BACKGROUND
the formation of bolting and flowering, marking the developmental transition from vegetative growth to flowering, is one of the most important developmental traits in plant life cycle.
appropriate bolting and flowering time is crucial to ensure reproductive success and high agricultural productivity for crop plants  <cit> .
in arabidopsis thaliana, some molecular and genetic studies have revealed that a complex gene-network has evolved to orchestrate the initiation of bolting and flowering and is regulated by diverse environmental and endogenous factors such as temperature, light signals, day length, developmental stage and plant hormones .
considerable studies in arabidopsis revealed that a set of genes involved in flowering control have been discovered and characterized and integrated into the genetic circuitry of flowering and multiple flowering pathways, including pathways of photoperiod, vernalization, aging, autonomous and gibberellin   <cit> .
the transition from vegetative growth to reproductive development is regulated by the interplays of central flowering genes involved in different flowering pathways  <cit> .
the pathways of autonomous and vernalization are integrated by flowering locus c , which is a main flowering repressor and encodes a mads-box transcription factor  <cit> .
the expression level of flc is correlated with plant flowering time and overexpression of flc causes late flowering  <cit> .
in addition, suppressor of overexpression of constans  <dig> , flowering locus t  and leafy  are showed to be convergence points for different flowering pathways and defined as flowering pathway integrators  <cit> .
in the past decade, a number of functional genes and regulatory pathways related to flowering time had also been discovered in many crops including rice  <cit> , maize  <cit> , strawberry  <cit>  and soybean  <cit> .
with the development of next-generation sequencing  technologies, several platforms such as illumina solexa, roche  <dig> and abi solid, have been proven to be powerful and efficient tools for advanced genomic researches, including the whole genome re-sequencing and transcriptome sequencing  <cit> .
recently, the advent of ngs-based rna sequencing  of transcriptome has provided an opportunity for large-scale discovery and profiling of functional genes involved in diverse biological processes  <cit> , such as floral scent biosynthesis in wintersweet  <cit> , discovery of disease-resistance genes  in camelina sativa  <cit> , biosynthesis and accumulation of lignin in celery  <cit>  and terpenoid biosynthesis in pitanga  <cit> .
rna-seq has also been used for the discovery of genes related to flowering time regulation and flower development in some plant species including bamboo  <cit> , eichhornia paniculata  <cit> , lagerstroemia indica  <cit> , ipomoea nil  <cit>  and sweet potato  <cit> .
however, genome-wide identification of flowering-related functional genes is still lacking in root vegetable crops.
radish  is an important annual or biennial root vegetable crop of brassicaceae family and belongs to ld  plant.
bolting and flowering are integral phases in the complete life cycle of radish.
premature bolting is a destructive problem that limits vegetative growth and reduces yield and quality of economic products in brassicaceae crops, especially the radish grown in spring.
the timing of bolting and flowering is of great importance for high productivity.
recently, the draft genomic sequences of radish have been assembled  <cit> , providing useful database for genomic research in radish.
moreover, some recent studies concerning the radish transcriptome assembly and analysis were reported, and a list of radish unique sequences were generated from various radish tissues  <cit> .
previous studies reported that several flowering genes have been isolated and characterized by expression profiling and transgenic approaches .
however, the genetic networks of bolting and flowering regulation in radish are poorly understood.
to characterize the radish leaf transcriptome and identify critical genes involved in bolting and flowering of radish, transcript sequences from radish leaves during different developmental phases were isolated and sequenced by illumina sequencing method.
the goal of this study was to obtain a complete set of assembled unigenes and transcripts in radish leaves, and to identify transition of bolting and flowering related genes involved in flowering-time regulatory networks.
the full-length cdna sequences of seven flowering related genes were isolated by t-a cloning and further compared with the assembled transcript sequences from radish leaf transcriptome.
moreover, expression patterns of seven isolated genes were validated by rt-qpcr analysis.
furthermore, a graphical network of bolting and flowering-time regulation was proposed.
these findings could provide a solid foundation for better understanding of bolting and flowering-time regulatory networks in radish, and provide significant insights into molecular genetic mechanisms underlying bolting and flowering regulation in brassicaceae vegetable crops.
RESULTS
illumina sequencing and de novo transcriptome assembly
rna-seq is a useful approach for obtaining a complete set of transcripts from certain plant tissue at specific developmental stage or under certain physiological condition  <cit> .
to obtain a comprehensive overview of radish leaf transcriptome, a cdna library from radish leaves of late-bolting ‘nau-lu127’ at different developmental stages was constructed and sequenced using illumina rna-seq.
a total of  <dig> , <dig> pair-end reads were generated from the radish leaf transcriptome, named ‘nau-lb’.
after the removal of adapter sequences, ambiguous reads and low quality reads,  <dig> , <dig> high-quality clean reads comprising of  <dig> , <dig>  nucleotides  were obtained with an average length of 90 nt and average gc content of  <dig>  % .
all clean reads were assembled into  <dig>  contigs using the trinity program  <cit> , with a mean length of 429 nt and an n <dig> length of 476 nt .
then, these contigs were assembled into  <dig>  unigenes with a total length of  <dig> ,520 nt, an average length of 802 nt and an n <dig> length of 1169 nt .
length distributions of assembled contigs and unigenes were analyzed and demonstrated in fig.
<dig> table  <dig> summary of illumina transcriptome sequencing from radish leaves
fig.
<dig> overview of the radish leaf transcriptome assembly.
the length distribution of assembled contigs  and unigenes , and the cds  and predicted proteins  by blastx alignment in radish
using the rna-seq platform and trinity program, a number of assembled unigenes were also generated from radish leaves and roots  <cit> .
zhang et al.
<cit>  reported that  <dig>  unigenes with an average length of 394 bp and an n <dig> of 422 bp was generated from radish leaves by transcriptome assembly.
wang et al.
<cit>  showed that  <dig>  unigenes with a mean length of 763 nt and an n <dig> of 1095 nt were obtained from radish root transcriptome.
to evaluate the quality of assembly results and transcriptome data, comparison analysis showed that the average length and n <dig> length of unigenes obtained in this study were longer than that in previous studies on radish, indicating these sequencing data were of high quality and sufficient quantity for further analysis.
to estimate the expression levels of assembled unigenes in radish leaf transcriptome, the fpkm  method  <cit>  was used for the calculation of unigene expression level.
the global detailed information of all the assembled unigenes was showed in additional file  <dig>  it is well known that the de novo transcriptome sequencing had been successfully applied for many vegetable crops including b. oleracea  <cit> , b. napus  <cit>  and celery  <cit> .
consistent with these studies, the transcript sequences from radish leaf transcriptome could provide a valuable resource for comprehensively identifying specific developmental processes, pathways and functional genes in radish.
functional annotation of radish leaf transcriptome
to assign putative gene functions and annotations of all the assembled unigenes, blastx alignment was performed with an e-value threshold of  <dig> e- <dig> against the public protein databases including ncbi non-redundant protein , swiss-prot, clusters of orthologous groups  and kyoto encyclopedia of genes and genomes .
a total of  <dig>  unigenes  were annotated and matched to one or more of the public protein databases .
the best aligning results were used to decide the sequence direction of each assembled unigene.
the size distributions for the coding sequence  and predicted proteins were analyzed and shown in fig.
<dig>  nevertheless, a small proportion of sequences  was unannotated and showed no matches to any above protein databases, which might represent radish-specific transcriptomic sequences in radish leaves.table  <dig> the annotations of radish leaf unigenes against the public databases
by sequence similarity searching against the ncbi nr database,  <dig>  annotated sequences  were detected and showed significant similarities to known proteins .
according to the e-value distribution of significant hits against the nr database,  <dig>  % of the matched sequences showed high homologies with the e-value <  <dig> e- <dig>  while  <dig>  % of the top hits had e-value in the range of  <dig> e- <dig> to  <dig> e- <dig> .
the similarity distribution of the top blast hits revealed that  <dig>  % of the matched sequences were in similarity higher than 80 % .
further analysis of homologies among different plant species showed that  <dig>  % of the annotated sequences matched to sequences from arabidopsis lyrata, followed by a. thaliana , thellungiella halophila  and b. napus  .
the species distribution revealed that most of annotated unigenes had high hits with sequences from the brassicaceae species, suggesting that the assembly and annotation of radish leaf transcriptome are correct and reliable.fig.
<dig> characteristics of sequence homology of radish unigenes against nr database.
a
e-value distribution of blast hits for each unigene with an e-value cutoff of  <dig> e- <dig>  b similarity distribution of the top blast hits for each unigene.
c species distribution of the top blast hits
go and cog classification
based on the blastx results against the nr database, blast2go program was performed to obtain gene ontology  functional annotations and categorizations of these assembled unigenes.
as a result,  <dig>  unique sequences were annotated and classified into  <dig> go classes including  <dig> biological processes,  <dig> cellular components and  <dig> molecular functions .
under the biological process category, ‘cellular process’ , ‘metabolic process’  and ‘single-organism process’  were prominently represented.
within these cellular components, ‘cell’ , ‘cell part’  and ‘organelle’  were the most highly represented categories.
for the molecular function category, the largest proportion of genes were clustered into ‘binding’ , ‘catalytic activity’  and ‘nucleic acid binding transcription factor activity’ .
additionally, only a few sequences were assigned to ‘virion’, ‘virion part’, ‘channel regulator activity’ and ‘translation regulator activity’ terms with less than ten sequences.fig.
<dig> go classification of the radish leaf transcriptome
to further predict and classify the possible functions of radish unigenes, these unique sequences from radish leaf transcriptome were aligned to the cog database.
the results showed that  <dig>  unigenes  were assigned to  <dig> cog categories .
of these, the largest category was ‘general function prediction only’ , followed by ‘transcription’ , ‘replication, recombination and repair’ , ‘posttranslational modification, protein turnover, chaperones’  and ‘signal transduction mechanisms’ ; whereas the clusters of ‘extracellular structures’  and ‘nuclear structure’  represented the smallest classifications.
these assigned functions of unigenes covered a wide range of go and cog classifications, indicating that the transcriptome data from radish leaves represented a broad variety of transcripts in radish.fig.
<dig> cog functional classification of the radish leaf transcriptome
identification of functional genes involved in circadian rhythm
to systematically study the complex biological functions of genes and identify active biological pathways in radish leaves, the assembled unigenes were mapped against the kegg database using blastx analysis with a cut-off e-value of  <dig> e- <dig>  in total,  <dig>  unigenes  had significant matches to the database and were assigned to  <dig> kegg pathways .
the most dominant pathways were ‘metabolic pathways’ , ‘biosynthesis of secondary metabolites’ , ‘plant hormone signal transduction’ , ‘plant-pathogen interaction’  and ‘rna transport’  .
the results were comparable with the previous transcriptome profiles in radish root, in which  <dig>  unigenes were clustered into  <dig> kegg pathways  <cit> .
significantly, pathway-based analysis revealed that a total of  <dig> transcript sequences representing  <dig> rhythm related candidate genes were identified for the ‘circadian rhythm-plant’ pathway  .
these rhythm related genes including constans , circadian clock associated  <dig> , early flowering  <dig> , late elongated hypocotyl  and gigantea , were involved in many rhythmic processes including photoperiodic flowering, uv-b protection and cell elongation .
in addition, several identified potential genes including lov kelch protein  <dig> , cryptochrome  <dig> , cryptochrome  <dig> , phytochrome a , phytochrome b , phytochrome d  and phytochrome e , were implicated in plant light signaling pathways  <cit> .fig.
<dig> the pathway of circadian rhythm in radish.
the circadian related genes identified in radish are in red.
arrows indicate positive regulation and bars indicate negative regulation
plant circadian clock as an internal timekeeper controls daily and seasonal changes of many rhythmic processes according to the day length and photoperiod  <cit> .
photoperiodic flowering control is linked to circadian clock by the transcriptional expression of co which modulates plant flowering time and initiates floral development .
co encoding a zinc finger protein is central component of photoperiodic flowering pathway and could integrate endogenous and environmental information to trigger plant flowering at proper time  <cit> .
it was reported that the expression of co was regulated by the circadian clock and the peak expression occurred at the end of the day under long day conditions  <cit> .
in the present study,  <dig> transcripts were annotated and identified to be homologous sequences of co .
understanding the roles of circadian-regulated genes responsible for controlling flowering could contribute to study the interplays of photoperiod and circadian clock.
identification of functional genes involved in flowering pathways in radish
functional genetic studies have evidenced that several coordinate flowering pathways form a complex genetic network and control the development transition of flowering  <cit> .
more than  <dig> flowering-related genes implicated in the complexity of flowering have been identified and characterized in model crop arabidopsis  <cit> .
in this study, to comprehensively identify the candidate genes putatively implicated in bolting and flowering regulation in radish, a local blastx similarity search was performed against a. thaliana flowering genes which were downloaded from ncbi database.
a total of  <dig> potential bolting and flowering-related genes including  <dig> transcript sequences were identified and shared higher similarity to the homologous genes from arabidopsis .
according to the reported flowering pathways and regulatory networks, these identified bolting and flowering-related genes were characterized and involved in various flowering pathways , including photoperiod/circadian clock, vernalization, autonomous, ga and aging pathways  <cit> .
the blast searching showed that  <dig> potential functional genes including constans-like  <dig> , tempranillo  <dig> , tem <dig>  terminal flower  <dig>  and flowering locus d , were identified and implicated in photoperiod pathway .
in addition, the pathways of vernalization, autonomous, ga and aging contained  <dig>   <dig>   <dig> and  <dig> candidate genes, respectively.
these results revealed that the known genetic flowering pathways and many critical flowering genes shared a high degree of conservation in radish and arabidopsis.table  <dig> the identified candidate genes involved in various flowering pathways of radish
abh1/ens
agl12
agl14
agl15
agl16
agl18
agl19
agl24
agl6
agl8/ful
ap1
ap2
ap3
arp4
arp6
arr3
ath1
atx1
cbf1
cbf2
cca1
ccr2
cdf1
cdf2
cdf3
co
col1
col2
col3
col4
col5
col9
cop1
cry1
cry2
cstf64
cstf77
ddf1
ddf2
ddl
ebs
efs
elf3
elf4
elf5
elf6
elf7
elf8
elf9
emf1
esd4
fca
fd
fes1
fie
fip1
fip2
flc
fld
flk
flm/maf1
flp1
fpa
fpf1
fri
ft
fve/msi4
fy
ga3
gai
gi
gid1a
gid1b
gid1c
gnc
gnl
hua2
hyl1/drb1
lcl1
ld
ldl2
lfy
lhp1
lhy
lkp2
lwd1/atan11
lwd2
mbd9
myb33
myb65
nf-ya1/hap2a
nf-ya4
nf-yb1
nf-yb2
pearli
phya
phyb
phyc
phyd
phye
pi
pie1
prc2/clf/emf2
prr3
prr7
prr9
ref6
rga
rgl1
rgl2
sdg10/swn
sep1/agl2
sep2/agl4
sep3/agl9
shp1/agl1
shp2/agl5
skb1
soc1/agl20
spa
spl
spl3
spl5
spl9
spy
suf4
svp/agl22
tem1
tem2
tfl2
tic
toe1
toe2
toe3
tps1
vel1
vin3
vip1
vip2
vrn1
vrn2
wnk1
moreover, to summarize and characterize the identified functional genes and different flowering pathways, a putative schematic network of bolting and flowering regulation in radish was proposed .
more importantly, many key players of flowering regulation including soc <dig>  co, ft and flc and several functional genes associated with floral meristem identity and flower development including apetala <dig> , sepallata1- <dig> , agamous-like  <dig>  and lfy, were identified in this study .
similar results were also found in previous genetic analysis studies on rice  <cit> , barley  <cit> , soybean  <cit> , maize  <cit>  and strawberry  <cit> .
these results provide essential information for functional analysis of bolting and flowering genes and regulatory pathways, and facilitate further genetically investigating in radish.fig.
<dig> the putative schematic network of bolting and flowering regulation in radish.
the genes in black were the flowering pathway integrators.
the mir <dig>  mir <dig>  mir <dig> and mir <dig> were identified in our previous study  <cit> .
arrows indicate positive regulation and bars indicate negative regulation
characterization of bolting and flowering genes and regulatory pathways in radish
studies on developmental switch of arabidopsis flowering have demonstrated that multiple different flowering pathways converge at several flowering pathway integrators including ft, soc <dig> and lfy to determine the flowering time  <cit> .
these integrators are regulated by two key upstream genes co and flc, which antagonistically control flowering  <cit> .
flc as a main flowering repressor integrates autonomous and vernalization pathways and negatively regulates the expressions of ft and soc <dig>  <cit> .
previous studies had identified the homologous genes of flc in b. rapa  <cit> , sweet potato  <cit>  and orange  <cit> .
in this study, three unique sequences of flc gene, cl <dig> contig <dig>  cl <dig> contig <dig> and cl <dig> contig <dig>  were identified and matched to the homologs of atflc .
after undergoing vernalization process, a long exposure to low temperature, the plants permit to initiate the phase transition of flowering.
while the process could be repressed by the high level of flc  <cit> , which requires its activator frigida , a plant-specific gene conferring vernalization response in arabidopsis  <cit> .
in addition, genomic analysis suggested that many genes were involved in vernalization pathway and played roles in flowering control  <cit> .
as expected, the vernalization-response genes including fri, frigida essential  <dig> , vernalization insenstive  <dig> , vernalization  <dig>  and vrn <dig>  were also identified in this study , which was consistent with previous studies on flowering gene discovery  <cit> .
additionally, we also found transcript sequences matching to the candidate genes of soc <dig> , agl <dig>  and lfy  .
both soc <dig> and agl <dig> belonged to mads-box genes, and their interaction directly regulated the expression of lfy and determined the flowering time  <cit> .
the family of mads-box transcription factor is a major group of regulators controlling floral transition in arabidopsis and act in floral organ specification and floral development in flowering plants .
our studies also identified some members of mads family such as agls, ap <dig>  ap <dig>  short vegetative phase  and flowering locus m , which might participate in the development and flowering regulation of radish .
moreover, radish transcripts which showed to be homologous genes in ga pathway included flowering promoting factor  <dig> , gibberellic acid insensitive , myb <dig>  myb <dig> and spindly  .
it was reported that myb <dig> and myb <dig> were the target genes of mir <dig> which could control the flowering time via the ga pathway  <cit> , while spy targeted by mir <dig> was a negative regulator of gibberellin signaling and functioned in mediating flowering time  <cit> .
furthermore, some bolting and flowering-related critical mirnas including mir <dig>  mir <dig>  mir <dig> and mir <dig> were also identified in our previous study  <cit>  .
these findings revealed that the transcriptome dataset of radish leaves could be used for the discovery and profiling of functional genes involved in various developmental pathways and processes in root vegetable crops.
isolation and validation of bolting and flowering-related genes
based on the annotation analysis and blast search of radish leaf transcriptomic sequences, the full-length sequences of functional genes related to flowering regulation were obtained by transcript sequence assembly.
to evaluate the quality of assembled sequences and annotation results from the ngs sequencing, the sequences of several flowering genes from t-a cloning and transcript assembly were subjected to comparative analysis.
the full-length cdna sequences of seven randomly selected bolting and flowering-related genes including agl <dig>  ap <dig>  fpf <dig>  luminidependens , vrn <dig>  soc <dig> and svp, were isolated from radish late-bolting line ‘nau-lu127’ using specific primers and sequenced by the sanger methods .
comparative analysis showed that the full length of these isolated gene sequences ranged from 365 bp to  <dig>  bp, and all of them contained the complete open reading frame  .
these assembled transcripts had more than 92 % coverage to the corresponding full-length genes, with more than 91 % similarity of orf.
these results revealed that the transcript sequences from radish leaf transcriptome could be used for the identification and isolation of more functional genes related to bolting and flowering in radish.table  <dig> sequence analysis of the isolated genes related to bolting and flowering in radish
rsagl24
rsap2
rsfpf1
rsld
rssoc1
rssvp
rsvrn2
remarkably, the assembled transcript sequence of soc <dig> had 100 % coverage and 100 % orf similarity to that from t-a cloning .
the role and function of soc <dig> is mainly promoting flowering and floral meristem identity, and the photoperiod, vernalization and ga pathways converge on soc <dig> to control the flowering time  <cit> .
phylogenetic relationship between rssoc <dig> and the homologous genes in other plants was analyzed using mega  <dig>  software .
the phylogenetic analysis indicated that the soc <dig> homologs were clustered into two subgroups, and rssoc <dig> shared the closest relationship with the bragl <dig> from b. rapa and was clearly separated from zmmads <dig>  a zmsoc1-like gene from zea mays.fig.
<dig> the phylogenetic analysis of rssoc <dig> and the homologous genes in other plants using mega  <dig>  software with neighbor-joining algorithm.
the abbreviations for the respective species names are as follows: at, arabidopsis thaliana; br, brassica rapa; gm, glycine max; fv, fragaria vesca; md, malus domestica; sl, solanum lycopersicum; vv, vitis vinifera; zm, zea mays
expression profiles by rt-qpcr
to investigate the spatial-temporal expression patterns of bolting and flowering-related genes, the relative expression levels of seven isolated candidate genes in various tissues and at different developmental stages were validated by rt-qpcr  analysis .
the expression patterns showed that seven selected genes related to bolting and flowering were differentially expressed in root, stem, leaf and flower of radish .
most of them showed a low expression in radish root, but the lowest expression levels of rsagl <dig>  rssoc <dig> and rssvp were in the flower.
the relative expression levels of rsagl <dig>  rssoc <dig>  rssvp and rsvrn <dig> were significantly higher in the leaf than in the other three tissues, whereas rsap <dig> was mainly expressed in the flower.
the transcript levels of rsfpf <dig> and rsld were higher in leaf and flower, and no significant difference was detected in expression of these two tissues.
the results indicate that these genes may play different roles in various tissues and thus affect the growth and development of radish.
the expression pattern differences of these genes were also observed in previous studies  <cit> .fig.
<dig> the spatial-temporal expression profiling of bolting and flowering-related genes by rt-qpcr analysis.
a the expression patterns of seven genes in different radish tissues.
b the expression patterns of seven genes at vegetative stage  and reproductive stage .
each bar shows the mean ± se of triplicate assays.
the values with different letters indicate significant differences at p <  <dig>  according to duncan’s multiple range tests
the expression profiling of these seven bolting and flowering-related genes also revealed that their expression patterns in radish leaves varied at vegetative stage and reproductive stage , which contributed to further explore their possible roles during radish bolting and flowering.
the expression levels of rsagl <dig> and rssoc <dig> were lower at vegetative stage and increased at reproductive stage; whereas the expression levels of these four genes including rsap <dig>  rsfpf <dig>  rssvp and rsvrn <dig>  were relative higher at vegetative stage and significantly decreased at reproductive stage.
the up- or down-expression of these genes were in agreement with previous analysis in other species  <cit> .
flowering pathway analysis indicated that seven selected genes were implicated in different flowering pathways .
among them, ap <dig> belonging to photoperiod pathway is regulated by mir <dig> and represses the bolting and flowering  <cit> ; while soc <dig> as a floral integrator negatively regulates flc expression and plays vital roles in promoting flowering  <cit> .
these findings revealed that these differentially expressed genes might participate in the transition from vegetative stage to bolting and flowering in radish.
additionally, four flower development-related genes including rsap <dig>  rssep <dig>  rsagl <dig> and rslfy, were selected for rt-qpcr analysis to explore their expression patterns during radish flower development and in different flower parts.
the expression levels of rssep <dig>  rsagl <dig> and rslfy were relative higher at whole flower, while no significant difference of rsap <dig> expression was showed at three stages of flower development .
the expression analysis revealed that four genes were differentially expressed in four flower parts , which was in consistent with previous studies  <cit> , suggesting that these genes may play important roles in flower organ identity.
importantly, ap <dig> belonging to a-class genes in the abc model of flower organ identity, determines the development of sepal and petal  <cit> .
the results showed that rsap <dig> expression level was relative higher in sepal and petal than in stamen and pistil , suggesting that ap <dig> gene is essential for sepal and petal development and specify the identity of the floral meristem  <cit> .fig.
<dig> the spatial-temporal expression profiling of flower development-related genes by rt-qpcr analysis.
a the relative expression levels of four genes at three stages of flower development.
b the relative expression levels of four genes at different flower parts .
each bar shows the mean ± se of triplicate assays.
the values with different letters indicate significant differences at p <  <dig>  according to duncan’s multiple range tests
CONCLUSIONS
to our knowledge, this is the first report on de novo transcriptome sequencing and assembly in late-bolting radish leaves using rna-seq technology.
a total of  <dig>  unigenes were assembled in radish leaf transcriptome and  <dig>  unique sequences were successfully annotated by go, cog and kegg databases.
a total of  <dig> candidate genes were involved in plant circadian rhythmic pathway.
moreover,  <dig> potential radish genes related to bolting and flowering-time regulation were identified by blast similarity search with a. thaliana flowering-related genes.
rt-qpcr analysis revealed that several flowering-related genes showed spatial-temporal expression patterns in various radish tissues and at different development stages.
the findings from this study provided insights into bolting and flowering-time regulatory networks in radish, and facilitated further dissecting molecular genetic mechanisms associated with bolting and flowering regulation in brassicaceae vegetable crops.

 the formation of bolting and flowering, marking the developmental transition from vegetative growth to flowering, is one of the most important developmental traits in plant life cycle. appropriate bolting and flowering time is crucial to ensure reproductive success and high agricultural productivity for crop plants. in arabidopsis thaliana, some molecular and genetic studies have revealed that a complex gene-network has evolved to orchestrate the initiation of bolting and flowering and is regulated by diverse environmental and endogenous factors such as temperature, light signals, day length, developmental stage and plant hormones. considerable studies in arabidopsis revealed that a set of genes involved in flowering control have been discovered and characterized and integrated into the genetic circuitry of flowering and multiple flowering pathways, including pathways of photoperiod, vernalization, aging, autonomous and gibberellin. the transition from vegetative growth to reproductive development is regulated by the interplays of central flowering genes involved in different flowering pathways. the pathways of autonomous and vernalization are integrated by flowering locus c, which is a main flowering repressor and encodes a mads-box transcription factor. the expression level of flc is correlated with plant flowering time and overexpression of flc causes late flowering. in addition, suppressor of overexpression of constans, flowering locus t and leafy are showed to be convergence points for different flowering pathways and defined as flowering pathway integrators. in the past decade, a number of functional genes and regulatory pathways related to flowering time had also been discovered in many crops including rice, maize, strawberry and soybean. with the development of next-generation sequencing technologies, several platforms such as illumina solexa, roche and abi solid, have been proven to be powerful and efficient tools for advanced genomic researches, including the whole genome re-sequencing and transcriptome sequencing. recently, the advent of ngs-based rna sequencing of transcriptome has provided an opportunity for large-scale discovery and profiling of functional genes involved in diverse biological processes, such as floral scent biosynthesis in wintersweet, discovery of disease-resistance genes in camelina sativa, biosynthesis and accumulation of lignin in celery and terpenoid biosynthesis in pitanga. rna-seq has also been used for the discovery of genes related to flowering time regulation and flower development in some plant species including bamboo, eichhornia paniculata, lagerstroemia indica, ipomoea nil and sweet potato. however, genome-wide identification of flowering-related functional genes is still lacking in root vegetable crops. radish is an important annual or biennial root vegetable crop of brassicaceae family and belongs to ld plant. bolting and flowering are integral phases in the complete life cycle of radish. premature bolting is a destructive problem that limits vegetative growth and reduces yield and quality of economic products in brassicaceae crops, especially the radish grown in spring. the timing of bolting and flowering is of great importance for high productivity. recently, the draft genomic sequences of radish have been assembled, providing useful database for genomic research in radish. moreover, some recent studies concerning the radish transcriptome assembly and analysis were reported, and a list of radish unique sequences were generated from various radish tissues. previous studies reported that several flowering genes have been isolated and characterized by expression profiling and transgenic approaches. however, the genetic networks of bolting and flowering regulation in radish are poorly understood. to characterize the radish leaf transcriptome and identify critical genes involved in bolting and flowering of radish, transcript sequences from radish leaves during different developmental phases were isolated and sequenced by illumina sequencing method. the goal of this study was to obtain a complete set of assembled unigenes and transcripts in radish leaves, and to identify transition of bolting and flowering related genes involved in flowering-time regulatory networks. the full-length cdna sequences of seven flowering related genes were isolated by t-a cloning and further compared with the assembled transcript sequences from radish leaf transcriptome. moreover, expression patterns of seven isolated genes were validated by rt-qpcr analysis. furthermore, a graphical network of bolting and flowering-time regulation was proposed. these findings could provide a solid foundation for better understanding of bolting and flowering-time regulatory networks in radish, and provide significant insights into molecular genetic mechanisms underlying bolting and flowering regulation in brassicaceae vegetable crops.  illumina sequencing and de novo transcriptome assembly rna-seq is a useful approach for obtaining a complete set of transcripts from certain plant tissue at specific developmental stage or under certain physiological condition. to obtain a comprehensive overview of radish leaf transcriptome, a cdna library from radish leaves of late-bolting ‘nau-lu127’ at different developmental stages was constructed and sequenced using illumina rna-seq. a total of, pair-end reads were generated from the radish leaf transcriptome, named ‘nau-lb’. after the removal of adapter sequences, ambiguous reads and low quality reads,, high-quality clean reads comprising of, nucleotides were obtained with an average length of 90 nt and average gc content of  %. all clean reads were assembled into contigs using the trinity program, with a mean length of 429 nt and an n length of 476 nt. then, these contigs were assembled into unigenes with a total length of,520 nt, an average length of 802 nt and an n length of 1169 nt. length distributions of assembled contigs and unigenes were analyzed and demonstrated in fig.  table summary of illumina transcriptome sequencing from radish leaves fig. overview of the radish leaf transcriptome assembly. the length distribution of assembled contigs and unigenes, and the cds and predicted proteins by blastx alignment in radish using the rna-seq platform and trinity program, a number of assembled unigenes were also generated from radish leaves and roots. zhang et al. reported that unigenes with an average length of 394 bp and an n of 422 bp was generated from radish leaves by transcriptome assembly. wang et al. showed that unigenes with a mean length of 763 nt and an n of 1095 nt were obtained from radish root transcriptome. to evaluate the quality of assembly results and transcriptome data, comparison analysis showed that the average length and n length of unigenes obtained in this study were longer than that in previous studies on radish, indicating these sequencing data were of high quality and sufficient quantity for further analysis. to estimate the expression levels of assembled unigenes in radish leaf transcriptome, the fpkm method was used for the calculation of unigene expression level. the global detailed information of all the assembled unigenes was showed in additional file it is well known that the de novo transcriptome sequencing had been successfully applied for many vegetable crops including b. oleracea, b. napus and celery. consistent with these studies, the transcript sequences from radish leaf transcriptome could provide a valuable resource for comprehensively identifying specific developmental processes, pathways and functional genes in radish. functional annotation of radish leaf transcriptome to assign putative gene functions and annotations of all the assembled unigenes, blastx alignment was performed with an e-value threshold of e- against the public protein databases including ncbi non-redundant protein, swiss-prot, clusters of orthologous groups and kyoto encyclopedia of genes and genomes. a total of unigenes were annotated and matched to one or more of the public protein databases. the best aligning results were used to decide the sequence direction of each assembled unigene. the size distributions for the coding sequence and predicted proteins were analyzed and shown in fig.  nevertheless, a small proportion of sequences was unannotated and showed no matches to any above protein databases, which might represent radish-specific transcriptomic sequences in radish leaves.table the annotations of radish leaf unigenes against the public databases by sequence similarity searching against the ncbi nr database, annotated sequences were detected and showed significant similarities to known proteins. according to the e-value distribution of significant hits against the nr database,  % of the matched sequences showed high homologies with the e-value <  e- while  % of the top hits had e-value in the range of e- to e-. the similarity distribution of the top blast hits revealed that  % of the matched sequences were in similarity higher than 80 %. further analysis of homologies among different plant species showed that  % of the annotated sequences matched to sequences from arabidopsis lyrata, followed by a. thaliana, thellungiella halophila and b. napus. the species distribution revealed that most of annotated unigenes had high hits with sequences from the brassicaceae species, suggesting that the assembly and annotation of radish leaf transcriptome are correct and reliable.fig. characteristics of sequence homology of radish unigenes against nr database. a e-value distribution of blast hits for each unigene with an e-value cutoff of e- b similarity distribution of the top blast hits for each unigene. c species distribution of the top blast hits go and cog classification based on the blastx results against the nr database, blast2go program was performed to obtain gene ontology functional annotations and categorizations of these assembled unigenes. as a result, unique sequences were annotated and classified into go classes including biological processes, cellular components and molecular functions. under the biological process category, ‘cellular process’, ‘metabolic process’ and ‘single-organism process’ were prominently represented. within these cellular components, ‘cell’, ‘cell part’ and ‘organelle’ were the most highly represented categories. for the molecular function category, the largest proportion of genes were clustered into ‘binding’, ‘catalytic activity’ and ‘nucleic acid binding transcription factor activity’. additionally, only a few sequences were assigned to ‘virion’, ‘virion part’, ‘channel regulator activity’ and ‘translation regulator activity’ terms with less than ten sequences.fig. go classification of the radish leaf transcriptome to further predict and classify the possible functions of radish unigenes, these unique sequences from radish leaf transcriptome were aligned to the cog database. the results showed that unigenes were assigned to cog categories. of these, the largest category was ‘general function prediction only’, followed by ‘transcription’, ‘replication, recombination and repair’, ‘posttranslational modification, protein turnover, chaperones’ and ‘signal transduction mechanisms’; whereas the clusters of ‘extracellular structures’ and ‘nuclear structure’ represented the smallest classifications. these assigned functions of unigenes covered a wide range of go and cog classifications, indicating that the transcriptome data from radish leaves represented a broad variety of transcripts in radish.fig. cog functional classification of the radish leaf transcriptome identification of functional genes involved in circadian rhythm to systematically study the complex biological functions of genes and identify active biological pathways in radish leaves, the assembled unigenes were mapped against the kegg database using blastx analysis with a cut-off e-value of e- in total, unigenes had significant matches to the database and were assigned to kegg pathways. the most dominant pathways were ‘metabolic pathways’, ‘biosynthesis of secondary metabolites’, ‘plant hormone signal transduction’, ‘plant-pathogen interaction’ and ‘rna transport’. the results were comparable with the previous transcriptome profiles in radish root, in which unigenes were clustered into kegg pathways. significantly, pathway-based analysis revealed that a total of transcript sequences representing rhythm related candidate genes were identified for the ‘circadian rhythm-plant’ pathway. these rhythm related genes including constans, circadian clock associated, early flowering, late elongated hypocotyl and gigantea, were involved in many rhythmic processes including photoperiodic flowering, uv-b protection and cell elongation. in addition, several identified potential genes including lov kelch protein, cryptochrome, cryptochrome, phytochrome a, phytochrome b, phytochrome d and phytochrome e, were implicated in plant light signaling pathways.fig. the pathway of circadian rhythm in radish. the circadian related genes identified in radish are in red. arrows indicate positive regulation and bars indicate negative regulation plant circadian clock as an internal timekeeper controls daily and seasonal changes of many rhythmic processes according to the day length and photoperiod. photoperiodic flowering control is linked to circadian clock by the transcriptional expression of co which modulates plant flowering time and initiates floral development. co encoding a zinc finger protein is central component of photoperiodic flowering pathway and could integrate endogenous and environmental information to trigger plant flowering at proper time. it was reported that the expression of co was regulated by the circadian clock and the peak expression occurred at the end of the day under long day conditions. in the present study, transcripts were annotated and identified to be homologous sequences of co. understanding the roles of circadian-regulated genes responsible for controlling flowering could contribute to study the interplays of photoperiod and circadian clock. identification of functional genes involved in flowering pathways in radish functional genetic studies have evidenced that several coordinate flowering pathways form a complex genetic network and control the development transition of flowering. more than flowering-related genes implicated in the complexity of flowering have been identified and characterized in model crop arabidopsis. in this study, to comprehensively identify the candidate genes putatively implicated in bolting and flowering regulation in radish, a local blastx similarity search was performed against a. thaliana flowering genes which were downloaded from ncbi database. a total of potential bolting and flowering-related genes including transcript sequences were identified and shared higher similarity to the homologous genes from arabidopsis. according to the reported flowering pathways and regulatory networks, these identified bolting and flowering-related genes were characterized and involved in various flowering pathways, including photoperiod/circadian clock, vernalization, autonomous, ga and aging pathways. the blast searching showed that potential functional genes including constans-like, tempranillo, tem terminal flower and flowering locus d, were identified and implicated in photoperiod pathway. in addition, the pathways of vernalization, autonomous, ga and aging contained and candidate genes, respectively. these results revealed that the known genetic flowering pathways and many critical flowering genes shared a high degree of conservation in radish and arabidopsis.table the identified candidate genes involved in various flowering pathways of radish abh1/ens agl12 agl14 agl15 agl16 agl18 agl19 agl24 agl6 agl8/ful ap1 ap2 ap3 arp4 arp6 arr3 ath1 atx1 cbf1 cbf2 cca1 ccr2 cdf1 cdf2 cdf3 co col1 col2 col3 col4 col5 col9 cop1 cry1 cry2 cstf64 cstf77 ddf1 ddf2 ddl ebs efs elf3 elf4 elf5 elf6 elf7 elf8 elf9 emf1 esd4 fca fd fes1 fie fip1 fip2 flc fld flk flm/maf1 flp1 fpa fpf1 fri ft fve/msi4 fy ga3 gai gi gid1a gid1b gid1c gnc gnl hua2 hyl1/drb1 lcl1 ld ldl2 lfy lhp1 lhy lkp2 lwd1/atan11 lwd2 mbd9 myb33 myb65 nf-ya1/hap2a nf-ya4 nf-yb1 nf-yb2 pearli phya phyb phyc phyd phye pi pie1 prc2/clf/emf2 prr3 prr7 prr9 ref6 rga rgl1 rgl2 sdg10/swn sep1/agl2 sep2/agl4 sep3/agl9 shp1/agl1 shp2/agl5 skb1 soc1/agl20 spa spl spl3 spl5 spl9 spy suf4 svp/agl22 tem1 tem2 tfl2 tic toe1 toe2 toe3 tps1 vel1 vin3 vip1 vip2 vrn1 vrn2 wnk1 moreover, to summarize and characterize the identified functional genes and different flowering pathways, a putative schematic network of bolting and flowering regulation in radish was proposed. more importantly, many key players of flowering regulation including soc co, ft and flc and several functional genes associated with floral meristem identity and flower development including apetala, sepallata1-, agamous-like and lfy, were identified in this study. similar results were also found in previous genetic analysis studies on rice, barley, soybean, maize and strawberry. these results provide essential information for functional analysis of bolting and flowering genes and regulatory pathways, and facilitate further genetically investigating in radish.fig. the putative schematic network of bolting and flowering regulation in radish. the genes in black were the flowering pathway integrators. the mir mir mir and mir were identified in our previous study. arrows indicate positive regulation and bars indicate negative regulation characterization of bolting and flowering genes and regulatory pathways in radish studies on developmental switch of arabidopsis flowering have demonstrated that multiple different flowering pathways converge at several flowering pathway integrators including ft, soc and lfy to determine the flowering time. these integrators are regulated by two key upstream genes co and flc, which antagonistically control flowering. flc as a main flowering repressor integrates autonomous and vernalization pathways and negatively regulates the expressions of ft and soc. previous studies had identified the homologous genes of flc in b. rapa, sweet potato and orange. in this study, three unique sequences of flc gene, cl contig cl contig and cl contig were identified and matched to the homologs of atflc. after undergoing vernalization process, a long exposure to low temperature, the plants permit to initiate the phase transition of flowering. while the process could be repressed by the high level of flc, which requires its activator frigida, a plant-specific gene conferring vernalization response in arabidopsis. in addition, genomic analysis suggested that many genes were involved in vernalization pathway and played roles in flowering control. as expected, the vernalization-response genes including fri, frigida essential, vernalization insenstive, vernalization and vrn were also identified in this study, which was consistent with previous studies on flowering gene discovery. additionally, we also found transcript sequences matching to the candidate genes of soc, agl and lfy. both soc and agl belonged to mads-box genes, and their interaction directly regulated the expression of lfy and determined the flowering time. the family of mads-box transcription factor is a major group of regulators controlling floral transition in arabidopsis and act in floral organ specification and floral development in flowering plants. our studies also identified some members of mads family such as agls, ap ap short vegetative phase and flowering locus m, which might participate in the development and flowering regulation of radish. moreover, radish transcripts which showed to be homologous genes in ga pathway included flowering promoting factor, gibberellic acid insensitive, myb myb and spindly. it was reported that myb and myb were the target genes of mir which could control the flowering time via the ga pathway, while spy targeted by mir was a negative regulator of gibberellin signaling and functioned in mediating flowering time. furthermore, some bolting and flowering-related critical mirnas including mir mir mir and mir were also identified in our previous study. these findings revealed that the transcriptome dataset of radish leaves could be used for the discovery and profiling of functional genes involved in various developmental pathways and processes in root vegetable crops. isolation and validation of bolting and flowering-related genes based on the annotation analysis and blast search of radish leaf transcriptomic sequences, the full-length sequences of functional genes related to flowering regulation were obtained by transcript sequence assembly. to evaluate the quality of assembled sequences and annotation results from the ngs sequencing, the sequences of several flowering genes from t-a cloning and transcript assembly were subjected to comparative analysis. the full-length cdna sequences of seven randomly selected bolting and flowering-related genes including agl ap fpf luminidependens, vrn soc and svp, were isolated from radish late-bolting line ‘nau-lu127’ using specific primers and sequenced by the sanger methods. comparative analysis showed that the full length of these isolated gene sequences ranged from 365 bp to  bp, and all of them contained the complete open reading frame. these assembled transcripts had more than 92 % coverage to the corresponding full-length genes, with more than 91 % similarity of orf. these results revealed that the transcript sequences from radish leaf transcriptome could be used for the identification and isolation of more functional genes related to bolting and flowering in radish.table sequence analysis of the isolated genes related to bolting and flowering in radish rsagl24 rsap2 rsfpf1 rsld rssoc1 rssvp rsvrn2 remarkably, the assembled transcript sequence of soc had 100 % coverage and 100 % orf similarity to that from t-a cloning. the role and function of soc is mainly promoting flowering and floral meristem identity, and the photoperiod, vernalization and ga pathways converge on soc to control the flowering time. phylogenetic relationship between rssoc and the homologous genes in other plants was analyzed using mega software. the phylogenetic analysis indicated that the soc homologs were clustered into two subgroups, and rssoc shared the closest relationship with the bragl from b. rapa and was clearly separated from zmmads a zmsoc1-like gene from zea mays.fig. the phylogenetic analysis of rssoc and the homologous genes in other plants using mega software with neighbor-joining algorithm. the abbreviations for the respective species names are as follows: at, arabidopsis thaliana; br, brassica rapa; gm, glycine max; fv, fragaria vesca; md, malus domestica; sl, solanum lycopersicum; vv, vitis vinifera; zm, zea mays expression profiles by rt-qpcr to investigate the spatial-temporal expression patterns of bolting and flowering-related genes, the relative expression levels of seven isolated candidate genes in various tissues and at different developmental stages were validated by rt-qpcr analysis. the expression patterns showed that seven selected genes related to bolting and flowering were differentially expressed in root, stem, leaf and flower of radish. most of them showed a low expression in radish root, but the lowest expression levels of rsagl rssoc and rssvp were in the flower. the relative expression levels of rsagl rssoc rssvp and rsvrn were significantly higher in the leaf than in the other three tissues, whereas rsap was mainly expressed in the flower. the transcript levels of rsfpf and rsld were higher in leaf and flower, and no significant difference was detected in expression of these two tissues. the results indicate that these genes may play different roles in various tissues and thus affect the growth and development of radish. the expression pattern differences of these genes were also observed in previous studies.fig. the spatial-temporal expression profiling of bolting and flowering-related genes by rt-qpcr analysis. a the expression patterns of seven genes in different radish tissues. b the expression patterns of seven genes at vegetative stage and reproductive stage. each bar shows the mean ± se of triplicate assays. the values with different letters indicate significant differences at p <  according to duncan’s multiple range tests the expression profiling of these seven bolting and flowering-related genes also revealed that their expression patterns in radish leaves varied at vegetative stage and reproductive stage, which contributed to further explore their possible roles during radish bolting and flowering. the expression levels of rsagl and rssoc were lower at vegetative stage and increased at reproductive stage; whereas the expression levels of these four genes including rsap rsfpf rssvp and rsvrn were relative higher at vegetative stage and significantly decreased at reproductive stage. the up- or down-expression of these genes were in agreement with previous analysis in other species. flowering pathway analysis indicated that seven selected genes were implicated in different flowering pathways. among them, ap belonging to photoperiod pathway is regulated by mir and represses the bolting and flowering; while soc as a floral integrator negatively regulates flc expression and plays vital roles in promoting flowering. these findings revealed that these differentially expressed genes might participate in the transition from vegetative stage to bolting and flowering in radish. additionally, four flower development-related genes including rsap rssep rsagl and rslfy, were selected for rt-qpcr analysis to explore their expression patterns during radish flower development and in different flower parts. the expression levels of rssep rsagl and rslfy were relative higher at whole flower, while no significant difference of rsap expression was showed at three stages of flower development. the expression analysis revealed that four genes were differentially expressed in four flower parts, which was in consistent with previous studies, suggesting that these genes may play important roles in flower organ identity. importantly, ap belonging to a-class genes in the abc model of flower organ identity, determines the development of sepal and petal. the results showed that rsap expression level was relative higher in sepal and petal than in stamen and pistil, suggesting that ap gene is essential for sepal and petal development and specify the identity of the floral meristem.fig. the spatial-temporal expression profiling of flower development-related genes by rt-qpcr analysis. a the relative expression levels of four genes at three stages of flower development. b the relative expression levels of four genes at different flower parts. each bar shows the mean ± se of triplicate assays. the values with different letters indicate significant differences at p <  according to duncan’s multiple range tests  to our knowledge, this is the first report on de novo transcriptome sequencing and assembly in late-bolting radish leaves using rna-seq technology. a total of unigenes were assembled in radish leaf transcriptome and unique sequences were successfully annotated by go, cog and kegg databases. a total of candidate genes were involved in plant circadian rhythmic pathway. moreover, potential radish genes related to bolting and flowering-time regulation were identified by blast similarity search with a. thaliana flowering-related genes. rt-qpcr analysis revealed that several flowering-related genes showed spatial-temporal expression patterns in various radish tissues and at different development stages. the findings from this study provided insights into bolting and flowering-time regulatory networks in radish, and facilitated further dissecting molecular genetic mechanisms associated with bolting and flowering regulation in brassicaceae vegetable crops. 
these results could provide a foundation for further investigating bolting and flowering regulatory networks in radish, and facilitate dissecting molecular genetic mechanisms underlying bolting and flowering in brassicaceae vegetable crops. in this study, to generate dataset of radish unigene sequences for large-scale gene discovery and functional pathway identification, a cdna library from mixed radish leaves at different developmental stages was subjected to high-throughput rna sequencing. among these, unigenes were successfully annotated by blast searching against the public protein databases. although several flowering regulatory pathways had been described in some plant species, no study on genetic networks of bolting and flowering regulation was performed in radish. functional classification and annotation indicated that and unique sequences were assigned to go terms and cog categories, respectively. kegg pathway analysis revealed that unigenes were classified into functional pathways, among which candidate genes related to plant circadian rhythm were identified. a total of million clean reads and contigs representing unigenes were obtained from the radish leaf transcriptome. in addition, seven critical bolting and flowering-related genes were isolated and profiled by t-a cloning and rt-qpcr analysis. the appropriate timing of bolting and flowering is pivotal for reproductive success in brassicaceae crops including radish.
 the appropriate timing of bolting and flowering is pivotal for reproductive success in brassicaceae crops including radish. although several flowering regulatory pathways had been described in some plant species, no study on genetic networks of bolting and flowering regulation was performed in radish. in this study, to generate dataset of radish unigene sequences for large-scale gene discovery and functional pathway identification, a cdna library from mixed radish leaves at different developmental stages was subjected to high-throughput rna sequencing.  a total of million clean reads and contigs representing unigenes were obtained from the radish leaf transcriptome. among these, unigenes were successfully annotated by blast searching against the public protein databases. functional classification and annotation indicated that and unique sequences were assigned to go terms and cog categories, respectively. kegg pathway analysis revealed that unigenes were classified into functional pathways, among which candidate genes related to plant circadian rhythm were identified. moreover, potential bolting and flowering-related genes involved in various flowering pathways were identified. in addition, seven critical bolting and flowering-related genes were isolated and profiled by t-a cloning and rt-qpcr analysis. finally, a schematic network model of bolting and flowering regulation and pathways was put forward in radish.  this study is the first report on systematic identification of bolting and flowering-related genes based on transcriptome sequencing and assembly in radish. these results could provide a foundation for further investigating bolting and flowering regulatory networks in radish, and facilitate dissecting molecular genetic mechanisms underlying bolting and flowering in brassicaceae vegetable crops. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords radishde novo assemblybolting and floweringtranscriptomeflowering regulatory networkhttp://dx.doi.org/ /501100001809national natural science foundation of china 31372064liu liwang national key technologies r & d program of china2012bad02b01liu liwang key technologies r & d program of jiangsu province, chinabe2013429liu liwang issue-copyright-statement© the author 2016 