BACKGROUND
chrysanthemum  is a popular ornamental plant worldwide  <cit> .
chrysanthemum plants are susceptible to damage when exposed to prolonged periods of low temperature; therefore, improving their tolerance to cold stress is perceived as an important breeding goal.
the high chromosome number and polyploidy of major ornamental species complicate the genetics and capacity for gene discovery  <cit> .
hence, c. nankingense has been considered as a convenient genomic model due to its simple diploid nature.
in addition, it displays better tolerance to low temperature as compared to the ornamental polyploid species  <cit> .
temperature is a major determinant of the geographical distribution and length of the growing season in most plant species  <cit> .
however, episodes of low temperature during the growing season cause a substantial loss in the yield of many temperate crops.
during chrysanthemum production, specifically in china, extreme low temperatures in early spring and winter, unusual freezing temperatures during late cold spring, and sudden frosts in fall often lead to growth arrest and block flower buds or inflorescence, which in turn result in significant economic losses every year  <cit> .
temperate plant species can acquire the ability to withstand a prolonged period of sub-zero temperature if they are previously exposed to a period of low temperature above 0°c; this phenomenon is known as low temperature acclimation  <cit> .
however, the molecular basis of cold acclimation  and low temperature/freezing tolerance in chrysanthemum has not yet been explored.
in the present study, we aim to fish out candidate genes underlying the process of ca and response to low /freezing temperature, which will help to elucidate the molecular basis of the cold response in c. nankingense, and then improve the chrysanthemum varieties cold tolerance.
in arabidopsis thaliana and some of the winter cereals, a variety of physiological, biochemical, and molecular changes are known to occur during the low temperature acclimation process  <cit> .
in these species, the physical state of the plasma membrane has been shown to be an important determinant of the plant's ability to sense changes in the air temperature .
membrane rigidification leads to an increase in the cytosolic concentration of the ca2+ ion  <cit> , which is regarded as a major regulator of low temperature responsive factor.
certain ca2+-dependent protein kinases have also been recognized as positive regulators  <cit> .
the mitogen-activated protein kinase  cascade participates in the low temperature signalling and low temperature tolerance  <cit> .
three candidate chilling response genes, encoding mapkkk , clc-d  and rlk  homologues are all up-regulated following chilling stress in maize  <cit> .
the cbf low temperature-response pathway is well conserved across a diversity of species and is a significant component of tolerance to sub-zero temperatures  <cit> .
the cbf pathway is positively regulated by the circadian clock components cca <dig> and lhy  <cit> , while the inducer of cbf expression  <dig>  protein acts upstream of cbf in the low temperature response pathway  <cit> .
a high expression of osmotically responsive gene   <dig> acts as a negative regulator of the low temperature response  <cit> .
salicylic acid  is also involved in the response to low temperature stress  <cit> .
rna processing and nucleocytoplasmic transport play crucial roles in plant stress  <cit> .
the rna-seq platform has become a key technology for quantifying the transcriptional response in nonmodel organisms or those with genome characteristics extremely difficult to whole-genome sequencing  <cit> .
in tea , this approach has enabled the recognition of  <dig>  differentially transcribed genes  induced during low temperature acclimation  <cit> .
a similar transcriptomic analysis of jatropha curcas identified over  <dig>  genes as being up- or down-regulated by low temperature  <cit> .
while in anthurium sp., the method of digital gene expression enabled  <dig> low temperature-inducible transcription factors  to be identified  <cit> .
here, the rna-seq platform based on illumina ngs technology was used to characterize the transcriptomic response to low temperature by comparing the different transcriptome of c. nankingense plants subjected to periods of sub-zero temperature with or without a prior low temperature acclimation, with a view to gaining a deeper insight into the molecular basis of this physiological adaptation.
in addition, the identified candidate genes will be useful for improving adaptation to low temperature and enhancing productivity and geographical distribution.
RESULTS
rna-seq libraries and reads mapping
an overview of the rna-seq reads derived from the six libraries using illumina hiseq™  <dig> platform is presented in table  <dig> and additional file 1: figure s <dig>  the raw sequence data have been deposited in the ncbi sequence read archive .
the number of clean reads per library ranged from  <dig>  to  <dig>  million, and the total number of nucleotides sequenced from  <dig> , <dig> to  <dig> , <dig> .
the proportion of clean reads was over 99% in each library .
overall, from library a to library ck,  <dig> , <dig>   <dig> , <dig>   <dig> , <dig>   <dig> , <dig>   <dig> , <dig>  and  <dig> , <dig> clean reads were obtained respectively.
correspondingly,  <dig> , <dig>   <dig> , <dig>   <dig> , <dig>   <dig> , <dig>   <dig> , <dig>  and  <dig> , <dig> total base-pairs  were generated.
the clean reads were mapped onto a reference gene database, which included all known c. nankingense unigene sequences.
raw sequence data were deposited in the ncbi sequence read archive database under the accession number srp <dig>  the proportion of unambiguously mapped reads per library ranged from  <dig> % in library c <dig> to  <dig> % , and the proportion of unique matches from  <dig> %  to  <dig> % .
as the number of reads increased, the identification rate of new genes slowed, which indicated the saturation around seven million reads  .table  <dig>
summary of mapping result
quantification of transcripts and identification of differentially transcribed genes
the quality of the rna-seq dataset is assessed by gene coverage, which is the percentage of a gene covered by reads.
this value is determined as the ratio of the base number in a gene covered by unique mapping reads to the total bases number of that gene.
the distribution of the six libraries was presented in additional file 3: figure s <dig>  in addition, transcript abundances for each gene  were calculated according to the method following mortazavi et al.
<cit> .
moreover, differential transcription was identified through pair-wise comparison between various libraries, by setting a threshold fdr of  <dig>  and a│log <dig> ratio│ of  <dig> based on the algorithm developed by audic et al.
<cit> .
from the seven comparisons, including treatment cka , ckb <dig> , ckb <dig> , ckc <dig> , ckc <dig> , ac <dig>  and ac <dig>  the results showed that a large number of dtgs were identified .
the number of dtgs detected was as follows: treatment cka,  <dig>  ; ckb <dig>   <dig> ; ckb <dig>   <dig> ; ckc <dig>   <dig>  ; ckc <dig>   <dig>  ; ac <dig>   <dig> ; and ac <dig>   <dig>  .
these results indicated that more dtgs were identified in the treatments, which underwent a prior ca , as compared to the treatments which didn’t undergo ca .
in addition, a smaller number of dtgs was found in a vs c <dig> and a vs c <dig> comparisons than in ck vs b <dig> and ck vs b <dig>  moreover, on extending the freezing treatment, fewer dtg were detected in a vs c <dig> comparison than in a vs c <dig>  however, contrary results were obtained in case of ck vs b <dig> and ck vs b <dig> comparisons.
based on the assumption that genes with similar expression patterns usually exhibit functional correlation, the consistency of the dtgs was checked by multiple comparisons clustering among the cka, ckc <dig> and ckc2; between the ckb <dig> and ckb <dig> treatments; and between the comparisons, a vs c <dig> and a vs c <dig>  a total of  <dig>  dtgs were observed in the first multiple comparison clustering, out of which, only three genes behaved inconsistently .
of the  <dig>  consistent dtgs,  <dig>  were up- and  <dig> were down-regulated .
in the second multiple comparison clustering,  <dig> dtgs  were obtained , and all were found to be consistent.
in the third comparison clustering between a vs c <dig> and a vs c <dig>   <dig> dtgs  showed consistency .figure  <dig>
the numbers of dtgs identified in comparisons between pairs of libraries.
go classification of differentially transcribed genes
in the treatment cka,  <dig>  of the  <dig>  dtgs could be assigned a go term; the equivalent number for other comparisons were as follows: treatment ckb <dig>  155/337; ckb <dig>  246/718; ckc <dig>   <dig> /3722; ckc <dig>   <dig> /4119; a vs c <dig>  60/194; and a vs c <dig>  30/ <dig> .
for ck vs a,  <dig> go classes fell into the categories “biological process”,  <dig> into “cellular component” and  <dig> into “molecular function”.
the equivalent distribution in ck vs b <dig> was  <dig>   <dig>  and 7; in ck vs b <dig>   <dig>   <dig> and 9; in both, ck vs c <dig> and ck vs c <dig>   <dig>   <dig>  and 11; in a vs c <dig>   <dig>   <dig>  and 5; and in a vs c <dig>   <dig>   <dig>  and  <dig>  the major classes of biological process among the dtgs in ck vs a comparison were “metabolic process”, “cellular process”, “single organism process”, “response to stimulus”, “localization”, “establishment localization”, “biological regulation” and “regulation of biological process”; the predominant cellular components were “cell”, “cell part”, “organelle” and “organelle part”; and for molecular function “binding”, “catalytic activity”, “transporter activity”, “nucleic acid binding transcription factor activity” and “antioxidant activity”.
only a few genes belonged to the categories “cell killing”, “electron carrier activity”, “positive regulation of biological process”, “extracellular matrix”, “receptor activity”, “cell junction”, “protein binding transcription factor activity” and “carbon utilization”.
the details of go classification of dtgs in ck vs a, and other comparisons are presented in figure  <dig>  plant hormone signal transduction pathways  were well represented, particularly those associated with auxin-mediated signalling.
low temperature sensing and signalling genes influenced by ca2+, as well as other protein kinases were also identified.
a number of tf families, genes encoding functional proteins and post-translational regulated genes were represented.figure  <dig>
gene ontology  classification of the dtgs identified in each comparison between a pair of libraries.
dtgs were annotated in three categories: biological process, cellular component and molecular function.
y-axis  represents the number of dtgs in each category; y-axis  represents the percentage of a specific category of dtgs within that main category.
panels a, b, c, d, e, f and g  represents dtgs in the comparison between library ck  and a    , library ck and b <dig>   , library ck and b <dig>   , library ck and c <dig>   , library ck and c <dig>   , library a and c <dig>  , and library a and c <dig>   respectively.
function annotation of dtgs using the kegg database
unigene kegg annotation was aimed at dtgs from the above comparisons.
in the ck vs a comparison,  <dig>  dtgs were assigned to the kegg database involved  <dig> pathways; for ck vs b <dig>   <dig> dtgs were assigned to  <dig> pathways; for ck vs b <dig>   <dig> dtgs were assigned to  <dig> pathways; for b <dig> vs b <dig>   <dig> dtgs were assigned to  <dig> pathways; for ck vs c <dig>   <dig>  dtgs were assigned to  <dig> pathways; for ck vs c <dig>   <dig>  dtgs were assigned to  <dig> pathways; for a vs c <dig>   <dig> dtgs were assigned to  <dig> pathways; and for a vs c <dig>   <dig> dtgs were assigned to  <dig> pathways.
the details of the kegg classification of the above comparisons are presented in additional file 16: table s <dig>  the major pathways identified were “metabolism”, “biosynthesis of secondary metabolites”, “hormone signal transduction”, “plant-pathogen interaction”, “spliceosome”, “phenylpropanoid biosynthesis”, “endocytosis” and “protein processing in the endoplasm”, “ribosome” and “starch and sucrose metabolism”.
genes involved in the response to low temperature
more dtgs were identified in the ck vs a, ck vs c <dig> and ck vs c <dig> comparisons than in the ck vs b <dig> and ck vs b <dig>  this observation demonstrated that many genes were involved in low temperature acclimation.
the dtgs revealed in the comparisons, a vs c <dig> and a vs c <dig>  were found to be involved in response to freezing treatment, when the plants were exposed to prior ca.
however, the dtgs obtained from the ck vs b <dig> and ck vs b <dig> comparisons participated in the response mechanism, when the plants didn’t undergo prior ca.
some of all the dtgs belonged to the group of genes involved in cold sensing and signal transduction pathways.
ca2+ signaling pathway, which is regarded as an important signal sensing and transduction pathway under stresses, includes many members, such as cation/calcium exchangers , calcium-binding proteins , calmodulin-like proteins , cbl-interacting protein kinases , calcium-dependent protein kinases  and calmodulin-binding receptor-like kinases .
the ck vs a comparison included three ccx, two cbp, five cml, three cipk, and one cbrlk genes; the equivalent order for the ck vs c <dig> comparison was, respectively, three, one, five, five and one; for ck vs c <dig>  three, zero, four, four and one; for ck vs b <dig>  zero, one, three, zero and zero, along with one sodium/calcium exchanger family protein gene ; for ck vs b <dig>  zero, zero, five, two, zero, along with one sce and one cdpk; for a vs c <dig>  zero, one, two, one, zero; and for a vs c <dig>  only one cbp was involved.
in the comparisons, ck vs a, ck vs c <dig>  and ck vs c <dig>  three ccx , one cml , and one cdpk  were all found to be up-regulated; in addition, two cmls , and one cdpk  show-ed down-regulation.
one cml  was found to be up-regulated in both comparisons, ck vs b <dig> and ck vs b <dig>  these findings indicated that the genes related to ca2+ signaling pathway played an essential role in the phenomenon of ca and freezing response without a prior ca; the complete details of dtgs involved in ca2+ signalling pathway are presented in table  <dig>  no dtgs associated with the mapk cascade was identified in the ck vs a comparison.
in contrast, one mapkkk and one mapk gene were found to be differentially transcribed in ck vs c <dig>  and one mapkkk was represented in ck vs c <dig> as well as in ck vs b <dig>  four mapkkk genes were observed in the ck vs b <dig> comparison, and one mapkkk gene was identified in both, a vs c <dig> and a vs c <dig>  the results showed that mapk cascade, especially mapkkk genes, participated in the plant’s response to freezing treatment, instead of ca.
therefore, all the dtgs involved in mapk cascade were found to be up-regulated .
a higher number of genes encoding serine/threonine-protein kinases were differentially transcribed in ck vs a, ck vs c <dig> and ck vs c <dig> than in ck vs b <dig> or ck vs b <dig> .
however, no dtg encoding this specific kinase was identified in the a vs c <dig> or a vs c <dig> comparisons.table  <dig>
the differential gene expression of genes involved in ca
2+
signalling pathway in each comparison
in the present study, members of various low tem-perature-responsive transcription factor  families were identified; and  <dig>   <dig> and  <dig> such genes were found to be differentially transcribed in the comparisons, ck vs a, ck vs c <dig> and ck vs c <dig>  respectively.
the major tf families presented were ap2/erf, bhlh, wrky and tcp, along with small numbers of myb, myc, nac, dof, and the trihelix family.
the ck vs b <dig> and ck vs b <dig> comparisons showed  <dig> and  <dig> tf dtgs, respectively.
the number of tf dtgs identified in the ck vs b <dig> or ck vs b <dig> comparison was lesser than the comparisons, ck vs a, ck vs c <dig>  or ck vs c <dig>  which were involved in the process of low temperature acclimation.
a larger number of tf dtgs were present in ck vs b <dig> than in the ck vs b <dig> comparison.
in addition, five  and seven tf dtgs  were found in the a vs c <dig> and a vs c <dig> comparisons, respectively.
the identified tf dtgs from the a vs c <dig> and a vs c <dig> comparisons, were involved in response to freezing treatment in plants with prior exposure to ca.
the differently transcribed tfs of the cka treatment, the a vs c <dig>  and a vs c <dig> comparisons are presented in table  <dig> table  <dig>
the differential gene expression of transcription factors  in each comparison
other relevant classes of protein, which featured as dtgs products, were dehydrin, lea  proteins, heat shock proteins .
the proteins involved in post-transcriptional regulation, such as ribosomal proteins and a dead-box atp-dependent rna helicase , were particularly found in the ck vs a, ck vs c <dig> and ck vs c <dig> comparisons.
yet, with respect to the above classes of proteins, only one lea was found in both, a vs c <dig> and a vs c <dig>  while two hsps were identified in the a vs c <dig> comparison.
the details of classes of protein of the cka treatment are presented in table  <dig> table  <dig>
the differential gene expression of genes encoding lea protein, hsps, and rna helicase in treatment cka
lea
hsps
verification of differential transcription using quantitative real time pcr
to further verify the expression profiles of genes in our illumina rna-seq results, we have performed a selection of  <dig> dtgs for their key roles in response to low temperature by qrt-pcr, these incorporated genes encoding serine/threonine-protein kinase, lea protein, dehydrin, a gibberellin-regulated protein, a jasmonate zim-domain protein, and a dead-box atp-dependent rh, along with a selection of tfs .
the qpcr outcomes in each case correlated closely with the transcript abundances estimated from the rna-seq output .figure  <dig>
qpcr validation for  <dig> dtgs identified by rna-seq in the comparison between ck and a.
discussion
global patterns of transcription in response to low temperature
the information available on the molecular basis of the response of chrysanthemum to low temperature is still meagre.
however, the development of next-generation sequencing technology provides a straightforward method for the identification of genes involved in this process, and we can try to elaborate the molecular mechanism underlying the response to low temperature.
over 77% of the reads in each of the six rna-seq libraries corresponded with known transcripts , a proportion which was as high as that achieved in a similar study of anthurium
<cit> .
the less than 23% of reads were unmapped probably as a result of unidentified transcripts  <cit> .
around  <dig>  dtgs were identified in each of the cka, ckc <dig> and ckc <dig> treatments .
as was also the case for camellia sinensis
<cit> , the majority of dtgs involved up- rather than down-regulation by the stress.
in both species, many of the dtgs comprised genes associated with low temperature sensing or signal transduction, low temperature-responsive tfs, stabilization of the plasma membrane and osmosensing-responsiveness.
low temperature sensing and signaling genes
low temperature stress-induced signals are directed to various pathways  <cit> .
ca2+ is well recognized as a messenger in stress signalling  <cit> , and is sensed by proteins of three main classes: cdpks, cams and cbls  <cit> .
a further important pathway is the mitogen-activated protein kinases  cascade  <cit> , while a number of receptor-like protein kinases  are known to be responsible for perceiving changes in the external environment and transducing the appropriate signal  <cit> .
the arabidopsis and rice genomes harbor, respectively,  <dig> and  <dig> cml-encoding genes  <cit> .
in rice, the transcription of the calmodulin-like osmsr <dig> gene is significantly up-regulated by a series of stresses including low temperature in different tissues at different developmental stages, and its heterologous expression in a. thaliana has suggested that the gene affects salinity and drought tolerance in an aba-dependent manner  <cit> .
in camellia sinensis exposed to low temperature, five calmodulin genes, two cdpk genes and one cbl gene were identified to be involved in signal transduction  <cit> .
here, five unigenes resembling cml  were identified as significant dtgs in the cka treatment, and three of these were also differentially transcribed in both the ckc <dig> and ckc <dig> treatments.
cbl-interacting protein kinases , which specifically interact with cbls, are thought to act as sensors since they lack any enzymatic activity  <cit> .
three dtgs with homology to cipk  were identified in the cka treatment.
both the ckc <dig> and ckc <dig> treatments also featured the same two dtgs , as well as the other three cipk homologues  in the former treatment, and two cipk homologues  in the latter.
a number of cdpks have been proven to participate in rapid abiotic stress and immune signaling responses  <cit> .
transgenic arabidopsis heterologously expressing the populus euphratica gene pecpk <dig> show an enhanced level of freezing tolerance, perhaps through the transgene's enhancement of the transcript abundance of the abiotic stress-responsive genes rd29b and cor15a
<cit> .
only one cdpk homologue  was identified as a dtg in the ckb <dig> treatment, but no other homologues featured as dtgs in any of the other treatments.
however, in a vs c <dig> comparison, with respect to ca2+ signalling pathway, only two cml genes  and one cipk gene  were identified as dtgs.
four mapkkk genes  were differently transcribed in the ckb <dig> treatment.
unigene36211_s2_ <dig> also featured in the ckb <dig> and ckc <dig> treatments, while a fifth mapk gene  was found to transcribe differently in the ckc <dig> treatment.
no one gene of mapk cascade was identified in the cka treatment.
in addition, many dtgs encoding serine/threonine-protein kinases were found in the cka, ckc <dig> and ckc <dig> treatments; however, no gene was detected in the comparisons, a vs c <dig> and a vs c <dig>  these findings provided evidence for the crucial role of ca2+ in the low temperature acclimation process in c. nankingense, and further proved that the mapk pathway and serine/threonine-protein kinases are more strongly involved in the response to freezing.
major classes of tf involved in the response to low temperature
transcriptional regulation of stress-responsive genes is a vital component of the response to both abiotic and biotic stress  <cit> .
five major tf classes  were identified as dtgs in the treatments involved in a process of low temperature acclimation.
the ap2/erf family, which a large group of plant-specific transcription factors, has been sub-divided into ap <dig>  rav, erf and dreb tfs  <cit> .
drebs control the aba-independent transcription of low temperature responsive genes in a. thaliana
<cit> ; the atdreb <dig> sub-family harbors six members  <cit> , of which dreb1a/cbf <dig>  dreb1b/cbf <dig> and dreb1c/cbf <dig> are the ones which respond most rapidly to low temperature.
a. thaliana plants constitutively expressing any one of these tfs display a heightened tolerance to freezing, drought and salinity  <cit> .
the dreb2b tf present in the desert-adapted plant eremosparton songoricum has been shown to enhance the tolerance of both yeast and tobacco against a variety of abiotic stresses.
the constitutive expression in tobacco of esdreb2b promotes the accumulation of proline in response to abiotic stress   <cit> .
here, the dreb <dig> homologue unigene73473_s2_ <dig> was up-regulated upon exposure to low temperature acclimation.
it was speculated that the dreb <dig> may be involved in the accumulation of proline in response to low temperature.
it is well known proline accumulates in many plant species in response to environmental stress  <cit> .
the constitutive expression of an ap <dig> tf has been shown to improve the tolerance of a. thaliana to low temperature, as well as to drought and high temperature  <cit> .
here, three ap2-like genes  were also all up-regulated by ca.
a number of other tfs, belonging to the wrky, bhlh, tcp, myb, myc, trihelix and b-zip families were also among the dtgs identified in treatments involving low temperature acclimation.
the class of b-zip transcription factors, abre binding proteins , can bind to abre and activate aba-dependent gene expression when plants are exposed to low temperature  <cit> .
many researches have also indicated that three families of transcription factors: wrky, bhlh and myb closely related to plant cold stress .
five of six wrkys, which were detected as dtgs, were down-regulated in ck vs a comparison.
however, three wrkys were all up-regulated in a vs c <dig>  and no wrky as dtg was found in a vs c <dig>  these findings suggested that wrky family played essential and different roles in ca and early freezing response.
most erfs also showed down-regulated in ck vs a comparison, while, one erf was detected up-regulated as dtg in a vs c1; on extending the freezing treatment, five erfs as dtgs were all up-regulated in a vs c <dig>  the erfs performing under different temperature treatment showed its roles in ca and cold tolerance.
few reports to date have indicated that tcp transcription factors involve in the response to cold stress.
low temperature responsive genes related to post-transcriptional regulation
post-transcriptional regulation  has been implicated in the process of low temperature acclimation  <cit> .
dead-box rhs are intimately associated with rna-mediated processes and are related to various rna metabolism events, including rna synthesis to rna degradation by means of catalyzing the atp-dependent unwinding of local rna secondary structures  <cit> .
the transcription of the genes encoding these proteins is known to be regulated by stress in both bacteria and plants .
in a. thaliana, rh <dig> has been associated with enhanced freezing tolerance, probably through its function as an rna chaperone  <cit> .
the product of rcf <dig>  a low temperature-inducible rh gene, is important for low temperature-responsive gene regulation and low temperature tolerance in plants through maintenance of normal pre-mrna splicing instead of regulating mrna export like a previously reported dead-box rh  regulates mrna export  <cit> .
it has been demonstrated that the functional roles and rna chaperone activity related to intron splicing in mitochondrial and chloroplast .
here, respectively, eleven, nine and twelve genes encoding dead-box rhs were up-regulated in the cka, ckc <dig> and ckc <dig> treatments, but none were differentially transcribed in either the ckb <dig> or the ckb <dig> treatment; this was taken to imply that dead-box rhs are activated during the process of low temperature acclimation.
in future, a major task is to clear how rna chaperones recognize substrate rnas and how they work with other proteins to regulate post-transcriptional rna metabolism in response to developmental and environmental condition  <cit> .
genes encoding functional proteins
besides protein factors involved in further regulation of signal transduction and gene expression such as transcription factor and protein kinase that probably function in stress response, various functional proteins featured as products of the dtgs, in particular, lea protein and dehydrin, lea proteins are accumulated during the late stage of seed maturation and under moisture deficient conditions, and act to protect higher plants from damage caused by abiotic stress.
when the maize gene zmlea <dig> is expressed in tobacco and yeast, it improves the plant's level of tolerance against both osmotic and oxidative stress  <cit> .
the wheat lea protein gene wci <dig> expressed heterologously in a. thaliana enhances freezing tolerance  <cit> .
here, seven lea protein genes were among the dtgs identified in the cka treatment, five in both the ckc <dig> and ckc <dig> treatment, none were present in either the ckb <dig> or the ckb <dig> treatment, and only one was identified in both comparisons of a vs c <dig> and a vs c <dig>  the implication was that lea proteins probably enhance low temperature tolerance through their participation in the low temperature acclimation process.
dehydrins constitute a group of plant proteins involved in tolerance to low temperature and drought  <cit> .
the thranscrips of genes encoding three dehydrins in e. globulus accumulate strongly in the stem and leaf tissue of acclimated plants, compared to non-acclimated  <cit> .
here, four genes encoding dehydrins were identified as dtgs in the cka, ckc <dig> and ckc <dig> treatments.
but none in either the ckb <dig> or ckb <dig> treatments, suggesting that, as with the lea protein genes, their contribution to low temperature tolerance is expressed during the low temperature acclimation process.
hsps were initially identified from their involvement in the response to high temperatures, but it is now recognized that many of them also respond to low temperature.
it is well known hsps can protect plants against stress by means of reestablishing normal protein conformation and thus cellular homeostasis.
five major hsp families have been conservely defined, based on their molecular weight: hsp100s, hsp90s, hsp70s, hsp60s and small hsps.
five hsp90-encoding dtgs  featured in the cka, ckc <dig> and ckc <dig> treatments.
hsp90s function as molecular chaperones during signal transduction, cell cycling, the stress response, and in protein folding, degradation and transport  <cit> .
in a. thaliana, expression of hsp <dig> is developmentally regulated, but it also responds to high and low temperature, as well as salinity  <cit> .
it is speculated that hsp <dig> as molecular chaperones play an important role in signal transduction and stress management in c. nankinginse.
a gene hsp70- encoding dtgs  featured in the cka, ckb <dig>  ckc <dig> and ckc <dig> treatments, which not found in the ckb <dig> treatment; and a number of small hsps, belonging to the  <dig> ,  <dig>  and  <dig>  families were also among the dtgs identified in treatments involving low temperature response.
in conclusion, it was clear that a large number of genes were induced when exposed to low temperature.
the complex gene networks involved a set of interactions between cold sensing and signaling genes, genes related to post-transcription regulation responding to cold stress, transcription factors and certain functional proteins.
in the present study, the dtgs identified as candidate genes in response to low temperature, require further investigation for a complete understanding of the molecular basis of cold response in c. nankingense.
this information will prove beneficial in molecular breeding programs for excellent chrysanthemum varieties.
CONCLUSIONS
an overview of the many changes to the c. nankingense transcriptome induced by exposure to low temperature has been provided.
many of the dtgs identified were involved in predictable classes of gene, encoding, for example, low temperature sensors and signaling molecules, tfs and certain functional proteins associated with the stress response.
the large number of dtgs identified confirms the complexity of the regulatory machinery involved in low temperature acclimation and low temperature/freezing tolerance.
establishing which of these many dtgs reflect the primary response to low temperature is necessary before the molecular basis of the response can be fully elaborated; such genes would represent candidates for intervention in the breeding of chrysanthemum varieties endowed with a heightened tolerance to low temperature stress.

 chrysanthemum is a popular ornamental plant worldwide. chrysanthemum plants are susceptible to damage when exposed to prolonged periods of low temperature; therefore, improving their tolerance to cold stress is perceived as an important breeding goal. the high chromosome number and polyploidy of major ornamental species complicate the genetics and capacity for gene discovery. hence, c. nankingense has been considered as a convenient genomic model due to its simple diploid nature. in addition, it displays better tolerance to low temperature as compared to the ornamental polyploid species. temperature is a major determinant of the geographical distribution and length of the growing season in most plant species. however, episodes of low temperature during the growing season cause a substantial loss in the yield of many temperate crops. during chrysanthemum production, specifically in china, extreme low temperatures in early spring and winter, unusual freezing temperatures during late cold spring, and sudden frosts in fall often lead to growth arrest and block flower buds or inflorescence, which in turn result in significant economic losses every year. temperate plant species can acquire the ability to withstand a prolonged period of sub-zero temperature if they are previously exposed to a period of low temperature above 0°c; this phenomenon is known as low temperature acclimation. however, the molecular basis of cold acclimation and low temperature/freezing tolerance in chrysanthemum has not yet been explored. in the present study, we aim to fish out candidate genes underlying the process of ca and response to low /freezing temperature, which will help to elucidate the molecular basis of the cold response in c. nankingense, and then improve the chrysanthemum varieties cold tolerance. in arabidopsis thaliana and some of the winter cereals, a variety of physiological, biochemical, and molecular changes are known to occur during the low temperature acclimation process. in these species, the physical state of the plasma membrane has been shown to be an important determinant of the plant's ability to sense changes in the air temperature. membrane rigidification leads to an increase in the cytosolic concentration of the ca2+ ion, which is regarded as a major regulator of low temperature responsive factor. certain ca2+-dependent protein kinases have also been recognized as positive regulators. the mitogen-activated protein kinase cascade participates in the low temperature signalling and low temperature tolerance. three candidate chilling response genes, encoding mapkkk, clc-d and rlk homologues are all up-regulated following chilling stress in maize. the cbf low temperature-response pathway is well conserved across a diversity of species and is a significant component of tolerance to sub-zero temperatures. the cbf pathway is positively regulated by the circadian clock components cca and lhy, while the inducer of cbf expression protein acts upstream of cbf in the low temperature response pathway. a high expression of osmotically responsive gene acts as a negative regulator of the low temperature response. salicylic acid is also involved in the response to low temperature stress. rna processing and nucleocytoplasmic transport play crucial roles in plant stress. the rna-seq platform has become a key technology for quantifying the transcriptional response in nonmodel organisms or those with genome characteristics extremely difficult to whole-genome sequencing. in tea, this approach has enabled the recognition of differentially transcribed genes induced during low temperature acclimation. a similar transcriptomic analysis of jatropha curcas identified over genes as being up- or down-regulated by low temperature. while in anthurium sp., the method of digital gene expression enabled low temperature-inducible transcription factors to be identified. here, the rna-seq platform based on illumina ngs technology was used to characterize the transcriptomic response to low temperature by comparing the different transcriptome of c. nankingense plants subjected to periods of sub-zero temperature with or without a prior low temperature acclimation, with a view to gaining a deeper insight into the molecular basis of this physiological adaptation. in addition, the identified candidate genes will be useful for improving adaptation to low temperature and enhancing productivity and geographical distribution.  rna-seq libraries and reads mapping an overview of the rna-seq reads derived from the six libraries using illumina hiseq™ platform is presented in table  and additional file 1: figure s the raw sequence data have been deposited in the ncbi sequence read archive. the number of clean reads per library ranged from to million, and the total number of nucleotides sequenced from, to,. the proportion of clean reads was over 99% in each library. overall, from library a to library ck,,,,,, and, clean reads were obtained respectively. correspondingly,,,,,, and, total base-pairs were generated. the clean reads were mapped onto a reference gene database, which included all known c. nankingense unigene sequences. raw sequence data were deposited in the ncbi sequence read archive database under the accession number srp the proportion of unambiguously mapped reads per library ranged from % in library c to %, and the proportion of unique matches from % to %. as the number of reads increased, the identification rate of new genes slowed, which indicated the saturation around seven million reads.table summary of mapping result quantification of transcripts and identification of differentially transcribed genes the quality of the rna-seq dataset is assessed by gene coverage, which is the percentage of a gene covered by reads. this value is determined as the ratio of the base number in a gene covered by unique mapping reads to the total bases number of that gene. the distribution of the six libraries was presented in additional file 3: figure s in addition, transcript abundances for each gene were calculated according to the method following mortazavi et al.. moreover, differential transcription was identified through pair-wise comparison between various libraries, by setting a threshold fdr of and a│log ratio│ of based on the algorithm developed by audic et al.. from the seven comparisons, including treatment cka, ckb, ckb, ckc, ckc, ac and ac the results showed that a large number of dtgs were identified. the number of dtgs detected was as follows: treatment cka,; ckb; ckb; ckc; ckc; ac; and ac. these results indicated that more dtgs were identified in the treatments, which underwent a prior ca, as compared to the treatments which didn’t undergo ca. in addition, a smaller number of dtgs was found in a vs c and a vs c comparisons than in ck vs b and ck vs b moreover, on extending the freezing treatment, fewer dtg were detected in a vs c comparison than in a vs c however, contrary results were obtained in case of ck vs b and ck vs b comparisons. based on the assumption that genes with similar expression patterns usually exhibit functional correlation, the consistency of the dtgs was checked by multiple comparisons clustering among the cka, ckc and ckc2; between the ckb and ckb treatments; and between the comparisons, a vs c and a vs c a total of dtgs were observed in the first multiple comparison clustering, out of which, only three genes behaved inconsistently. of the consistent dtgs, were up- and were down-regulated. in the second multiple comparison clustering, dtgs were obtained, and all were found to be consistent. in the third comparison clustering between a vs c and a vs c dtgs showed consistency.figure the numbers of dtgs identified in comparisons between pairs of libraries. go classification of differentially transcribed genes in the treatment cka, of the dtgs could be assigned a go term; the equivalent number for other comparisons were as follows: treatment ckb 155/337; ckb 246/718; ckc /3722; ckc /4119; a vs c 60/194; and a vs c 30/. for ck vs a, go classes fell into the categories “biological process”, into “cellular component” and into “molecular function”. the equivalent distribution in ck vs b was and 7; in ck vs b and 9; in both, ck vs c and ck vs c and 11; in a vs c and 5; and in a vs c and the major classes of biological process among the dtgs in ck vs a comparison were “metabolic process”, “cellular process”, “single organism process”, “response to stimulus”, “localization”, “establishment localization”, “biological regulation” and “regulation of biological process”; the predominant cellular components were “cell”, “cell part”, “organelle” and “organelle part”; and for molecular function “binding”, “catalytic activity”, “transporter activity”, “nucleic acid binding transcription factor activity” and “antioxidant activity”. only a few genes belonged to the categories “cell killing”, “electron carrier activity”, “positive regulation of biological process”, “extracellular matrix”, “receptor activity”, “cell junction”, “protein binding transcription factor activity” and “carbon utilization”. the details of go classification of dtgs in ck vs a, and other comparisons are presented in figure  plant hormone signal transduction pathways were well represented, particularly those associated with auxin-mediated signalling. low temperature sensing and signalling genes influenced by ca2+, as well as other protein kinases were also identified. a number of tf families, genes encoding functional proteins and post-translational regulated genes were represented.figure gene ontology classification of the dtgs identified in each comparison between a pair of libraries. dtgs were annotated in three categories: biological process, cellular component and molecular function. y-axis represents the number of dtgs in each category; y-axis represents the percentage of a specific category of dtgs within that main category. panels a, b, c, d, e, f and g represents dtgs in the comparison between library ck and a, library ck and b, library ck and b, library ck and c, library ck and c, library a and c, and library a and c respectively. function annotation of dtgs using the kegg database unigene kegg annotation was aimed at dtgs from the above comparisons. in the ck vs a comparison, dtgs were assigned to the kegg database involved pathways; for ck vs b dtgs were assigned to pathways; for ck vs b dtgs were assigned to pathways; for b vs b dtgs were assigned to pathways; for ck vs c dtgs were assigned to pathways; for ck vs c dtgs were assigned to pathways; for a vs c dtgs were assigned to pathways; and for a vs c dtgs were assigned to pathways. the details of the kegg classification of the above comparisons are presented in additional file 16: table s the major pathways identified were “metabolism”, “biosynthesis of secondary metabolites”, “hormone signal transduction”, “plant-pathogen interaction”, “spliceosome”, “phenylpropanoid biosynthesis”, “endocytosis” and “protein processing in the endoplasm”, “ribosome” and “starch and sucrose metabolism”. genes involved in the response to low temperature more dtgs were identified in the ck vs a, ck vs c and ck vs c comparisons than in the ck vs b and ck vs b this observation demonstrated that many genes were involved in low temperature acclimation. the dtgs revealed in the comparisons, a vs c and a vs c were found to be involved in response to freezing treatment, when the plants were exposed to prior ca. however, the dtgs obtained from the ck vs b and ck vs b comparisons participated in the response mechanism, when the plants didn’t undergo prior ca. some of all the dtgs belonged to the group of genes involved in cold sensing and signal transduction pathways. ca2+ signaling pathway, which is regarded as an important signal sensing and transduction pathway under stresses, includes many members, such as cation/calcium exchangers, calcium-binding proteins, calmodulin-like proteins, cbl-interacting protein kinases, calcium-dependent protein kinases and calmodulin-binding receptor-like kinases. the ck vs a comparison included three ccx, two cbp, five cml, three cipk, and one cbrlk genes; the equivalent order for the ck vs c comparison was, respectively, three, one, five, five and one; for ck vs c three, zero, four, four and one; for ck vs b zero, one, three, zero and zero, along with one sodium/calcium exchanger family protein gene; for ck vs b zero, zero, five, two, zero, along with one sce and one cdpk; for a vs c zero, one, two, one, zero; and for a vs c only one cbp was involved. in the comparisons, ck vs a, ck vs c and ck vs c three ccx, one cml, and one cdpk were all found to be up-regulated; in addition, two cmls, and one cdpk show-ed down-regulation. one cml was found to be up-regulated in both comparisons, ck vs b and ck vs b these findings indicated that the genes related to ca2+ signaling pathway played an essential role in the phenomenon of ca and freezing response without a prior ca; the complete details of dtgs involved in ca2+ signalling pathway are presented in table  no dtgs associated with the mapk cascade was identified in the ck vs a comparison. in contrast, one mapkkk and one mapk gene were found to be differentially transcribed in ck vs c and one mapkkk was represented in ck vs c as well as in ck vs b four mapkkk genes were observed in the ck vs b comparison, and one mapkkk gene was identified in both, a vs c and a vs c the results showed that mapk cascade, especially mapkkk genes, participated in the plant’s response to freezing treatment, instead of ca. therefore, all the dtgs involved in mapk cascade were found to be up-regulated. a higher number of genes encoding serine/threonine-protein kinases were differentially transcribed in ck vs a, ck vs c and ck vs c than in ck vs b or ck vs b. however, no dtg encoding this specific kinase was identified in the a vs c or a vs c comparisons.table the differential gene expression of genes involved in ca 2+ signalling pathway in each comparison in the present study, members of various low tem-perature-responsive transcription factor families were identified; and and such genes were found to be differentially transcribed in the comparisons, ck vs a, ck vs c and ck vs c respectively. the major tf families presented were ap2/erf, bhlh, wrky and tcp, along with small numbers of myb, myc, nac, dof, and the trihelix family. the ck vs b and ck vs b comparisons showed and tf dtgs, respectively. the number of tf dtgs identified in the ck vs b or ck vs b comparison was lesser than the comparisons, ck vs a, ck vs c or ck vs c which were involved in the process of low temperature acclimation. a larger number of tf dtgs were present in ck vs b than in the ck vs b comparison. in addition, five and seven tf dtgs were found in the a vs c and a vs c comparisons, respectively. the identified tf dtgs from the a vs c and a vs c comparisons, were involved in response to freezing treatment in plants with prior exposure to ca. the differently transcribed tfs of the cka treatment, the a vs c and a vs c comparisons are presented in table  table the differential gene expression of transcription factors in each comparison other relevant classes of protein, which featured as dtgs products, were dehydrin, lea proteins, heat shock proteins. the proteins involved in post-transcriptional regulation, such as ribosomal proteins and a dead-box atp-dependent rna helicase, were particularly found in the ck vs a, ck vs c and ck vs c comparisons. yet, with respect to the above classes of proteins, only one lea was found in both, a vs c and a vs c while two hsps were identified in the a vs c comparison. the details of classes of protein of the cka treatment are presented in table  table the differential gene expression of genes encoding lea protein, hsps, and rna helicase in treatment cka lea hsps verification of differential transcription using quantitative real time pcr to further verify the expression profiles of genes in our illumina rna-seq results, we have performed a selection of dtgs for their key roles in response to low temperature by qrt-pcr, these incorporated genes encoding serine/threonine-protein kinase, lea protein, dehydrin, a gibberellin-regulated protein, a jasmonate zim-domain protein, and a dead-box atp-dependent rh, along with a selection of tfs. the qpcr outcomes in each case correlated closely with the transcript abundances estimated from the rna-seq output.figure qpcr validation for dtgs identified by rna-seq in the comparison between ck and a. discussion global patterns of transcription in response to low temperature the information available on the molecular basis of the response of chrysanthemum to low temperature is still meagre. however, the development of next-generation sequencing technology provides a straightforward method for the identification of genes involved in this process, and we can try to elaborate the molecular mechanism underlying the response to low temperature. over 77% of the reads in each of the six rna-seq libraries corresponded with known transcripts, a proportion which was as high as that achieved in a similar study of anthurium. the less than 23% of reads were unmapped probably as a result of unidentified transcripts. around dtgs were identified in each of the cka, ckc and ckc treatments. as was also the case for camellia sinensis, the majority of dtgs involved up- rather than down-regulation by the stress. in both species, many of the dtgs comprised genes associated with low temperature sensing or signal transduction, low temperature-responsive tfs, stabilization of the plasma membrane and osmosensing-responsiveness. low temperature sensing and signaling genes low temperature stress-induced signals are directed to various pathways. ca2+ is well recognized as a messenger in stress signalling, and is sensed by proteins of three main classes: cdpks, cams and cbls. a further important pathway is the mitogen-activated protein kinases cascade, while a number of receptor-like protein kinases are known to be responsible for perceiving changes in the external environment and transducing the appropriate signal. the arabidopsis and rice genomes harbor, respectively, and cml-encoding genes. in rice, the transcription of the calmodulin-like osmsr gene is significantly up-regulated by a series of stresses including low temperature in different tissues at different developmental stages, and its heterologous expression in a. thaliana has suggested that the gene affects salinity and drought tolerance in an aba-dependent manner. in camellia sinensis exposed to low temperature, five calmodulin genes, two cdpk genes and one cbl gene were identified to be involved in signal transduction. here, five unigenes resembling cml were identified as significant dtgs in the cka treatment, and three of these were also differentially transcribed in both the ckc and ckc treatments. cbl-interacting protein kinases, which specifically interact with cbls, are thought to act as sensors since they lack any enzymatic activity. three dtgs with homology to cipk were identified in the cka treatment. both the ckc and ckc treatments also featured the same two dtgs, as well as the other three cipk homologues in the former treatment, and two cipk homologues in the latter. a number of cdpks have been proven to participate in rapid abiotic stress and immune signaling responses. transgenic arabidopsis heterologously expressing the populus euphratica gene pecpk show an enhanced level of freezing tolerance, perhaps through the transgene's enhancement of the transcript abundance of the abiotic stress-responsive genes rd29b and cor15a. only one cdpk homologue was identified as a dtg in the ckb treatment, but no other homologues featured as dtgs in any of the other treatments. however, in a vs c comparison, with respect to ca2+ signalling pathway, only two cml genes and one cipk gene were identified as dtgs. four mapkkk genes were differently transcribed in the ckb treatment. unigene36211_s2_ also featured in the ckb and ckc treatments, while a fifth mapk gene was found to transcribe differently in the ckc treatment. no one gene of mapk cascade was identified in the cka treatment. in addition, many dtgs encoding serine/threonine-protein kinases were found in the cka, ckc and ckc treatments; however, no gene was detected in the comparisons, a vs c and a vs c these findings provided evidence for the crucial role of ca2+ in the low temperature acclimation process in c. nankingense, and further proved that the mapk pathway and serine/threonine-protein kinases are more strongly involved in the response to freezing. major classes of tf involved in the response to low temperature transcriptional regulation of stress-responsive genes is a vital component of the response to both abiotic and biotic stress. five major tf classes were identified as dtgs in the treatments involved in a process of low temperature acclimation. the ap2/erf family, which a large group of plant-specific transcription factors, has been sub-divided into ap rav, erf and dreb tfs. drebs control the aba-independent transcription of low temperature responsive genes in a. thaliana; the atdreb sub-family harbors six members, of which dreb1a/cbf dreb1b/cbf and dreb1c/cbf are the ones which respond most rapidly to low temperature. a. thaliana plants constitutively expressing any one of these tfs display a heightened tolerance to freezing, drought and salinity. the dreb2b tf present in the desert-adapted plant eremosparton songoricum has been shown to enhance the tolerance of both yeast and tobacco against a variety of abiotic stresses. the constitutive expression in tobacco of esdreb2b promotes the accumulation of proline in response to abiotic stress. here, the dreb homologue unigene73473_s2_ was up-regulated upon exposure to low temperature acclimation. it was speculated that the dreb may be involved in the accumulation of proline in response to low temperature. it is well known proline accumulates in many plant species in response to environmental stress. the constitutive expression of an ap tf has been shown to improve the tolerance of a. thaliana to low temperature, as well as to drought and high temperature. here, three ap2-like genes were also all up-regulated by ca. a number of other tfs, belonging to the wrky, bhlh, tcp, myb, myc, trihelix and b-zip families were also among the dtgs identified in treatments involving low temperature acclimation. the class of b-zip transcription factors, abre binding proteins, can bind to abre and activate aba-dependent gene expression when plants are exposed to low temperature. many researches have also indicated that three families of transcription factors: wrky, bhlh and myb closely related to plant cold stress. five of six wrkys, which were detected as dtgs, were down-regulated in ck vs a comparison. however, three wrkys were all up-regulated in a vs c and no wrky as dtg was found in a vs c these findings suggested that wrky family played essential and different roles in ca and early freezing response. most erfs also showed down-regulated in ck vs a comparison, while, one erf was detected up-regulated as dtg in a vs c1; on extending the freezing treatment, five erfs as dtgs were all up-regulated in a vs c the erfs performing under different temperature treatment showed its roles in ca and cold tolerance. few reports to date have indicated that tcp transcription factors involve in the response to cold stress. low temperature responsive genes related to post-transcriptional regulation post-transcriptional regulation has been implicated in the process of low temperature acclimation. dead-box rhs are intimately associated with rna-mediated processes and are related to various rna metabolism events, including rna synthesis to rna degradation by means of catalyzing the atp-dependent unwinding of local rna secondary structures. the transcription of the genes encoding these proteins is known to be regulated by stress in both bacteria and plants. in a. thaliana, rh has been associated with enhanced freezing tolerance, probably through its function as an rna chaperone. the product of rcf a low temperature-inducible rh gene, is important for low temperature-responsive gene regulation and low temperature tolerance in plants through maintenance of normal pre-mrna splicing instead of regulating mrna export like a previously reported dead-box rh regulates mrna export. it has been demonstrated that the functional roles and rna chaperone activity related to intron splicing in mitochondrial and chloroplast. here, respectively, eleven, nine and twelve genes encoding dead-box rhs were up-regulated in the cka, ckc and ckc treatments, but none were differentially transcribed in either the ckb or the ckb treatment; this was taken to imply that dead-box rhs are activated during the process of low temperature acclimation. in future, a major task is to clear how rna chaperones recognize substrate rnas and how they work with other proteins to regulate post-transcriptional rna metabolism in response to developmental and environmental condition. genes encoding functional proteins besides protein factors involved in further regulation of signal transduction and gene expression such as transcription factor and protein kinase that probably function in stress response, various functional proteins featured as products of the dtgs, in particular, lea protein and dehydrin, lea proteins are accumulated during the late stage of seed maturation and under moisture deficient conditions, and act to protect higher plants from damage caused by abiotic stress. when the maize gene zmlea is expressed in tobacco and yeast, it improves the plant's level of tolerance against both osmotic and oxidative stress. the wheat lea protein gene wci expressed heterologously in a. thaliana enhances freezing tolerance. here, seven lea protein genes were among the dtgs identified in the cka treatment, five in both the ckc and ckc treatment, none were present in either the ckb or the ckb treatment, and only one was identified in both comparisons of a vs c and a vs c the implication was that lea proteins probably enhance low temperature tolerance through their participation in the low temperature acclimation process. dehydrins constitute a group of plant proteins involved in tolerance to low temperature and drought. the thranscrips of genes encoding three dehydrins in e. globulus accumulate strongly in the stem and leaf tissue of acclimated plants, compared to non-acclimated. here, four genes encoding dehydrins were identified as dtgs in the cka, ckc and ckc treatments. but none in either the ckb or ckb treatments, suggesting that, as with the lea protein genes, their contribution to low temperature tolerance is expressed during the low temperature acclimation process. hsps were initially identified from their involvement in the response to high temperatures, but it is now recognized that many of them also respond to low temperature. it is well known hsps can protect plants against stress by means of reestablishing normal protein conformation and thus cellular homeostasis. five major hsp families have been conservely defined, based on their molecular weight: hsp100s, hsp90s, hsp70s, hsp60s and small hsps. five hsp90-encoding dtgs featured in the cka, ckc and ckc treatments. hsp90s function as molecular chaperones during signal transduction, cell cycling, the stress response, and in protein folding, degradation and transport. in a. thaliana, expression of hsp is developmentally regulated, but it also responds to high and low temperature, as well as salinity. it is speculated that hsp as molecular chaperones play an important role in signal transduction and stress management in c. nankinginse. a gene hsp70- encoding dtgs featured in the cka, ckb ckc and ckc treatments, which not found in the ckb treatment; and a number of small hsps, belonging to the, and families were also among the dtgs identified in treatments involving low temperature response. in conclusion, it was clear that a large number of genes were induced when exposed to low temperature. the complex gene networks involved a set of interactions between cold sensing and signaling genes, genes related to post-transcription regulation responding to cold stress, transcription factors and certain functional proteins. in the present study, the dtgs identified as candidate genes in response to low temperature, require further investigation for a complete understanding of the molecular basis of cold response in c. nankingense. this information will prove beneficial in molecular breeding programs for excellent chrysanthemum varieties.  an overview of the many changes to the c. nankingense transcriptome induced by exposure to low temperature has been provided. many of the dtgs identified were involved in predictable classes of gene, encoding, for example, low temperature sensors and signaling molecules, tfs and certain functional proteins associated with the stress response. the large number of dtgs identified confirms the complexity of the regulatory machinery involved in low temperature acclimation and low temperature/freezing tolerance. establishing which of these many dtgs reflect the primary response to low temperature is necessary before the molecular basis of the response can be fully elaborated; such genes would represent candidates for intervention in the breeding of chrysanthemum varieties endowed with a heightened tolerance to low temperature stress. 
a major constraint affecting the quality and productivity of chrysanthemum is the unusual period of low temperature occurring during early spring, late autumn, and winter. herein, we used rna-seq platform to characterize the transcriptomic response to low temperature by comparing different transcriptome of chrysanthemum nankingense plants and subjecting them to a period of sub-zero temperature, with or without a prior low temperature acclimation. the large number of dtgs identified in this study, confirmed the complexity of the regulatory machinery involved in the processes of low temperature acclimation and low temperature/freezing tolerance. the differential transcription of dtgs was validated using quantitative rt-pcr. the differentially transcribed genes were identified as low temperature sensing and signalling genes, transcription factors, functional proteins associated with the abiotic response, and low temperature-responsive genes involved in post-transcriptional regulation. six separate rna-seq libraries were generated from the rna samples of leaves and stems from six different temperature treatments, including one cold acclimation, two freezing treatments without prior ca, two freezing treatments with prior ca and the control. yet, there has been no systematic investigation on the genes underlying the response to low temperature in chrysanthemum. transcriptomerna sequencinglow temperature toleranceornamental plantissue-copyright-statement© the author 2014
 a major constraint affecting the quality and productivity of chrysanthemum is the unusual period of low temperature occurring during early spring, late autumn, and winter. yet, there has been no systematic investigation on the genes underlying the response to low temperature in chrysanthemum. herein, we used rna-seq platform to characterize the transcriptomic response to low temperature by comparing different transcriptome of chrysanthemum nankingense plants and subjecting them to a period of sub-zero temperature, with or without a prior low temperature acclimation.  six separate rna-seq libraries were generated from the rna samples of leaves and stems from six different temperature treatments, including one cold acclimation, two freezing treatments without prior ca, two freezing treatments with prior ca and the control. at least seven million clean reads were obtained from each library. over 77% of the reads could be mapped to sets of c. nankingense unigenes established previously. the differentially transcribed genes were identified as low temperature sensing and signalling genes, transcription factors, functional proteins associated with the abiotic response, and low temperature-responsive genes involved in post-transcriptional regulation. the differential transcription of dtgs was validated using quantitative rt-pcr.  the large number of dtgs identified in this study, confirmed the complexity of the regulatory machinery involved in the processes of low temperature acclimation and low temperature/freezing tolerance. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords transcriptomerna sequencinglow temperature toleranceornamental plantissue-copyright-statement© the author 2014 