BACKGROUND
with the rapid development of the national economy and increasing integration in the international community, the demand for flowers is changing from high yield to high quality in recent years.
among the many qualities that influence flowers, flower colour is one of the most important indicators that determines its ornamental quality.
ornamental plants with novel flower colours can effectively stimulate the senses and psychology of consumers, as well as have a broader market appeal  <cit> , such as the blue rose  and yellow violet   <cit> .
thus, breeding new flower cultivars with rare colours has been an important target for flower breeders.
herbaceous peony  is a traditional flower in china that belongs to the paeoniaceae family and has been a well-known symbol of prosperity.
in china, it enjoys a high status as a traditional flower and has shared the name with “the king and minister of flowers” with the tree peony   <cit> .
until recently, there has been a large germplasm resource of p. lactiflora with more than  <dig> cultivars worldwide, and the flower colour characteristics vary widely among different cultivars, which can be divided into nine flower colour categories, i.e., red, pink, white, blue, purple, green, yellow, black and double colour  <cit> .
among these flower colours, yellow, which represents gold, suggests riches and honour, as well as reveals the king demeanour in flowers.
in addition, the commercial price of yellow p. lactiflora cultivars is usually approximately  <dig> times more than purple or red cultivars.
whereas, yellow cultivars of p. lactiflora are very rare, where currently, there is only one pure yellow cultivar, namely ‘huangjinlun’  <cit> .
this limitation can not highly meet the needs of consumers.
thus, breeding new p. lactiflora cultivars with yellow flowers has been an important aim, although it has been challenging using selective breeding and hybridisation breeding.
therefore, understanding of the biochemical and molecular mechanisms of yellow characteristic formation in p. lactiflora, identifying its key genes, and the ability to modify flower colour using modern genetic engineering are important to breed new p. lactiflora varieties in the future.
however, very limited genomic resources are available for p. lactiflora, with only  <dig> nucleotide sequences deposited in genbank until  <dig> january  <dig>  which has restricted the development of modern breeding technologies.
transcriptome sequencing  is a highly efficient and conventional molecular biology research method based on next-generation sequencing technology  <cit> .
in recent years, the continuous development and improvement of this technology, combined with a more mature commercial operation, have provided new approaches and designs in the study of functional genomics of different plants and have made transcriptomics a new starting point and breakthrough point in plant research .
compared with the gene chip technique, which is based on the premise of a large number of genes or molecular database information, rna-seq can be studied in non-model organisms without corresponding genome sequences as a reference .
moreover, transcriptome study enables researchers to understand plant gene functions and structures at a specific stage and is more convenient to reveal the molecular mechanisms of specific biological processes  <cit> .
recently, rna-seq has become widely applied in many plants such as cucumber   <cit> , chinese cabbage   <cit> , blueberry   <cit> , peach   <cit> , camelina   <cit> , etc.
to the best of our knowledge, colour formation has been intensely investigated in fruits using rna-seq, and some information has been obtained regarding unigenes, such as sweet orange   <cit>  and chinese bayberry   <cit> .
however, in flowers, rna-seq has been used in in-depth studies of colour formation only in wintersweet   <cit>  and safflower   <cit> , and the flavonoid biosynthetic pathway has been annotated in these plants which is widespread in regulating flower colour formation of plants.
in model plants, this biosynthetic pathway can be divided into three stages  <cit> .
the first stage is the conversion of phenylalanine to coumarate-coa by phenylalanine ammonialyase gene , cinnamate 4-hydroxylase gene  and 4-coumarate coa ligase gene .
the second stage is the formation of dihydroflavonol by one molecule of coumarate-coa and three molecules of malonyl-coa catalyzed by chalcone synthase gene , chalcone isomerase gene , flavanone 3-hydroxylase gene , flavonoid 3′-hydroxylase gene , flavonoid 3′,5′-hydroxylase gene  and flavonol synthase gene .
the third stage is the formation of various anthocyanidins by dihydroflavonols catalyzed by dihydroflavonol 4-reductase gene  and anthocyanidin synthase gene .
the synthesized anthocyanidins are then modified through a series of glycosylation and methylation steps to form stable anthocyanins catalyzed by anthocyanidin 3-o-glucosyltransferase gene , anthocyanidin 5-o-glucosyltransferase gene  and methyl transferase gene .
however, there has been limited related information on p. lactiflora, and these studies provide very useful references for the study of yellow formation in p. lactiflora.
it is well known that plant materials with obvious differences are essential in the study of gene function.
furthermore, genetic mutants that control relative characteristics are the best materials to study gene function.
in the present study, a flower colour chimaera cultivar ‘jinhui’ with a consistent genetic background red outer-petal and yellow inner-petal was used as the experimental material including four developmental stages , and illumina sequencing was adopted to analyse their difference of transcriptome.
based on this, the expression patterns of differentially expressed genes  identified in the flavonoid biosynthetic pathway were analysed using real-time quantitative polymerase chain reaction , and qualitative and quantitative analysis of flavonoids was performed using high-performance liquid chromatograph -electrospray ionization -mass spectrometry  technology, thereby revealing the biochemical and molecular mechanisms of yellow formation in p. lactiflora.
taken together, these results would provide a foundation for breeding yellow p. lactiflora varieties.
RESULTS
colour indices
to obtain the same genetic background, a flower colour chimaera cultivar ‘jinhui’ with red outer-petal and yellow inner-petal was used to study the mechanism of yellow formation in p. lactiflora .
firstly, their colour indices were measured.
on one hand, the flower colours were defined according to the royal horticultural society color chart .
during flower development, the colour gradation of the outer-petal ranged from 164a, 73a and 75b to 75c, while that of the inner-petal changed from 11a, 11b and 11c to 11d.
on the other hand, flower colours were expressed as l*, a* and b*.
in a uniform colour space, l* represented lightness, a* represented the ratio of red/magenta and green, and b* represented the ratio of yellow and blue  <cit> .
in four different developmental stages, l* values of the outer-petal and inner-petal were all gradually increased which was always lower in the outer-petal compared to the inner-petal; however, the b* value of the outer-petal and a* value of the inner-petal decreased, which all tended to zero.
these colour indices showed that the colours of the outer-petal and inner-petal were pure red and yellow in s <dig>  respectively, and then faded gradually with flower development.
in addition, the flower diameter was also measured, such that the whole flower and inner-petal were similar and had constantly increased from s <dig> to s <dig>  taken together, these measured results were consistent with the visual results.figure  <dig>
figures of
p. lactiflora
flower developmental stages, flower qualitative trait and color variables.
s <dig> , flower-bud stage; s <dig> , initiating bloom stage; s <dig> , bloom stage; s <dig> , wither stage.
rna-seq and de novo assembly
to deeply elucidate the molecular mechanism underlying yellow colour formation, rna-seq analysis were performed using the illumina platform.
each library of the outer-petal and inner-petal produced  <dig> , <dig> and  <dig> , <dig> total raw reads, following cleaning and quality checks, and  <dig> , <dig> and  <dig> , <dig> total clean reads were obtained with a single read length of 90 nt and a q <dig> percentage  over  <dig> % .
these data showed that the rna-seq quality was better, which can be used for further analysis.table  <dig>
output statistics of rna-seq of
p. lactiflora
outer-petal and inner-petal
subsequently, short reads from the outer-petal and inner-petal were assembled into  <dig>  and  <dig>  contigs, which contained an average length of  <dig> and 337 nt, together with n <dig> length  of  <dig> and 664 nt, respectively.
after linking the contigs together,  <dig>  and  <dig>  unigenes with an average length of  <dig> and 617 nt of the outer-petal and inner-petal were identified, respectively, and their n <dig> values were all 1047 nt.
finally,  <dig>  non-redundant all-unigenes were obtained according to splicing and reducing the redundancy of the unigenes sequence in the outer-petal and inner-petal, and its average length and n <dig> value were 763 nt and 1219 nt, respectively .
among these, there were  <dig>  unigenes with a length of  <dig> to 500 nt, which accounted for  <dig> %, and excluding n rate reached 100%.
all these results showed that the assembly quality was sufficiently high for gene function analysis.
in addition, the distributions of the reads in all-unigenes were analysed, and these results showed that the number of reads in the 3′ and 5′ ends from all-unigenes was relatively low, particularly in the 3′ and 5′ tail ends.
however, the number of reads located in  <dig>  to  <dig>  was relatively high, and its distribution was balanced .table  <dig>
data assembly of transcriptome contig and unigene in
p. lactiflora
outer-petal and inner-petal
functional annotation
due to the lack of a complete genome sequence in p. lactiflora,  <dig>  all-unigenes were blasted against six public databases, i.e., non-redundant protein database , swiss-prot protein database , kyoto encyclopedia of genes and genomes database , cluster of orthologous groups of proteins database , gene ontology database  and non-redundant nucleotide database .
these results revealed that  <dig>  all-unigenes were annotated, which accounted for  <dig> %.
of the all-unigenes,  <dig> ,  <dig>  and  <dig>  all-unigenes could be annotated to nr, nt and go databases, accounting for  <dig> %,  <dig> % and  <dig> %, respectively, whereas in the swiss-prot, kegg and cog databases, only  <dig> ,  <dig>  and  <dig>  all-unigenes were annotated, which accounted for  <dig> %,  <dig> % and  <dig> %.
for the nr annotation, the ratio of the all-unigenes distributed in 10-15 < e-value < 10- <dig> and 0 < e-value < 10- <dig> with  <dig> % and  <dig> % was maximum;  <dig> % of all-unigenes showed 60-95% similarity; and  <dig>  all-unigene sequences shared a similarity with available plant sequences from vitis vinifera, p. persica, ricinus communis, populus trichocarpa, fragaria vesca subsp.
vesca, c. sativus and glycine max, including up to  <dig> % similarity with vitis vinifera .
when compared with the cog database,  <dig>  all-unigenes were divided into  <dig> different functional classifications, and the functional types comprehensively involved most of the life activities .
among these types, the maximum number of all-unigenes was categorised as general function prediction only , followed by transcription , replication, recombination and repair  and posttranslational modification, protein turnover, chaperones , while the minimum number was classified to nuclear structure .figure  <dig>
figure of cog categories of the annotated all-unigenes.
comparative analysis of degs and go functional annotation between the outer-petal and inner-petal
to identify key genes controlling yellow formation in p. lactiflora, the transcriptomes of the outer-petal and inner-petal were comparatively analysed based on the outer-petal as the control group.
as shown in figure  <dig>   <dig>  degs were obtained, among which, the expression levels of  <dig>  degs  were up-regulated and the expression levels of  <dig>  degs  were down-regulated.
furthermore, abundant blue scatters indicated no difference in expression between the outer-petal and inner-petal.
on the basis of sequence homology, these  <dig>  degs could be classified into three categories in go assignments: biological process, cellular component and molecular function, and they embraced  <dig>   <dig> and  <dig> functional classifications, respectively .
in the biological process category, cellular process  and metabolic process  accounted for the major proportion.
in the cellular component category,  <dig>  and  <dig>  degs  were enriched in the cell and cell part.
under the molecular function category, catalytic activity  and binding  were dominant functions.figure  <dig>
figure of degs in
p. lactiflora
outer-petal and inner-petal.
red scatters indicate up-regulated degs, green scatters indicate down-regulated degs, and blue scatters indicate no difference degs in expression between the outer-petal and inner-petal.
figure of go categories of degs in
p. lactiflora
outer-petal and inner-petal.
functional classification using kegg
the pathway database was the major component of kegg, which contained most of the metabolic pathways representing information on the molecular interaction and reaction networks  <cit> .
in this study,  <dig>  degs were subsequently analysed in the kegg pathway database, where  <dig>  degs  were assigned to  <dig> kegg pathways and only  <dig> pathways met the condition of q-value ≤  <dig>  .
among these pathways, metabolic pathways demonstrated the largest number of degs , followed by biosynthesis of secondary metabolites , plant-pathogen interaction , plant hormone signal transduction , endocytosis , starch and sucrose metabolism  and glycerophospholipid metabolism .
many studies revealed that colour development of the p. lactiflora petal was closely related to flavonoids , and our previous study also confirmed this conclusion  <cit> .
as expected, five pathways related to colour development were identified, namely phenylpropanoid biosynthesis , flavonoid biosynthesis , flavone and flavonol biosynthesis , isoflavonoid biosynthesis  and anthocyanin biosynthesis , and they all belonged to the flavonoid metabolic pathway which could be establish connection taking flavonoid biosynthesis as the ligament.figure  <dig>
interactive pathways during the outer-petal and inner-petal development.
blue part is flavonoid metabolic pathway related to colour development.
expression analysis of candidate degs related to colour development
according to the genes annotated in kegg and the results of flower colour formation obtained in many plants  <cit> ,  <dig> degs were selected for expression analysis .
among these degs, pal, chi, f3h, f3′h, dfr, ans, 3gt and 5gt had been registered in genbank, and their accession numbers were jq <dig>  jn <dig>  jq <dig>  jq <dig>  jq <dig>  jq <dig>  jq <dig> and jq <dig>  respectively.
however, f3′5′h and fls were first found in p. lactiflora.
among them, more than one unigene was annotated as the same enzyme, which might be because these unigenes represented different members of the same gene family, different fragments of a single transcript, or both, except pal and f3′5′h
<cit> .
as shown in figure  <dig>  q-pcr was used to study their expression patterns in four different developmental stages of the outer-petal and inner-petal.
single gene expression level analyses indicated that although the expressions of all  <dig> degs were detected during flower development, but their expression levels were different.
among the  <dig> degs, the expression levels of plf3h and plf3′h were the highest, and downstream pldfr and pl5gt exhibited the lowest expression levels, while the other degs ranged between the two genes listed above.
in terms of the overall trend, the expression levels of the  <dig> degs were similar during the outer-petal and inner-petal development except in some stages.
for example, the expression levels of plfls and pl3gt increased until they reached the maximum level during flower development, the other  <dig> degs all showed a downward trend, and they only had some differences in quantity between the outer-petal and inner-petal.
for different petals, the first key gene in flavonoid metabolism plpal; pldfr controlling the formation of leucoanthocyanidin; and pl3gt together with pl5gt, which catalyses the formation of the complex aglycone of anthocyanin composition, all had higher expression levels in the outer-petal compared to the inner-petal in the four different developmental stages, whereas f3′5′h showed an opposite trend.table  <dig>
candidate degs involved in
p. lactiflora
colour development
pal
chi
f3h
f3′h
f3′5′h
fls
dfr
ans
3gt
5gt
no.
all indicates the total number of degs, no.
up indicates the number of degs in the inner-petal compared with the outer-petal in s <dig>  no.
down indicates the number of degs in the inner-petal compared with the outer-petal in s <dig>
expression analysis of  <dig> candidate degs related to colour development in
p. lactiflora
.
qualitative and quantitative analysis of flavonoids
to examine whether flavonoid accumulation in p. lactiflora petals was consistent with the expression patterns of candidate degs related to colour development, qualitative and quantitative analysis of flavonoids were performed using hplc-esi-msn in the same samples used in the expression analysis.
on the basis of the ultraviolet–visible absorption characteristics, anthocyanins, anthoxanthins and chalcones were detected under the wavelength of 525 nm, 350 nm and 365 nm, respectively.
as shown in figure  <dig>  the chromatographic peaks of the outer-petal and inner-petal were identical, although there were only some differences in the peak area.
the hplc retention time and ultraviolet–visible spectral properties together with mass spectrometric data of identified main chromatographic peaks were listed in additional file 4: table s <dig>  moreover, two  and seven  peaks were identified at 525 nm and 350 nm, namely cyanidin- <dig> -di-o-glucoside, peonidin- <dig> -di-o-glucoside, kaempferol di-hexoside, kaempferol-3-o-malonylglucoside-7-o-glucoside, quercetin-3-o-galactoside, luteolin-7-o-galloylglucoside, luteolin-7-o-glucoside, isorhamnetin-3-o-glucoside and flavone c-glycoside, respectively.figure  <dig>
hplc chromatograms of
p. lactiflora
anthocyanins  and anthoxanthins  in s <dig>   indicates hplc chromatograms of outer-petal;  indicates hplc chromatograms of inner-petal;  indicates hplc chromatograms of  and  merge together.
a1-a <dig> indicates identified anthocyanins; f1-f <dig> indicates identified anthoxanthins.
relevant standards were used as the references for the relative quantitative analysis of flavonoids, the results showed that there were tremendous differences in both the two different petals and also during different developmental stages of the same petals .
for the composition, peonidin- <dig> -di-o-glucoside was the main component of anthocyanins in p. lactiflora outer-petal and inner-petal, which accounted for approximately  <dig> % and  <dig> % of the total anthocyanin contents in the outer-petal and inner-petal, respectively.
in addition, the main component of anthoxanthins in p. lactiflora outer-petal and inner-petal was luteolin-7-o-glucoside, which comprised approximately  <dig> % and  <dig> % of the total anthoxanthin contents in the outer-petal and inner-petal, respectively, which was followed by kaempferol di-hexoside , luteolin-7-o-galloylglucoside  and flavone c-glycoside .
concerning the content of composition, the results showed that the content of each composition in the outer-petal was basically higher compared to the inner-petal, particularly for a <dig> and a <dig> of anthocyanins.
in s <dig>  the contents of a <dig> and a <dig> in the outer-petal were  <dig> times as much as that of the inner-petal and increased to  <dig> times in s <dig>  in the case of different developmental stages, each anthocyanin and anthoxanthin composition in the outer-petal were basically decreased from s <dig> to s <dig>  and slightly increased in s <dig>  however, the compositions in the inner-petal were always decreased with flower development, except f <dig>  f <dig> and f <dig> in s <dig>  for total content, the total contents of anthocyanins, anthoxanthins and flavonoids in the outer-petal were all higher compared to the inner-petal, and specifically, the difference in anthocyanins was achieved by a multiple of  <dig>  meanwhile, the total contents of anthocyanins, anthoxanthins and flavonoids in the outer-petal and inner-petal all presented a downward trend as flower development, although there was an increase in the outer-petal at s <dig>  furthermore, the co-pigment index , which determined whether had the co-pigment effect between anthoxanthins and anthocyanins demonstrated an increasing tendency in both the outer-petal and inner-petal.
in the outer-petal, the ci had increased from  <dig>  in s <dig> to  <dig>  in s <dig>  in contrast, and its developmental difference in the inner-petal was more significant and increased from  <dig>  in s <dig> to  <dig>  in s <dig> .table  <dig>
contents of anthocyanin and anthoxanthin compositions in
p. lactiflora
petals during flower development
s <dig> , flower-bud stage; s <dig> , initiating bloom stage; s <dig> , bloom stage; s <dig> , wither stage.
total content of anthocyanins, anthoxanthins and flavonoids, and co-pigmentation index in
p. lactiflora
outer-petal and inner-petal during flower development.
discussion
p. lactiflora is an important ornamental plant with a cultivation history of more than 4000 years in china.
due to abundant flower colours, variable flower shapes, sharp flower fragrances and attractive flower poses, p. lactiflora exhibits a very high ornamental value and rich landscaping application value, which has become increasingly favoured by people worldwide.
however, compared with plants in which genome sequencing has been completed, such as arabidopsis  heynh.)
<cit> , mei   <cit> , lotus   <cit> , spider flower  iltis)  <cit>  and carnation   <cit> , a weak molecular basis and an incompletely clear genetic background in p. lactiflora severely limit its in-depth study.
transcriptome refers to the identity of each expressed gene and its expression level for cells in a particular state, whose expression largely controls the phenotype of an organism.
unlike the genome, the transcriptome is more dynamic and provides more valuable reference information for cell function interpretation, but its study is more inexpensive  <cit> .
due to overcoming technological limitations, transcriptome studies have been extensively developed in plants .
moreover, according to a different purpose of the study, rna-seq can be divided into two methods: on one hand, different experimental materials are mixed equally to sequence, which can obtain the largest number of unique transcripts  <cit> ; on the other hand, different experimental materials are sequenced separately, which can isolate tissue-specific expression genes  <cit> .
wang et al.
aimed to elucidate the transcriptome profiling of radish  root and identify the critical gene response to lead  stress, two radish root cdna samples, where one untreated control and the other pb-stressed with pb  <dig> at 1000 mg were separately sequenced, and  <dig>  degs were obtained  <cit> .
in this study, to characterize the transcriptome of p. lactiflora red outer-petal and yellow inner-petal and identify the critical genes mediating yellow formation, illumina sequencing was firstly employed to the outer-petal and inner-petal with significantly different colours separately.
each library of the outer-petal and inner-petal produced  <dig> , <dig>  and  <dig> , <dig>  total clean nucleotides with a q <dig> percentage over  <dig> % in which the quantity and quality were all relatively high compared with other plants, such as the apple tree  <cit>  and m. rubra
<cit> .
subsequently,  <dig>  and  <dig>  unigenes with an average length of  <dig> and 617 nt were found in the outer-petal and inner-petal, and  <dig>  degs were identified, which provided a large amount of information for molecular biological research in p. lactiflora.
colour formation is a complex process that includes many metabolic pathways and gene expression.
in the transcriptome study of colour, xu et al.
speculated that enhanced photosynthesis and reduced downstream gene expression in the carotenoid biosynthetic pathway were the key reason for sweet orange red-flesh mutant lycopene accumulation, which controlled red formation  <cit> .
recently, two varieties of flesh in four developmental stages were used to perform rna-seq by this research group, which further confirmed the correlation between carotenoid metabolism and red colour formation in sweet orange red-flesh mutant  <cit> .
in m. rubra, feng et al.
aimed to obtain a general overview of the transcriptome, four libraries containing mixed tissues, and three different developmental fruits were designed for rna-seq  <cit> .
this study identified  <dig>  unigenes, generated interactive pathways during bayberry fruit ripening, and found that the expression levels of flavonoid biosynthetic genes were overall increased, which was consistent with flavonoid accumulation and more intense colour.
to the best of our knowledge in flower colour, transcriptome studies had been performed only on c. praecox
<cit>  and c. tinctorius
<cit> .
in the transcriptome database of c. praecox,  <dig>  unigenes could be annotated in  <dig> different metabolic pathways.
furthermore,  <dig> unigenes with an average length of 677 bp were associated with the metabolic pathway of flower colour, including the flavonoid biosynthetic pathway , flavone and flavonol biosynthetic pathway  and anthocyanin biosynthetic pathway , which was convenient for gene cloning and function identification  <cit> .
otherwise, the flavonoid biosynthetic pathway , flavone and flavonol biosynthetic pathway  and anthocyanin biosynthetic pathway  were identified according to the rna-seq comparison of red and white c. tinctorius.
moreover, the expression levels of  <dig> flavonoid biosynthetic pathway-related candidate unigenes were analysed, indicating that chs  was very important for c. tinctorius flavonoid synthesis  <cit> .
in the present study,  <dig>  degs in the outer-petal and inner-petal had been annotated to  <dig> metabolic pathways, and there were  <dig> metabolic pathways enriched degs, which revealed the formation process complexity of different petals.
among these pathways, the flavonoid metabolic pathway containing phenylpropanoid biosynthesis, flavonoid biosynthesis, anthocyanin biosynthesis, isoflavonoid biosynthesis, flavone and flavonol biosynthesis was annotated, which validated the conclusion that flavonoids were the pigment of p. lactiflora petal  <cit> .
moreover, carotenoid biosynthesis and zeatin biosynthesis were also found in metabolic pathways, which might be resulted from incomplete petalody stamens.
interestingly,  <dig> glucide metabolic pathways, including pentose and glucuronate interconversions, starch and sucrose metabolism, glycolysis/gluconeogenesis, other glycan degradation, fructose and mannose metabolism, amino sugar and nucleotide sugar metabolism and galactose metabolism were annotated.
these pathways were not only associated with the growth and development of petals but were also related to the synthetic substrates of anthocyanins.
flavonoid biosynthesis, which was controlled by many genes  <cit> , and key structural genes in the flavonoid metabolic pathway were screened in this study.
interestingly, the well-known key chalcone synthase gene  catalyzing the formation of the chalcone derivative could not be found, which was the committed step leading to the biosynthesis of flavonoids, isoflavonoids and anthocyanins  <cit> .
this might be the reason that the expression of plchs in the inner-petal was not inhibited, and its level was not different with that in the outer-petal, which converted upstream materials to downstream materials in large quantity.
in the expression analysis of the  <dig> candidate degs, the expression of upstream plchi was also not inhibited in the inner-petal, instead its level was higher than that in the outer-petal, which was opposite to our previous results  <cit> , indicating that chalcone was not accumulated in the inner-petal and did not cause its yellow colour.
furthermore, the expression levels of downstream pldfr, plans, pl3gt and pl5gt in the inner-petal were all lower compared to the outer-petal, but this difference did not reach hundreds-fold as our previous results  <cit> , suggesting that anthocyanins were also produced in the inner-petal, just its content was relatively low.
flower colour was mainly dependent on the kinds of pigment, the content and its distribution in the petals  <cit> .
the generalised yellow flower from pale yellow to dark yellow could be divided into multiple grades according to its colour depth.
among these colours, the pigment of the yellow flower perceived by the naked eye was relatively complicated, which was related to flavonoids and carotenoids, this conclusion had been fully proved by previous transcriptome studies .
previous studies on the determination of p. lactiflora petal pigment composition showed that a great deal of chalcone and a small amount of anthoxanthin accumulation were the cause of its yellow petal formation  <cit> .
similarly, the biochemical mechanism of yellow characteristic formation in p. suffruticosa was the same, and kaempferol, quercetin, isorhamnetin, chrysoeriol, apigenin and luteolin were identified as the main anthoxanthins  <cit> .
this was mainly because anthocyanins were responsible for red, whereas chalcones and anthoxanthins were yellow.
in this study, chalcones could not be detected in the yellow inner-petal, whereas a large number of anthoxanthins  and a few anthocyanins  were identified, which was not consistent with previous results  <cit> .
although anthocyanins were in the yellow inner-petal, the co-pigmentation effect of anthoxanthins in the inner-petal was significant and completely covered the red effect of anthocyanins due to the increase of ci from  <dig>  in s <dig> to  <dig>  in s <dig>  in addition, compared with the red outer-petal, the contents of anthoxanthins and anthocyanins were relatively lower in the yellow inner-petal.
these physiological results obtained perfect explanation using key gene expression analysis.
due to the massive expression of upstream plpal and plchi, particularly plchi, a large number of upstream materials in the inner-petal were formed and transformed to the downstream, rather than in the accumulation of chalcones.
with the expression of plf3h, plf3′h, plf3′5′h and plfls, a variety of anthoxanthins was formed and accumulated.
furthermore, a small amount of anthocyanins was produced, which was combined with low expression levels of downstream pldfr, plans, pl3gt and pl5gt, and eventually made the inner-petal appear yellow.
in addition, because the expression level of the first key gene, plpal, in the inner-petal was always lower than that in the outer-petal, combined with lower plfls, pldfr, plans, pl3gt and pl5gt expression, the accumulation of anthoxanthins and anthocyanins in the inner-petal was lower compared to the outer-petal.
taken together, these results provided an understanding of the biochemical and molecular mechanisms of yellow characteristic formation in p. lactiflora, which might lay a theoretical foundation for breeding new yellow p. lactiflora varieties.
CONCLUSIONS
this study showed that rna-seq analysis based on the high throughput sequencing technology provided an efficient approach for identifying critical genes in p. lactiflora.
in the metabolic pathways related to yellow formation, flavonoid metabolic pathway and glucide metabolic pathway were identified, and plpal, plfls, pldfr, plans, pl3gt and pl5gt were selected as potential candidates involved in flavonoid metabolic pathway, which induced inhibition of anthocyanin biosynthesis mediated yellow formation in p. lactiflora.

 with the rapid development of the national economy and increasing integration in the international community, the demand for flowers is changing from high yield to high quality in recent years. among the many qualities that influence flowers, flower colour is one of the most important indicators that determines its ornamental quality. ornamental plants with novel flower colours can effectively stimulate the senses and psychology of consumers, as well as have a broader market appeal, such as the blue rose and yellow violet. thus, breeding new flower cultivars with rare colours has been an important target for flower breeders. herbaceous peony is a traditional flower in china that belongs to the paeoniaceae family and has been a well-known symbol of prosperity. in china, it enjoys a high status as a traditional flower and has shared the name with “the king and minister of flowers” with the tree peony. until recently, there has been a large germplasm resource of p. lactiflora with more than cultivars worldwide, and the flower colour characteristics vary widely among different cultivars, which can be divided into nine flower colour categories, i.e., red, pink, white, blue, purple, green, yellow, black and double colour. among these flower colours, yellow, which represents gold, suggests riches and honour, as well as reveals the king demeanour in flowers. in addition, the commercial price of yellow p. lactiflora cultivars is usually approximately times more than purple or red cultivars. whereas, yellow cultivars of p. lactiflora are very rare, where currently, there is only one pure yellow cultivar, namely ‘huangjinlun’. this limitation can not highly meet the needs of consumers. thus, breeding new p. lactiflora cultivars with yellow flowers has been an important aim, although it has been challenging using selective breeding and hybridisation breeding. therefore, understanding of the biochemical and molecular mechanisms of yellow characteristic formation in p. lactiflora, identifying its key genes, and the ability to modify flower colour using modern genetic engineering are important to breed new p. lactiflora varieties in the future. however, very limited genomic resources are available for p. lactiflora, with only nucleotide sequences deposited in genbank until january which has restricted the development of modern breeding technologies. transcriptome sequencing is a highly efficient and conventional molecular biology research method based on next-generation sequencing technology. in recent years, the continuous development and improvement of this technology, combined with a more mature commercial operation, have provided new approaches and designs in the study of functional genomics of different plants and have made transcriptomics a new starting point and breakthrough point in plant research. compared with the gene chip technique, which is based on the premise of a large number of genes or molecular database information, rna-seq can be studied in non-model organisms without corresponding genome sequences as a reference. moreover, transcriptome study enables researchers to understand plant gene functions and structures at a specific stage and is more convenient to reveal the molecular mechanisms of specific biological processes. recently, rna-seq has become widely applied in many plants such as cucumber, chinese cabbage, blueberry, peach, camelina, etc. to the best of our knowledge, colour formation has been intensely investigated in fruits using rna-seq, and some information has been obtained regarding unigenes, such as sweet orange and chinese bayberry. however, in flowers, rna-seq has been used in in-depth studies of colour formation only in wintersweet and safflower, and the flavonoid biosynthetic pathway has been annotated in these plants which is widespread in regulating flower colour formation of plants. in model plants, this biosynthetic pathway can be divided into three stages. the first stage is the conversion of phenylalanine to coumarate-coa by phenylalanine ammonialyase gene, cinnamate 4-hydroxylase gene and 4-coumarate coa ligase gene. the second stage is the formation of dihydroflavonol by one molecule of coumarate-coa and three molecules of malonyl-coa catalyzed by chalcone synthase gene, chalcone isomerase gene, flavanone 3-hydroxylase gene, flavonoid 3′-hydroxylase gene, flavonoid 3′,5′-hydroxylase gene and flavonol synthase gene. the third stage is the formation of various anthocyanidins by dihydroflavonols catalyzed by dihydroflavonol 4-reductase gene and anthocyanidin synthase gene. the synthesized anthocyanidins are then modified through a series of glycosylation and methylation steps to form stable anthocyanins catalyzed by anthocyanidin 3-o-glucosyltransferase gene, anthocyanidin 5-o-glucosyltransferase gene and methyl transferase gene. however, there has been limited related information on p. lactiflora, and these studies provide very useful references for the study of yellow formation in p. lactiflora. it is well known that plant materials with obvious differences are essential in the study of gene function. furthermore, genetic mutants that control relative characteristics are the best materials to study gene function. in the present study, a flower colour chimaera cultivar ‘jinhui’ with a consistent genetic background red outer-petal and yellow inner-petal was used as the experimental material including four developmental stages, and illumina sequencing was adopted to analyse their difference of transcriptome. based on this, the expression patterns of differentially expressed genes identified in the flavonoid biosynthetic pathway were analysed using real-time quantitative polymerase chain reaction, and qualitative and quantitative analysis of flavonoids was performed using high-performance liquid chromatograph -electrospray ionization -mass spectrometry technology, thereby revealing the biochemical and molecular mechanisms of yellow formation in p. lactiflora. taken together, these results would provide a foundation for breeding yellow p. lactiflora varieties.  colour indices to obtain the same genetic background, a flower colour chimaera cultivar ‘jinhui’ with red outer-petal and yellow inner-petal was used to study the mechanism of yellow formation in p. lactiflora. firstly, their colour indices were measured. on one hand, the flower colours were defined according to the royal horticultural society color chart. during flower development, the colour gradation of the outer-petal ranged from 164a, 73a and 75b to 75c, while that of the inner-petal changed from 11a, 11b and 11c to 11d. on the other hand, flower colours were expressed as l*, a* and b*. in a uniform colour space, l* represented lightness, a* represented the ratio of red/magenta and green, and b* represented the ratio of yellow and blue. in four different developmental stages, l* values of the outer-petal and inner-petal were all gradually increased which was always lower in the outer-petal compared to the inner-petal; however, the b* value of the outer-petal and a* value of the inner-petal decreased, which all tended to zero. these colour indices showed that the colours of the outer-petal and inner-petal were pure red and yellow in s respectively, and then faded gradually with flower development. in addition, the flower diameter was also measured, such that the whole flower and inner-petal were similar and had constantly increased from s to s taken together, these measured results were consistent with the visual results.figure figures of p. lactiflora flower developmental stages, flower qualitative trait and color variables. s, flower-bud stage; s, initiating bloom stage; s, bloom stage; s, wither stage. rna-seq and de novo assembly to deeply elucidate the molecular mechanism underlying yellow colour formation, rna-seq analysis were performed using the illumina platform. each library of the outer-petal and inner-petal produced, and, total raw reads, following cleaning and quality checks, and, and, total clean reads were obtained with a single read length of 90 nt and a q percentage over %. these data showed that the rna-seq quality was better, which can be used for further analysis.table output statistics of rna-seq of p. lactiflora outer-petal and inner-petal subsequently, short reads from the outer-petal and inner-petal were assembled into and contigs, which contained an average length of and 337 nt, together with n length of and 664 nt, respectively. after linking the contigs together, and unigenes with an average length of and 617 nt of the outer-petal and inner-petal were identified, respectively, and their n values were all 1047 nt. finally, non-redundant all-unigenes were obtained according to splicing and reducing the redundancy of the unigenes sequence in the outer-petal and inner-petal, and its average length and n value were 763 nt and 1219 nt, respectively. among these, there were unigenes with a length of to 500 nt, which accounted for %, and excluding n rate reached 100%. all these results showed that the assembly quality was sufficiently high for gene function analysis. in addition, the distributions of the reads in all-unigenes were analysed, and these results showed that the number of reads in the 3′ and 5′ ends from all-unigenes was relatively low, particularly in the 3′ and 5′ tail ends. however, the number of reads located in to was relatively high, and its distribution was balanced.table data assembly of transcriptome contig and unigene in p. lactiflora outer-petal and inner-petal functional annotation due to the lack of a complete genome sequence in p. lactiflora, all-unigenes were blasted against six public databases, i.e., non-redundant protein database, swiss-prot protein database, kyoto encyclopedia of genes and genomes database, cluster of orthologous groups of proteins database, gene ontology database and non-redundant nucleotide database. these results revealed that all-unigenes were annotated, which accounted for %. of the all-unigenes,, and all-unigenes could be annotated to nr, nt and go databases, accounting for %, % and %, respectively, whereas in the swiss-prot, kegg and cog databases, only, and all-unigenes were annotated, which accounted for %, % and %. for the nr annotation, the ratio of the all-unigenes distributed in 10-15 < e-value < 10- and 0 < e-value < 10- with % and % was maximum; % of all-unigenes showed 60-95% similarity; and all-unigene sequences shared a similarity with available plant sequences from vitis vinifera, p. persica, ricinus communis, populus trichocarpa, fragaria vesca subsp. vesca, c. sativus and glycine max, including up to % similarity with vitis vinifera. when compared with the cog database, all-unigenes were divided into different functional classifications, and the functional types comprehensively involved most of the life activities. among these types, the maximum number of all-unigenes was categorised as general function prediction only, followed by transcription, replication, recombination and repair and posttranslational modification, protein turnover, chaperones, while the minimum number was classified to nuclear structure.figure figure of cog categories of the annotated all-unigenes. comparative analysis of degs and go functional annotation between the outer-petal and inner-petal to identify key genes controlling yellow formation in p. lactiflora, the transcriptomes of the outer-petal and inner-petal were comparatively analysed based on the outer-petal as the control group. as shown in figure  degs were obtained, among which, the expression levels of degs were up-regulated and the expression levels of degs were down-regulated. furthermore, abundant blue scatters indicated no difference in expression between the outer-petal and inner-petal. on the basis of sequence homology, these degs could be classified into three categories in go assignments: biological process, cellular component and molecular function, and they embraced and functional classifications, respectively. in the biological process category, cellular process and metabolic process accounted for the major proportion. in the cellular component category, and degs were enriched in the cell and cell part. under the molecular function category, catalytic activity and binding were dominant functions.figure figure of degs in p. lactiflora outer-petal and inner-petal. red scatters indicate up-regulated degs, green scatters indicate down-regulated degs, and blue scatters indicate no difference degs in expression between the outer-petal and inner-petal. figure of go categories of degs in p. lactiflora outer-petal and inner-petal. functional classification using kegg the pathway database was the major component of kegg, which contained most of the metabolic pathways representing information on the molecular interaction and reaction networks. in this study, degs were subsequently analysed in the kegg pathway database, where degs were assigned to kegg pathways and only pathways met the condition of q-value ≤ . among these pathways, metabolic pathways demonstrated the largest number of degs, followed by biosynthesis of secondary metabolites, plant-pathogen interaction, plant hormone signal transduction, endocytosis, starch and sucrose metabolism and glycerophospholipid metabolism. many studies revealed that colour development of the p. lactiflora petal was closely related to flavonoids, and our previous study also confirmed this conclusion. as expected, five pathways related to colour development were identified, namely phenylpropanoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, isoflavonoid biosynthesis and anthocyanin biosynthesis, and they all belonged to the flavonoid metabolic pathway which could be establish connection taking flavonoid biosynthesis as the ligament.figure interactive pathways during the outer-petal and inner-petal development. blue part is flavonoid metabolic pathway related to colour development. expression analysis of candidate degs related to colour development according to the genes annotated in kegg and the results of flower colour formation obtained in many plants, degs were selected for expression analysis. among these degs, pal, chi, f3h, f3′h, dfr, ans, 3gt and 5gt had been registered in genbank, and their accession numbers were jq jn jq jq jq jq jq and jq respectively. however, f3′5′h and fls were first found in p. lactiflora. among them, more than one unigene was annotated as the same enzyme, which might be because these unigenes represented different members of the same gene family, different fragments of a single transcript, or both, except pal and f3′5′h. as shown in figure  q-pcr was used to study their expression patterns in four different developmental stages of the outer-petal and inner-petal. single gene expression level analyses indicated that although the expressions of all degs were detected during flower development, but their expression levels were different. among the degs, the expression levels of plf3h and plf3′h were the highest, and downstream pldfr and pl5gt exhibited the lowest expression levels, while the other degs ranged between the two genes listed above. in terms of the overall trend, the expression levels of the degs were similar during the outer-petal and inner-petal development except in some stages. for example, the expression levels of plfls and pl3gt increased until they reached the maximum level during flower development, the other degs all showed a downward trend, and they only had some differences in quantity between the outer-petal and inner-petal. for different petals, the first key gene in flavonoid metabolism plpal; pldfr controlling the formation of leucoanthocyanidin; and pl3gt together with pl5gt, which catalyses the formation of the complex aglycone of anthocyanin composition, all had higher expression levels in the outer-petal compared to the inner-petal in the four different developmental stages, whereas f3′5′h showed an opposite trend.table candidate degs involved in p. lactiflora colour development pal chi f3h f3′h f3′5′h fls dfr ans 3gt 5gt no. all indicates the total number of degs, no. up indicates the number of degs in the inner-petal compared with the outer-petal in s no. down indicates the number of degs in the inner-petal compared with the outer-petal in s expression analysis of candidate degs related to colour development in p. lactiflora. qualitative and quantitative analysis of flavonoids to examine whether flavonoid accumulation in p. lactiflora petals was consistent with the expression patterns of candidate degs related to colour development, qualitative and quantitative analysis of flavonoids were performed using hplc-esi-msn in the same samples used in the expression analysis. on the basis of the ultraviolet–visible absorption characteristics, anthocyanins, anthoxanthins and chalcones were detected under the wavelength of 525 nm, 350 nm and 365 nm, respectively. as shown in figure  the chromatographic peaks of the outer-petal and inner-petal were identical, although there were only some differences in the peak area. the hplc retention time and ultraviolet–visible spectral properties together with mass spectrometric data of identified main chromatographic peaks were listed in additional file 4: table s moreover, two and seven peaks were identified at 525 nm and 350 nm, namely cyanidin- -di-o-glucoside, peonidin- -di-o-glucoside, kaempferol di-hexoside, kaempferol-3-o-malonylglucoside-7-o-glucoside, quercetin-3-o-galactoside, luteolin-7-o-galloylglucoside, luteolin-7-o-glucoside, isorhamnetin-3-o-glucoside and flavone c-glycoside, respectively.figure hplc chromatograms of p. lactiflora anthocyanins and anthoxanthins in s indicates hplc chromatograms of outer-petal; indicates hplc chromatograms of inner-petal; indicates hplc chromatograms of and merge together. a1-a indicates identified anthocyanins; f1-f indicates identified anthoxanthins. relevant standards were used as the references for the relative quantitative analysis of flavonoids, the results showed that there were tremendous differences in both the two different petals and also during different developmental stages of the same petals. for the composition, peonidin- -di-o-glucoside was the main component of anthocyanins in p. lactiflora outer-petal and inner-petal, which accounted for approximately % and % of the total anthocyanin contents in the outer-petal and inner-petal, respectively. in addition, the main component of anthoxanthins in p. lactiflora outer-petal and inner-petal was luteolin-7-o-glucoside, which comprised approximately % and % of the total anthoxanthin contents in the outer-petal and inner-petal, respectively, which was followed by kaempferol di-hexoside, luteolin-7-o-galloylglucoside and flavone c-glycoside. concerning the content of composition, the results showed that the content of each composition in the outer-petal was basically higher compared to the inner-petal, particularly for a and a of anthocyanins. in s the contents of a and a in the outer-petal were times as much as that of the inner-petal and increased to times in s in the case of different developmental stages, each anthocyanin and anthoxanthin composition in the outer-petal were basically decreased from s to s and slightly increased in s however, the compositions in the inner-petal were always decreased with flower development, except f f and f in s for total content, the total contents of anthocyanins, anthoxanthins and flavonoids in the outer-petal were all higher compared to the inner-petal, and specifically, the difference in anthocyanins was achieved by a multiple of meanwhile, the total contents of anthocyanins, anthoxanthins and flavonoids in the outer-petal and inner-petal all presented a downward trend as flower development, although there was an increase in the outer-petal at s furthermore, the co-pigment index, which determined whether had the co-pigment effect between anthoxanthins and anthocyanins demonstrated an increasing tendency in both the outer-petal and inner-petal. in the outer-petal, the ci had increased from in s to in s in contrast, and its developmental difference in the inner-petal was more significant and increased from in s to in s.table contents of anthocyanin and anthoxanthin compositions in p. lactiflora petals during flower development s, flower-bud stage; s, initiating bloom stage; s, bloom stage; s, wither stage. total content of anthocyanins, anthoxanthins and flavonoids, and co-pigmentation index in p. lactiflora outer-petal and inner-petal during flower development. discussion p. lactiflora is an important ornamental plant with a cultivation history of more than 4000 years in china. due to abundant flower colours, variable flower shapes, sharp flower fragrances and attractive flower poses, p. lactiflora exhibits a very high ornamental value and rich landscaping application value, which has become increasingly favoured by people worldwide. however, compared with plants in which genome sequencing has been completed, such as arabidopsis heynh.), mei, lotus, spider flower iltis) and carnation, a weak molecular basis and an incompletely clear genetic background in p. lactiflora severely limit its in-depth study. transcriptome refers to the identity of each expressed gene and its expression level for cells in a particular state, whose expression largely controls the phenotype of an organism. unlike the genome, the transcriptome is more dynamic and provides more valuable reference information for cell function interpretation, but its study is more inexpensive. due to overcoming technological limitations, transcriptome studies have been extensively developed in plants. moreover, according to a different purpose of the study, rna-seq can be divided into two methods: on one hand, different experimental materials are mixed equally to sequence, which can obtain the largest number of unique transcripts; on the other hand, different experimental materials are sequenced separately, which can isolate tissue-specific expression genes. wang et al. aimed to elucidate the transcriptome profiling of radish root and identify the critical gene response to lead stress, two radish root cdna samples, where one untreated control and the other pb-stressed with pb at 1000 mg were separately sequenced, and degs were obtained. in this study, to characterize the transcriptome of p. lactiflora red outer-petal and yellow inner-petal and identify the critical genes mediating yellow formation, illumina sequencing was firstly employed to the outer-petal and inner-petal with significantly different colours separately. each library of the outer-petal and inner-petal produced, and, total clean nucleotides with a q percentage over % in which the quantity and quality were all relatively high compared with other plants, such as the apple tree and m. rubra. subsequently, and unigenes with an average length of and 617 nt were found in the outer-petal and inner-petal, and degs were identified, which provided a large amount of information for molecular biological research in p. lactiflora. colour formation is a complex process that includes many metabolic pathways and gene expression. in the transcriptome study of colour, xu et al. speculated that enhanced photosynthesis and reduced downstream gene expression in the carotenoid biosynthetic pathway were the key reason for sweet orange red-flesh mutant lycopene accumulation, which controlled red formation. recently, two varieties of flesh in four developmental stages were used to perform rna-seq by this research group, which further confirmed the correlation between carotenoid metabolism and red colour formation in sweet orange red-flesh mutant. in m. rubra, feng et al. aimed to obtain a general overview of the transcriptome, four libraries containing mixed tissues, and three different developmental fruits were designed for rna-seq. this study identified unigenes, generated interactive pathways during bayberry fruit ripening, and found that the expression levels of flavonoid biosynthetic genes were overall increased, which was consistent with flavonoid accumulation and more intense colour. to the best of our knowledge in flower colour, transcriptome studies had been performed only on c. praecox and c. tinctorius. in the transcriptome database of c. praecox, unigenes could be annotated in different metabolic pathways. furthermore, unigenes with an average length of 677 bp were associated with the metabolic pathway of flower colour, including the flavonoid biosynthetic pathway, flavone and flavonol biosynthetic pathway and anthocyanin biosynthetic pathway, which was convenient for gene cloning and function identification. otherwise, the flavonoid biosynthetic pathway, flavone and flavonol biosynthetic pathway and anthocyanin biosynthetic pathway were identified according to the rna-seq comparison of red and white c. tinctorius. moreover, the expression levels of flavonoid biosynthetic pathway-related candidate unigenes were analysed, indicating that chs was very important for c. tinctorius flavonoid synthesis. in the present study, degs in the outer-petal and inner-petal had been annotated to metabolic pathways, and there were metabolic pathways enriched degs, which revealed the formation process complexity of different petals. among these pathways, the flavonoid metabolic pathway containing phenylpropanoid biosynthesis, flavonoid biosynthesis, anthocyanin biosynthesis, isoflavonoid biosynthesis, flavone and flavonol biosynthesis was annotated, which validated the conclusion that flavonoids were the pigment of p. lactiflora petal. moreover, carotenoid biosynthesis and zeatin biosynthesis were also found in metabolic pathways, which might be resulted from incomplete petalody stamens. interestingly, glucide metabolic pathways, including pentose and glucuronate interconversions, starch and sucrose metabolism, glycolysis/gluconeogenesis, other glycan degradation, fructose and mannose metabolism, amino sugar and nucleotide sugar metabolism and galactose metabolism were annotated. these pathways were not only associated with the growth and development of petals but were also related to the synthetic substrates of anthocyanins. flavonoid biosynthesis, which was controlled by many genes, and key structural genes in the flavonoid metabolic pathway were screened in this study. interestingly, the well-known key chalcone synthase gene catalyzing the formation of the chalcone derivative could not be found, which was the committed step leading to the biosynthesis of flavonoids, isoflavonoids and anthocyanins. this might be the reason that the expression of plchs in the inner-petal was not inhibited, and its level was not different with that in the outer-petal, which converted upstream materials to downstream materials in large quantity. in the expression analysis of the candidate degs, the expression of upstream plchi was also not inhibited in the inner-petal, instead its level was higher than that in the outer-petal, which was opposite to our previous results, indicating that chalcone was not accumulated in the inner-petal and did not cause its yellow colour. furthermore, the expression levels of downstream pldfr, plans, pl3gt and pl5gt in the inner-petal were all lower compared to the outer-petal, but this difference did not reach hundreds-fold as our previous results, suggesting that anthocyanins were also produced in the inner-petal, just its content was relatively low. flower colour was mainly dependent on the kinds of pigment, the content and its distribution in the petals. the generalised yellow flower from pale yellow to dark yellow could be divided into multiple grades according to its colour depth. among these colours, the pigment of the yellow flower perceived by the naked eye was relatively complicated, which was related to flavonoids and carotenoids, this conclusion had been fully proved by previous transcriptome studies. previous studies on the determination of p. lactiflora petal pigment composition showed that a great deal of chalcone and a small amount of anthoxanthin accumulation were the cause of its yellow petal formation. similarly, the biochemical mechanism of yellow characteristic formation in p. suffruticosa was the same, and kaempferol, quercetin, isorhamnetin, chrysoeriol, apigenin and luteolin were identified as the main anthoxanthins. this was mainly because anthocyanins were responsible for red, whereas chalcones and anthoxanthins were yellow. in this study, chalcones could not be detected in the yellow inner-petal, whereas a large number of anthoxanthins and a few anthocyanins were identified, which was not consistent with previous results. although anthocyanins were in the yellow inner-petal, the co-pigmentation effect of anthoxanthins in the inner-petal was significant and completely covered the red effect of anthocyanins due to the increase of ci from in s to in s in addition, compared with the red outer-petal, the contents of anthoxanthins and anthocyanins were relatively lower in the yellow inner-petal. these physiological results obtained perfect explanation using key gene expression analysis. due to the massive expression of upstream plpal and plchi, particularly plchi, a large number of upstream materials in the inner-petal were formed and transformed to the downstream, rather than in the accumulation of chalcones. with the expression of plf3h, plf3′h, plf3′5′h and plfls, a variety of anthoxanthins was formed and accumulated. furthermore, a small amount of anthocyanins was produced, which was combined with low expression levels of downstream pldfr, plans, pl3gt and pl5gt, and eventually made the inner-petal appear yellow. in addition, because the expression level of the first key gene, plpal, in the inner-petal was always lower than that in the outer-petal, combined with lower plfls, pldfr, plans, pl3gt and pl5gt expression, the accumulation of anthoxanthins and anthocyanins in the inner-petal was lower compared to the outer-petal. taken together, these results provided an understanding of the biochemical and molecular mechanisms of yellow characteristic formation in p. lactiflora, which might lay a theoretical foundation for breeding new yellow p. lactiflora varieties.  this study showed that rna-seq analysis based on the high throughput sequencing technology provided an efficient approach for identifying critical genes in p. lactiflora. in the metabolic pathways related to yellow formation, flavonoid metabolic pathway and glucide metabolic pathway were identified, and plpal, plfls, pldfr, plans, pl3gt and pl5gt were selected as potential candidates involved in flavonoid metabolic pathway, which induced inhibition of anthocyanin biosynthesis mediated yellow formation in p. lactiflora. 
subsequently, the expression patterns of candidate differentially expressed genes involved in the flavonoid metabolic pathway were examined, and flavonoids were qualitatively and quantitatively analysed. the flavonoid metabolic pathway and glucide metabolic pathway were identified as relatived yellow formation in p. lactiflora, plpal, plfls, pldfr, plans, pl3gt and pl5gt were selected as potential candidates involved in flavonoid metabolic pathway, which inducing inhibition of anthocyanin biosynthesis mediated yellow formation in p. lactiflora. herbaceous peony is a traditional flower in china and a wedding attractive flower in worldwide. anthocyaninsflower colourflavonoidsherbaceous peonytranscriptomeyellowissue-copyright-statement© the author 2014 this study could lay a theoretical foundation for breeding new yellow p. lactiflora varieties. in its flower colour, yellow is the rarest which is ten times the price of the other colours. in this study, two cdna libraries generated from p. lactiflora chimaera with red outer-petal and yellow inner-petal were sequenced using an illumina hiseq™ platform., and, total raw reads from red outer-petal and yellow inner-petal cdna libraries were generated, which were assembled into and unigenes with an average length of and 617 nt, respectively. in addition, all-unigenes were differentially expressed between the red outer-petal and yellow inner-petal, with up-regulated and down-regulated all-unigenes, and the flavonoid metabolic pathway related to colour development was identified using the kyoto encyclopedia of genes and genomes database. numerous anthoxanthins and a few anthocyanins were detected in the yellow inner-petal, which were all lower than those in the red outer-petal due to the low expression levels of the phenylalanine ammonialyase gene, flavonol synthase gene, dihydroflavonol 4-reductase gene, anthocyanidin synthase gene, anthocyanidin 3-o-glucosyltransferase gene and anthocyanidin 5-o-glucosyltransferase gene. however, the breeding of new yellow p. lactiflora varieties using genetic engineering is severely limited due to the little-known biochemical and molecular mechanisms underlying its characteristic formation. transcriptome sequencing analysis based on the high throughput sequencing technology was an efficient approach to identify critical genes in p. lactiflora and other non-model plants. moreover, non-redundant all-unigenes were obtained, with all-unigenes annotated in public databases.
 herbaceous peony is a traditional flower in china and a wedding attractive flower in worldwide. in its flower colour, yellow is the rarest which is ten times the price of the other colours. however, the breeding of new yellow p. lactiflora varieties using genetic engineering is severely limited due to the little-known biochemical and molecular mechanisms underlying its characteristic formation.  in this study, two cdna libraries generated from p. lactiflora chimaera with red outer-petal and yellow inner-petal were sequenced using an illumina hiseq™ platform., and, total raw reads from red outer-petal and yellow inner-petal cdna libraries were generated, which were assembled into and unigenes with an average length of and 617 nt, respectively. moreover, non-redundant all-unigenes were obtained, with all-unigenes annotated in public databases. in addition, all-unigenes were differentially expressed between the red outer-petal and yellow inner-petal, with up-regulated and down-regulated all-unigenes, and the flavonoid metabolic pathway related to colour development was identified using the kyoto encyclopedia of genes and genomes database. subsequently, the expression patterns of candidate differentially expressed genes involved in the flavonoid metabolic pathway were examined, and flavonoids were qualitatively and quantitatively analysed. numerous anthoxanthins and a few anthocyanins were detected in the yellow inner-petal, which were all lower than those in the red outer-petal due to the low expression levels of the phenylalanine ammonialyase gene, flavonol synthase gene, dihydroflavonol 4-reductase gene, anthocyanidin synthase gene, anthocyanidin 3-o-glucosyltransferase gene and anthocyanidin 5-o-glucosyltransferase gene.  transcriptome sequencing analysis based on the high throughput sequencing technology was an efficient approach to identify critical genes in p. lactiflora and other non-model plants. the flavonoid metabolic pathway and glucide metabolic pathway were identified as relatived yellow formation in p. lactiflora, plpal, plfls, pldfr, plans, pl3gt and pl5gt were selected as potential candidates involved in flavonoid metabolic pathway, which inducing inhibition of anthocyanin biosynthesis mediated yellow formation in p. lactiflora. this study could lay a theoretical foundation for breeding new yellow p. lactiflora varieties. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords anthocyaninsflower colourflavonoidsherbaceous peonytranscriptomeyellowissue-copyright-statement© the author 2014 