum, solanum tuberosum, elaeis guineensis, brassica napus, soybean, and maize; these studies were mainly focused on calli or embryogenic calli, and involved techniques such as illumina sequencing, massively parallel signature sequencing, est analysis, microarray analysis, and suppression subtraction hybridization. no research has been performed on the longan transcriptome. to assist in the identification, quantification, and classification of genes expressed in longan embryogenic callus, we generated a global transcriptome from longan ec using high-throughput illumina rna sequencing, and analyzed functions, classification, and metabolic pathways of the resulting unigenes using bioinformatics. we then comparatively analyzed expression patterns to reveal selected unigenes participating in longan se. the resulting assembled and annotated transcriptome should serve as a highly useful resource for the identification of genes involved in longan se.  illumina sequencing, de novo assembly, and sequence analysis of the d. longan transcriptome to obtain a global overview of the longan ec transcriptome, we constructed a cdna library from a longan ec rna sample. using an illumina hiseq sequencing system,, clean reads were obtained after removing low-quality reads and adaptor sequences. q n, and gc percentages were %, %, and %, respectively. using the soapdenovo assembly program, all high-quality reads were assembled into contigs longer than bp, with a median length of bp and an n of bp. the size distribution of these contigs is shown in additional file the length of contigs ranged from to bp; contigs were longer than bp, and the remaining were mainly between 200– bp in length. using a paired-end sequencing strategy, contigs from the same transcript can be identified and the distances between these contigs evaluated. scaffolds, with a median length of bp and an n of bp, were generated. length distributions of the resulting scaffolds were as follows: 100– bp, 500– bp, 1000– bp, and > bp. then, the ratio of gap length to length of scaffold was analyzed; scaffolds had no gap at all, had gap lengths less than 10% and only exhibited gap lengths ranging from 20–40% of the total length. finally, paired-end reads were used again for gap filling of scaffolds to generate unigenes with the smallest number of ns. unigenes, with an average length of bp and an n of bp, were constructed from the scaffolds. unigenes with lengths ranging from 100– bp, 500– bp, and 1000– bp accounted for %, %, and % of the total, respectively; in addition, unigenes were ≥ bp long. of the unigenes, % had no gap and % had gap lengths less than 10% of the total length. protein coding region prediction of the d. longan transcriptome to determine the function of longan embryogenic unigenes, blastx alignment between unigenes and nr, swiss-prot, kegg and cog protein databases was carried out, and the results were used to predict unigene transcriptional orientations and coding regions. a total of unigenes were identified in the longan ec library, with unigenes remaining unidentified. for validation and annotation of gene names, cds, and predicted proteins, all assembled unigenes were first searched against nr and swiss-prot databases using blastx. in total, % of the putative protein unigenes showed significant similarity to known plant proteins in the databases. the distribution of unigenes with homologous matches was 200– bp, 600– bp, 1100– bp, and > bp. furthermore, unigene cdss had no gaps at all and exhibited gap lengths less than 10% of the total length. the coding region unigenes were translated into amino sequences using a standard codon table. there were unigenes coding for polypeptides approximately aa long and with polypeptide lengths ranging from to aa. in addition, there were a few unigenes with polypeptide lengths greater than aa; these included unigenes coding for zinc finger family protein, vacuolar protein sorting-associated protein 13c, auxin transport protein, wd g-beta repeats, phosphatidylinositol-4-phosphate 5-kinase family protein, and caax amino terminal protease family protein. the remaining unigenes with no homologs in the above databases were scanned again using estscan. putative protein unigenes were identified, with no gaps. putative protein unigenes with lengths ranging from 200– bp accounted for % of these; other approximate lengths represented were bp, bp, and bp. of the putative protein unigenes identified using estscan, % translated to polypeptide sequences about aa long. in total, % of putative protein coding unigenes were annotated by homology analysis using nr and swiss- prot databases or estscan predictions. with respect to plant growth and developmental functions, analysis of unigenes longer than bp showed that at least unigenes of embryogenesis-related genes were expressed in longan ec. among them, pentatricopeptide repeat-containing protein genes were the most dominant group, followed by emb family genes, and then mee family genes. surprisingly, in addition to the embryogenesis-related genes mentioned above, many reproductive growth-related genes were also expressed in ec, including genes related to flowering, meiosis, floral organ development, female and male gametophyte development, embryo sac development, ovule development, endosperm development, pollen tube growth, inflorescence meristem growth, floral organ number control, petal loss, and tapetum formation. furthermore, some vegetative growth-related genes, such as those related to apical meristem growth, root growth, and mycorrhizal formation, were also expressed in ec. go functional annotation and classification of the d. longan transcriptome to functionally categorize d.longan expressed genes, gene ontology terms were assigned to assembled unigenes. based on blastx hits against the nr database, blast2go and wego were used to obtain go annotations and classifications according to molecular function, biological process, and cellular component ontologies. based on nr annotations, unigenes were assigned to the three main go categories and sub-categories, which included cellular process, metabolic process, death, development process, cell, organelle, antioxidant activity, catalytic activity, binding, enzyme regulator activity, transcription regulator activity, and translation regulator activity. of the three main go categories, cellular component was the most dominant category, followed by biological process and molecular function. the biological process category was divided into sub-categories. among them, metabolic processes were the most highly represented, followed by cellular processes and biological regulation. only a few unigenes were assigned into sub-categories such as development process, death, growth, and immune system process. the cellular component category included unigenes in sub-categories, including cell, organelle, macromolecular complex, envelope, extracellular region, and membrane-enclosed lumen. with respect to molecular function, unigenes could be sub-categorized into functional groups. these included unigenes assigned to binding, followed by catalytic activity and transporter activity. in addition, a few unigenes were associated with transcription regulator activity, structural molecule activity, molecular transducer activity, translation regulator activity, antioxidant activity, enzyme regulator activity, nutrient reservoir activity, and metallochaperone activity. cog functional annotation and classification of the d. longan transcriptome the clusters of orthologous groups database is based on a set of coding proteins with complete genomes and information about systematic evolutionary relationships of bacteria, algae, and eukaryotes. all longan unigenes were searched against the cog database to predict and classify by possible function. overall, unigenes were assigned to cog categories. of the cog categories, the cluster for general function prediction associated with basic physiological and metabolic functions represented the largest group, followed by replication, recombination and repair, transcription, post-translational modification, protein turnover, chaperones, signal transduction mechanisms, carbohydrate transport and metabolism, translation, ribosomal structure and biogenesis, and amino acid transport and metabolism. a few unigenes were assigned to cell motility, nuclear structure, and extracellular structures. in addition, of longan unigenes were assigned into the function unknown cluster. kegg functional classification of the d. longan transcriptome the kyoto encyclopedia of genes and genomes database can be used to analyze gene products of metabolic processes and related cellular processes and to further research the genetics of biologically complex behaviors. to identify biological pathways in d.longan, unigenes were compared against the kegg database using blastx; as a result, unigenes were assigned to kegg pathways. among the kegg pathways, the pathways most represented by unigenes were metabolic pathways, primarily starch and sucrose metabolism, purine metabolism, pyrimidine metabolism, ubiquitin mediated proteolysis, glycolysis/gluconeogenesis, cysteine and methionine metabolism, and pyruvate metabolism. in contrast, only a few unigenes were assigned to thiamine metabolism, riboflavin metabolism, biotin metabolism, vitamin b metabolism, c5-branched dibasic acid metabolism, and caffeine metabolism. in addition, of longan unigenes mapped to the plant-pathogen interaction pathway, illustrating that many disease-resistance genes are expressed in longan ec. furthermore, unigenes were classified into biosynthesis of secondary metabolites pathways, including stilbenoid, diarylheptanoid and gingerol biosynthesis, flavonoid biosynthesis, zeatin biosynthesis, carotenoid biosynthesis, biosynthesis of unsaturated fatty acids, ubiquinone and other terpenoid-quinone biosynthesis, terpenoid backbone biosynthesis, steroid biosynthesis, diterpenoid biosynthesis, glucosinolate biosynthesis, flavone and flavonol biosynthesis, tropane, piperidine and pyridine alkaloid biosynthesis, brassinosteroid biosynthesis, folate biosynthesis, and anthocyanin biosynthesis. in addition to the pathways mentioned above, many longan unigenes were associated with genetic information processing involving spliceosome, ribosome, rna degradation, nucleotide excision repair, rna polymerase, dna replication, base excision repair, homologous recombination, mismatch repair, protein processing in endoplasmic reticulum, and protein export. finally, longan unigenes were also involved in carbon fixation in photosynthetic organisms, photosynthesis, and photosynthesis- antenna proteins. taken together, the annotated longan unigenes provided valuable information for investigating specific processes, functions, and pathways involved in longan ec development, and allowed identification of novel genes in non-model organisms. gene validation and expression analysis during longan se using quantitative real-time pcr to experimentally confirm that unigenes obtained from sequencing and computational analysis were indeed expressed, unigenes longer than bp, including embryogenesis-related genes, reproductive growth-related genes, and vegetative growth-related genes, were selected for qpcr analysis across the six sequential developmental stages of longan se: friable-embryogenic callus, incomplete compact pro-embryogenic cultures, globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonary embryos. based on the analyzed qpcr data, all selected unigenes were expressed at varying levels in different embryogenic tissues. mrh npgr ppr ref npg swp and vln were expressed throughout the different tissue culture developmental stages, although no significantly expression profiles were observed. expression levels of emb emb frp, spk vln aah, and swp were low in te, and high in he and ce, while bel1-like exhibited the highest expression in te. ghmp ppr and npgr were highly expressed in icpec, ghmp emb and swa showed strong expression in he, moderate expression in ec, and weak expression in icpec. eda and swa were highly expressed in ec. these results confirm that differential expressions of these unigenes have potential roles during longan se. discussion feasibility of illumina paired-end sequencing and assembly for non-model species with unsequenced genomes such as longan understanding the dynamics of plant transcriptomes is helpful for studying the complexity of transcriptional regulation and its impact on phenotype. transcriptome sequencing is one of the most important tools for gene discovery and expression pattern identification, but traditional est sequencing based on the sanger method is time-consuming and costly. because of their high throughput, accuracy, and low cost, next-generation sequencing technologies, such as illumina/solexa, and mpss, have been used successfully for plant genomic and transcriptomic analyses in many organisms. in this study, approximately million clean reads were obtained from longan ec using illumina hiseq sequencing and assembled into unigenes, more than that reported for plants such as s.indicum, t.chinensis, c.sinensis, g. hirsutum and i.batatas using the same technology. compared with previous studies, these sequences produced shorter unigenes than those assembled from sesamum, taxus, i.batatas, poncirus trifoliata and jatropha curcas, but longer than those generated from c.sinensis, fagopyrum, and maize. more importantly, of the assembled unigenes were longer than bp. these results demonstrate that illumina sequencing technology can be an effective tool for gene discovery in non- model organisms. moreover, % of the putative protein unigenes showed significant similarity to known plant proteins in databases, a higher percentage than that reported for s.indicum, i.batatas, and epimedium sagittatum. on the other hand, average unigene length in our study was shorter than that obtained for most plant species, and there were many unassembled reads; difficulties with the de novo transcriptome assembly may be attributed to various factors, such as short sequence fragments, assembly options, genes expressed at low levels, repetitive sequences, alternative splicing, and lack of a reference genome. quantity and variety of genes expressed in longan ec plant callus transcriptomes have been obtained for a number of species, including rice, populus, a.thaliana, g.hirsutum, s.tuberosum and e.guineensis. in this study, unigenes from longan ec were assembled, more than the number reported from calli of rice in one study, g. hirsutum and s.tuberosum, but less than from rice in another study, populus, and a.thaliana. the above studies failed to obtain global expression profiles of callus genes, either because there was insufficient transcriptome information or because no further analysis was conducted. studies have shown that plant se is morphologically and molecularly similar to zygotic embryogenesis. just as a plant zygotic embryo, the most complex plant organ, epitomizes the entire plant, so too a plant somatic embryo can be considered to represent an entire plant. in our study, we uncovered unigenes, the majority of which reflected expression of genes required for plant in vitro embryogenesis, such as pentatricopeptide repeat proteins and embryo defective family genes. a previous study has shown that mutations of different pprs have distinct impacts on embryo morphogenesis. in our study, ppr unigenes longer than bp were identified, and two highly abundent ppr unigenes were further confirmed and found to be expressed throughout longan se. ppr1_unigene was highly expressed in icpec and he, while ppr2_unigene mrna was abundant in ec and icpec. these results suggest the involvement of these genes during early developmental stages of longan se. in arabidopsis, emb genes have been confirmed to be required for normal embryo development, with emb displaying aberrant cell organization and undergoing morphological arrest before the globular-heart transition. in our study, three emb unigenes longer than bp—emb1_unigene emb2_unigene and emb3_unigene1123—were verified and strongly expressed in he and ce. in addition, emb1_unigene was also highly expressed in ge and moderately detectable in ec and te, while emb2_unigene and emb3_unigene exhibited moderate expression in ge and te. high expression levels of these selected embs in longan he and ce indicate their possible roles in the development of longan se. surprisingly, however, there were also many unigenes expressed in ec associated with reproductive growth characteristics, and vegetative growth. in our study, reproductive growth-related genes longer than bp were confirmed by qpcr during the developmental stages of longan se. for example, the fertility-related frp, was strongly expressed in he and ce, and also weakly in te. genes related to pollen growth—npgr, npgr, and vln —displayed ubiquitous but weak expression during longan se. npgr2_unigene was highly expressed in icpec, and vln2_unigene was accumulated in he and ce, but barely detectable in te. swa1/ essential for gametogenesis in arabidopsis, were also differentially expressed during longan se; while they were both highly expressed in ec, while swa1_unigene was also expressed in he. the ghmp kinase enzyme family, a primary determinant of sexual fate in caenorhabditis elegans, was also detected in our study. ghmp1_unigene expression was high in he, and ghmp2_unigene transcripts accumulated in icpec. bel1-like, is required for cytokinin and auxin signaling during ovule development in arabidopsis, was expressed highly in te and moderately in he and ce, but was barely detectable in icpec; this suggests it may play a major role in longan se during late embryonic stages. eda related to embryo sac development, was strongly expressed in ec, te, and ce. these results all demonstrate that these reproductive growth-related genes also play roles during longan se development. five vegetative growth-related genes were also chosen for qpcr analysis across the six sequential developmental stages of longan se. these genes included mrh, which is likely involved in polarized growth of root hairs in arabidopsis, ref, which plays divergent roles in the regulation of arabidopsis flowering, and swp, which plays an important role in defining the duration of cell proliferation. all of these genes were expressed at varied levels in different embryogenic tissues, suggesting their wide involvement in various developmental stages during longan se. in particular, swp1_unigene was highly expressed during late stages of longan se, but was barely detectable in ge. in addition, spk, required for normal cell shape control and tissue development, and aah, related to nitrogen fixation, were both strongly expressed in he and ce. the number and variety of expressed genes in ec suggests that ec practically reflects the entire se profile, and even that of the entire plant. in addition, ec might be considered as a “gene pool” for isolating various plant target genes, such as genes related to flowering, pollen development, root and shoot growth, and plant-pathogen interactions. this ec dataset provides new candidates with possible roles in longan somatic embryogenesis. in arabidopsis, the largest number of unannotated signatures was found in callus:, compared with in inflorescences, in leaves, in roots, and in siliques. in our study, we also found many unigenes with unknown function, demonstrating how little is known about the biology of undifferentiated plant cells. thus, the ec transcriptome can be used to more effectively discover new genes in longan. using solexa sequencing, 27% of identified unigenes in populus euphratica callus were found to be differentially expressed in response to salt stress; these genes were mainly involved in transport, transcription, cellular communication, and metabolism. during arabidopsis callus development, genes were found to be up-regulated and to be down-regulated. the most highly up-regulated genes encoded an unknown protein and acireductone dioxygenase, and the most highly down-regulated genes included a dr protease inhibitor, two peroxidase genes, two pearli genes, and two that encoded subtilases. in rice callus cells, expressed genes were identified using the microarray suite detection algorithm. these studies demonstrate that a large number of genes involved in various biological and metabolic pathways are expressed in plant ec. in our study, unigenes were assigned to kegg pathways, including metabolic pathways, plant-pathogen interactions, biosynthesis of secondary metabolites, and photosynthesis and genetic information processing-related pathways. these results lay a foundation for further identification of longan ec-related genes.  in summary, our study generated the first large-scale transcriptome dataset of longan ec. in addition, the types and quantities of genes expressed in longan, as well as their functions, classification, and metabolic pathways, were revealed for the first time. twenty-three unigenes related to embryogenesis and reproductive and vegetative growth were differentially expressed in various embryogenic cultures, indicating their possible roles in longan se. this transcriptome dataset provides new insights into molecular processes in d. longan. 
in addition, unigenes mapped to kegg pathways, with the categories of metabolic pathways, plant-pathogen interaction, biosynthesis of secondary metabolites, and genetic information processing being well represented. longan is a tropical/subtropical fruit tree of great economic importance in southeast asia. furthermore, unigenes related to embryogenesis and reproductive and vegetative growth were validated by quantitative real time pcr in samples from different stages of longan somatic embryogenesis; their differentially expressions in the various embryogenic cultures indicated their possible roles in longan se. using blastx, unigenes were found to have significant similarity with accessions in nr and swiss- prot databases. analyses of unigenes ≥ bp revealed embryogenesis-related unigenes as well as numerous unigenes expressed in ec associated with functions of reproductive growth, such as flowering, gametophytogenesis, and fertility, and vegetative growth, such as root and shoot growth. progress in understanding molecular mechanisms of longan embryogenesis, which is the primary influence on fruit quality and yield, is slowed by lack of transcriptomic and genomic information. the quantity and variety of expressed ec genes identified in this study is sufficient to serve as a global transcriptome dataset for longan ec and to provide more molecular resources for longan functional genomics.
 longan is a tropical/subtropical fruit tree of great economic importance in southeast asia. progress in understanding molecular mechanisms of longan embryogenesis, which is the primary influence on fruit quality and yield, is slowed by lack of transcriptomic and genomic information. illumina second generation sequencing, which is suitable for generating enormous numbers of transcript sequences that can be used for functional genomic analysis of longan.  in this study, a longan embryogenic callus cdna library was sequenced using an illumina hiseq system. a total of, clean reads comprising gb of nucleotides were assembled into unigenes of 448-bp mean length, with unigenes ≥ bp accounting for % of the total. using blastx, unigenes were found to have significant similarity with accessions in nr and swiss- prot databases. of these, unigenes were assigned to go sub-categories and unigenes were classified into cog sub-groups. in addition, unigenes mapped to kegg pathways, with the categories of metabolic pathways, plant-pathogen interaction, biosynthesis of secondary metabolites, and genetic information processing being well represented. analyses of unigenes ≥ bp revealed embryogenesis-related unigenes as well as numerous unigenes expressed in ec associated with functions of reproductive growth, such as flowering, gametophytogenesis, and fertility, and vegetative growth, such as root and shoot growth. furthermore, unigenes related to embryogenesis and reproductive and vegetative growth were validated by quantitative real time pcr in samples from different stages of longan somatic embryogenesis; their differentially expressions in the various embryogenic cultures indicated their possible roles in longan se.  the quantity and variety of expressed ec genes identified in this study is sufficient to serve as a global transcriptome dataset for longan ec and to provide more molecular resources for longan functional genomics. 