Transcriptome analysis of carnation (Dianthus caryophyllus L.) based on next-generation sequencing technology
© Tanase et al.; licensee BioMed Central Ltd. 2012
Received: 6 January 2012
Accepted: 2 July 2012
Published: 2 July 2012
Carnation (Dianthus caryophyllus L.), in the family Caryophyllaceae, can be found in a wide range of colors and is a model system for studies of flower senescence. In addition, it is one of the most important flowers in the global floriculture industry. However, few genomics resources, such as sequences and markers are available for carnation or other members of the Caryophyllaceae. To increase our understanding of the genetic control of important characters in carnation, we generated an expressed sequence tag (EST) database for a carnation cultivar important in horticulture by high-throughput sequencing using 454 pyrosequencing technology.
We constructed a normalized cDNA library and a 3’-UTR library of carnation, obtaining a total of 1,162,126 high-quality reads. These reads were assembled into 300,740 unigenes consisting of 37,844 contigs and 262,896 singlets. The contigs were searched against an Arabidopsis sequence database, and 61.8% (23,380) of them had at least one BLASTX hit. These contigs were also annotated with Gene Ontology (GO) and were found to cover a broad range of GO categories. Furthermore, we identified 17,362 potential simple sequence repeats (SSRs) in 14,291 of the unigenes. We focused on gene discovery in the areas of flower color and ethylene biosynthesis. Transcripts were identified for almost every gene involved in flower chlorophyll and carotenoid metabolism and in anthocyanin biosynthesis. Transcripts were also identified for every step in the ethylene biosynthesis pathway.
We present the first large-scale sequence data set for carnation, generated using next-generation sequencing technology. The large EST database generated from these sequences is an informative resource for identifying genes involved in various biological processes in carnation and provides an EST resource for understanding the genetic diversity of this plant.
KeywordsCarnation Dianthus caryopyllus L. Next-generation sequencing technology SSR Transcriptome
Carnation (Dianthus caryophyllus L.) is one of the most popular cut flowers, and hundreds of cultivars are grown around the world. Dianthus is a genus of about 300 species in the Caryophyllaceae family. Several species, including Dianthus caryophyllus D. barbatus D. chinensis D. plumarius D. superbus, and their hybrids are widely used as horticultural cultivars . The many flower varieties of carnation are divided into three groups (standards, sprays, and pot carnations) based on plant form, flower size, and flower shape. Standards have a single large flower per stem, whereas sprays have a larger number of smaller flowers; both types are used for cut flowers . Pot carnation is a dwarf with many small flowers that is used as a potted plant. Most carnation cultivars are diploid (2n = 2x = 30), although some species of Dianthus are tetraploid or hexaploid [3–6]. According to the Plant C-values Database (http://data.kew.org/cvalues/), the total genome size (C-value) in carnation is 613 Mb (1.23 pg/2 C), which is four times that of the model plant Arabidopsis (0.30 pg/2 C) . The genome of carnation is very small compared with those of other ornamental flowers, such as Antirrhinum majus (1,568 Mb), Chrysanthemum morifolium (9,384 Mb), Ipomoea nil (Pharbitis nil) (1,127 Mb), Lilium longiflorum (34,496 Mb), Petunia hybrida (1,642 Mb), Rosa hybrida (1,127 Mb), and Tulipa gesneriana (26,093 Mb).
Carnation cultivars are developed to be highly heterozygous so as to avoid the effects of inbreeding depression . Most commercially important cultivars are hybrids that are propagated vegetatively. Carnation cultivars have been bred for attractive characteristics such as flower color, flower size, fragrance, and flower longevity. Carnation cultivars have a wide range of colors, including red, yellow, white, green, and brown. In addition, some flowers show marginal variegation, flecks, or sectors . Recently, transgenic carnations with blue or violet flowers have been developed by the introduction of a heterologous flavonoid 3’, 5’-hydroxylase gene [10–12].
The vase life of cut flowers is one of the most important ornamental traits, because it affects consumer satisfaction and repeat purchasing. Carnation is a typical ethylene-sensitive flower [13, 14], and its flower life is normally short (about 7 days) if preservatives are not used . In the ethylene biosynthesis pathway, the conversion of S-adenosylmethionine (AdoMet) to 1-aminocyclopropane-1-carboxylate (ACC) and of ACC to ethylene are catalyzed by ACC synthase (ACS) and ACC oxidase (ACO), respectively. Transgenic carnations containing an antisense ACO gene exhibited low ethylene production and delayed petal senescence . When the Arabidopsis etr1-1 gene, capable of conferring ethylene insensitivity, was introduced into carnation, the transgenic carnation plants had reduced ethylene sensitivity caused by suppression of ACO expression, which prolonged flower life . On the other hand, by repeated selection for lines with longer vase life, Onozaki et al.  produced two carnation cultivars (named ‘Miracle Rouge’ and ‘Miracle Symphony’) with improved vase life in which expression of three ethylene biosynthesis genes (DcACS1 DcACS2, and DcACO1) was suppressed in flowers of both cultivars, which resulted in extremely low levels of ethylene production [18, 19].
Expressed sequence tag (EST) sequencing is essential for functional genomics studies: it has been used to identify novel genes from a broad range of organisms and to provide an indication of gene expression levels in specific tissues. Currently, there are more than 69 million ESTs in the database (dbEST) at NCBI. Since the development of high-throughput DNA sequencing technologies, analyses using next-generation sequencers have been performed in cereals, legumes, and fruits, and large amounts of EST data have been submitted to various DNA databases. These studies have revealed that high-throughput DNA sequencing is a cost-effective approach to analyzing the ESTs of both model plants and non-model plants. Surprisingly, in Arabidopsis, at least 60 transcripts which did not exist in previous EST collections were identified by next-generation sequencing . Furthermore, large-scale EST collection facilitates the design of microarrays and the high-throughput identification of simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNPs).
To identify the genes related to flower quality and important agronomic traits such as disease resistance, extensive gene expression profiling would be extremely valuable, but only 669 carnation ESTs were available on the NCBI website (http://www.ncbi.nlm.nih.gov/) at the early June 2012. Other genomics resources, such as markers and genomic sequences have yet to be developed for carnation. To improve the DNA sequence information available for carnation, we performed large-scale transcriptome sequencing of carnation using a next-generation sequencer (a Roche 454 GS FLX) and obtained more than 300,000 transcripts. This work will make a significant contribution toward plant physiology, biotechnology, and molecular genetics studies in carnation.
Results and discussion
EST sequencing and assembly
Summary of carnation transcripts data
Total high-quality reads
Reads in contigs
Total unique sequences (contigs plus singlets)
Size distribution of 454 sequencing reads after removal of adaptor sequences
Normalized cDNA library
Number of reads
Number of reads
SSR marker discovery
Number of di-, tri-, tetra-, and pentanucleotide simple sequence repeats (SSRs) identified in 1,417,410 reads obtained by 454 sequencing
Number of dinucleotide SSRs
Number of trinucleotide SSRs
Other trinucleotide repeats
(<5% of each one)
Number of tetranucleotide SSRs
Other tetranucleotide repeats
(<5% of each one)
Number of pentanucleotide SSRs
Other pentanucleotide repeats
(<5% of each one)
Very few genetic markers for horticulturally important characters in the major ornamentals, including carnation, have been identified . To our knowledge, only a few studies have reported SSR marker development in carnation [21, 31–33]. Smulders et al. [32, 33] developed 8 SSR markers from the EMBL database and evaluated the genetic diversity in Dianthus species. These SSR markers were also used for constructing a genetic linkage map of carnation . Kimura et al.  developed a set of 13 SSR markers and demonstrated their usefulness for genetic identification and hybridity confirmation of interspecific crosses in Dianthus species. Recently, a comprehensive set of 4,323 SSR primer pairs, representing 178 unique marker loci in 16 linkage groups, was developed and experimentally validated for carnation ; one of these loci was identified as a quantitative trait locus for carnation bacterial wilt resistance. In general, SSRs derived from ESTs are tightly linked with functional genes that may control useful characters. Furthermore, SSR markers can contribute to the construction of genetic linkage maps, genetic identification, and parentage analysis in Dianthus species.
Transcripts related to flower color
Red and yellow petal colors in higher plants are generally produced by anthocyanins and carotenoids, respectively, but species belonging to the order Caryophyllales show unique pigment composition in their flowers. In most of the Caryophyllales, red and yellow petal colors are derived from betalains; most of them accumulate neither anthocyanin nor carotenoids in their flowers. Carnation is an exception in that it accumulates anthocyanins and can express red and pink colors. The yellow petal color of carnation cultivars is derived from chalcone, a yellow flavonoid, rather than from carotenoids. Although chlorophylls are generally absent from the flowers of most plants, some carnation cultivars accumulate chlorophylls in their petals and have a green flower phenotype. It will therefore be interesting to investigate the expression of genes involved in the metabolism of these pigments in members of the Caryophyllales. The carnation EST database will provide useful information for future studies at the molecular level.
Carotenoid and chlorophyll metabolism
Carotenoid catabolism produces diverse apocarotenoid compounds that are essential for plant growth and reproduction . One category of these compounds (abscisic acid and strigolactone) is categorized as a plant hormone, and the others provide fruits and flowers with aromas and colors for attracting pollinators and seed dispersers. Such bioactive apocarotenoids are produced when carotenoids are cleaved by carotenoid cleavage dioxygenase (CCD). Analysis of the genome sequence of Arabidopsis led to the definition of nine clades of dioxygenases . Five of these, the 9-cis epoxycarotenoid dioxygenases (NCEDs; NCED2, NCED3, NCED5, NCED6, and NCED9) are involved in the synthesis of the plant hormone abscisic acid. The remaining four are involved in the synthesis of the plant hormone strigolactone (CCD7 and CCD8), in aroma formation (CCD1), and in the regulation of carotenoid content in the flower (CCD4). The carnation transcripts database contained sequences corresponding to two types of NCEDs, which showed high sequence similarity to NCED2 and NCED5, and one type of CCD, which showed high sequence similarity to CCD1.
Chlorophylls and carotenoids are essential pigments that play important roles in photosynthesis. In ‘Francesco’ carnation, high levels of these pigments were found in the leaves but not in the flowers (data not shown). Thus, the transcripts related to carotenoid and chlorophyll biosynthesis might have been derived from leaves. On the other hand, chlorophyll degradation is generally activated during leaf senescence. The absence of transcripts for some chlorophyll degrading enzymes might be explained by the fact that RNA was obtained from flowers and developing leaves but not from senescent leaves.
Anthocyanidins are modified by glycosylation and acylation, to form anthocyanins. These modifications play important roles in changing flower color, increasing water solubility, and enhancing pigment stability. Recently, two types of glucosyltransferase have been identified and characterized in carnation [45, 46]. Here, we found multiple transcripts encoding anthocyanidin glucosyltransferase and anthocyanin acyltransferase in the database. Thus, the carnation transcripts database will contribute to further investigations into the diversity of anthocyanin modification mechanisms.
Although the betalain biosynthesis pathway is poorly understood, several enzymes involved in this pathway have been identified and characterized [12, 47]. Among them, an transcripts encoding dihydroxyphenylalanine (DOPA) dioxygenase was found in the transcripts database; further investigation will be necessary to verify if a part of the betalain biosynthesis pathway is active in the carnation flower.
Ethylene biosynthesis and signaling
Distribution of carnation transcripts in the ethylene signaling pathway
Number of contigs
(ETHYLENE INSENSITIVE3-like) EIL
Ethylene-responsive-element–binding factor (ERF)
During flower senescence in carnation, a burst of ethylene production occurring in the gynoecium is followed by ethylene delivered to the petals, though the identity of the trigger signal molecule is still unknown. Autocatalytic ethylene production is induced by the signal, which in turn initiates downstream events in the senescence process such as lipid peroxidation and proteolytic activity [53, 54]. Therefore, there is much interest in the regulation of senescence by the expression of genes related to ethylene biosynthesis. In many ethylene-sensitive flowers, ACS and ACO are key steps in ethylene production, and transcript levels of the corresponding genes are rapidly upregulated at the ethylene burst stage [49, 50, 54]. These findings suggest that ACS and ACO gene expression is transcriptionally regulated in carnation.
As mentioned in the Background section, the improved cultivars ‘Miracle Rouge’ and ‘Miracle Symphony’ have very long flower life (average 18 days) and show much lower ethylene production than normal cultivars . In these improved cultivars, the expression levels of DcACS1 DcACS2 and DcACO1 were low throughout the experimental period , but sequencing of genomic DNA did not detect any mutations in these genes (Tanase, unpublished). On the other hand, custom-made cDNA microarrays of carnation showed that some transcripts encoding transcription factors, including EIN3-like (EIL) transcription factors, a putative MYB-like protein, a zinc finger protein, a MYC-type protein, and MADS-box proteins, were upregulated during flower senescence . In tomato, the MADS-box protein RIN (ripening inhibitor) regulates fruit ripening through direct activation of LeACS2[55, 56]. Other transcription factors such as TOMATO AGAMOUS-LIKE 1 MADS-box protein and tomato HD-Zip homeobox protein, which regulate fruit ripening, probably control the expression of ethylene biosynthesis genes [57, 58]. Our carnation database included many contigs related to transcription factor activity (11% of the Molecular Function ESTs) in the GO function analysis (Figure 1). Thus, the carnation transcripts database will contribute to further investigations into the regulation of ethylene biosynthesis and senescence programs in flowers.
In this study, an EST database was developed to enable broad characterization of the carnation transcriptome. We detected 17,362 potential simple sequence repeats (SSRs) in 14,291 unigenes and identified transcripts corresponding to genes associated with carotenoid biosynthesis, chlorophyll biosynthesis and degradation, anthocyanin (flavonoid) biosynthesis, and ethylene biosynthesis and signaling. This collection of transcripts from carnation will be useful for the annotation of the forthcoming carnation genome sequence and provide a remarkable resource for genomics studies in Caryophyllaceae.
Plant materials and RNA extraction
Carnation (Dianthus caryophyllus L.) cultivar ‘Francesco’ was grown under natural daylight conditions in a greenhouse as described previously . Each tissue was harvested from three plants. The following plant tissues were used: flower bud, flower (day 0 [full open flower], 3 days after full open, 8 days after full open, 4-h ethylene treated [10 μl·l–1, 20-h ethylene treated [10 μl·l-1), young and adult leaves, and stem (with shoot apex). Flowers contained sepals, petals, stamens and pistils. Tissues were immediately frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RNA concentration was estimated using an ND-1000 spectrophotometer (NanoDrop) (Thermo Scientific, Wilmington, DE, USA) and RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
cDNA library construction for 454 sequencing
For 454 sequencing, we made a normalized cDNA library and a 3’ cDNA library in cooperation with Takara Bio (Otsu, Shiga, Japan). RNA isolated from each tissue was combined in equal proportions in a single pool in an attempt to maximize the diversity of transcriptional units sampled. The Clontech SMART system (Clontech, Mountain View, CA, USA) was used for cDNA synthesis from the total RNA.
To construct the normalized cDNA library, the cDNA was normalized by digestion with a duplex-specific nuclease. The normalized cDNA was amplified under the following conditions: 95°C for 20 s, followed by 25 cycles of 95°C for 5 s and 68°C for 8 min. The PCR primers were as follows: TD-5-P2 primer, 5’-GAGTGGCCATTACGGCCGGG-3’; biotinylated (T18)VN B-adaptor oligo, 5’-biotin-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGTTTTTTTTTTTTTTTTTTVN-3’. After purification, the quantity of amplified cDNA was estimated using an ND-1000 spectrophotometer (NanoDrop) and the quality was evaluated using an Agilent 2100 Bioanalyzer. Approximately 5 μg of amplified cDNA was sheared into small fragments about 800 bp in length using an Acoustic Solubilizer (Covaris, Woburn, MA, USA). The cDNA library was constructed according to the manufacturer’s instructions in the Roche GS FLX Titanium General Library Preparation Method Manual.
For the 3’ cDNA library, we used the modified method of Eveland et al. (2008) . Approximately 10 μg of amplified cDNA was sheared into small fragments about 800 bp in length with an Acoustic Solubilizer (Covaris). The cDNA fragments were selected by size, 400–1000 bp, using gel-cut and eluting them. The 3’ ends of the fragments were purified by using streptavidin-coated magnetic beads. Titanium A-adaptors (Roche, Basel, Switzerland) were ligated to the purified 3’ fragments, and the single-stranded 3’ cDNA was treated with 100 mM NaOH, neutralized, and purified. The quality of the 3’ cDNA library was assessed as described above for the normalized cDNA library. The 454 sequencing was performed according to the manufacturer’s instructions in the Roche GS FLX Titanium Sequencing Method Manual.
To construct the cDNA library for Sanger sequencing, poly(A)+ RNA from aerial part of carnation plant was purified using Oligotex-dT30 Super (Nippon Roche, Tokyo, Japan), and cDNA was synthesized by using a cDNA synthesis kit (Agilent Technologies) according to the manufacturer’s instructions. The size selection of cDNA and cloning into a pBluescript II SK– plasmid were performed as previously described . For generation of ESTs, plasmid DNAs were prepared from the colonies and sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA). The reaction mixtures were run on an automated DNA sequencer (ABI PRISM 3730; Applied Biosystems).
Both the 454 sequences and Sanger sequences were trimmed of adaptor and low-quality sequence regions. All sequences were assembled and annotated by BLASTN searches of the NCBI database.
dditionally, the non-redundant set of consensus cDNA sequences represented by two or more reads (37,844 sequences) was annotated by BLAST searches of Arabidopsis cDNA databases. Functional classifications of these sequences were based on GO terms from the GO Slim classification in TAIR (http://www.arabidopsis.org).
Assembled transcripts of Carnation (Dianthus caryophyllus L.) were submitted to the Mass Submission System of DDBJ with the accession numbers FX296474 to FX334317.
Detection of SSR markers
All of the assembled sequences from the 454 reads were used for detection of SSRs. SSRs in the total unique putative transcripts were detected by using the MISA program (http://pgrc.ipk-gatersleben.de/misa/), which accepts FASTA-formatted sequence files. Sequences containing di-, tri-, tetra-, and pentanucleotide repeats were selected.
This study was supported by a grant from the project “Development of Innovative Crops through the Molecular Analysis of Useful Genes” of the National Agriculture and Food Research Organization (NARO), Japan.
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