Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology

Abstract

Background: Chrysanthemum morifolium is one of the most economically valuable ornamental plants worldwide. Chrysanthemum is an allohexaploid plant with a large genome that is commercially propagated by vegetative reproduction. New cultivars with different floral traits, such as color, morphology, and scent, have been generated mainly by classical cross-breeding and mutation breeding. However, only limited genetic resources and their genome information are available for the generation of new floral traits.

Results: To obtain useful information about molecular bases for floral traits of chrysanthemums, we read expressed sequence tags (ESTs) of chrysanthemums by high-throughput sequencing using the 454 pyrosequencing technology. We constructed normalized cDNA libraries, consisting of full-length, 3'-UTR, and 5'-UTR cDNAs derived from various tissues of chrysanthemums. These libraries produced a total number of 3,772,677 high-quality reads, which were assembled into 213,204 contigs. By comparing the data obtained with those of full genome-sequenced species, we confirmed that our chrysanthemum contig set contained the majority of all expressed genes, which was sufficient for further molecular analysis in chrysanthemums.

Conclusion: We confirmed that our chrysanthemum EST set (contigs) contained a number of contigs that encoded transcription factors and enzymes involved in pigment and aroma compound metabolism that was comparable to that of other species. This information can serve as an informative resource for identifying genes involved in various biological processes in chrysanthemums. Moreover, the findings of our study will contribute to a better understanding of the floral characteristics of chrysanthemums including the myriad cultivars at the molecular level.

Keywords: Chrysanthemum morifolium; Expressed sequence tag; Next-generation sequencing technology; Transcription factor; Transcriptome.

Fig. 1

A photograph of a flower of the Chrysanthemum morifolium cultivar ‘Sei-Marine’ used in this study

Fig. 2

Classification of transcripts into functional categories according to Arabidopsis Gene Ontology. a cellular component; b molecular function; and c biological process

Fig. 3

Classification of DXS genes. a Phylogenetic tree of DXS genes of Arabidopsis and chrysanthemum. According to our definition, same-colored contigs are classified into the same cluster. b–d Amino-acid alignment of the contigs in each cluster. Contigs in the same cluster share very high sequence homology

Fig. 4

The concept of the clusters. Contigs within the same cluster top-hit to the same Arabidopsis gene with BLAST E-value <1.0e-05 and very high sequence homology (BLAST E-value <1.0e-100) with at least one contig constituting the cluster. In this diagram, 28 contigs are consolidated into three clusters

Fig. 5

Phylogenetic tree of chrysanthemum class-B MADS-box proteins. Deduced amino-acid sequences of class-B proteins found in the chrysanthemum EST data (Cluster ID of our data) and those in other plant species (Additional file 2: Table S2) were compared and a phylogenetic tree was constructed using the neighbor-joining method. For the phylogenetic analysis, a chrysanthemum contig that was the most homologous to the Arabidopsis ortholog at the amino-acid levels was used as a representative of the clusters in this study

Fig. 6

Distribution of chrysanthemum ESTs in the carotenoid biosynthesis pathway. Each enzyme name is followed, in parentheses, by the number of contigs homologous to gene families that encode this enzyme. PSY, phytoene synthase; PDS, phytoene desaturase; Z-ISO, 15-cis-ζ-CRTISO; ZDS, ζ-carotene desaturase; CRTISO, carotenoid isomerase; LCYB, lycopene β-cyclase; LCYE, lycopene ε-cyclase; CHYB, β-ring hydroxylase; CHYE, ε-ring hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; and NSY, neoxanthin synthase

Fig. 7

A phylogenetic tree of the chrysanthemum CCD4. Deduced amino-acid sequences of CCD4 found in the chrysanthemum EST database (indicated by EST IDs) and those previously identified in the chrysanthemum cultivar ‘Jimba’ (indicated by GenBank accession numbers in Additional file 4: Table S4) were compared, and a phylogenetic tree was constructed using the neighbor-joining method. 4a, 4b, and 4c indicates CCD4a, CCD4b, and CCD4c subfamilies, respectively

Fig. 8

Distribution of chrysanthemum ESTs in the terpenoid biosynthetic pathway. Each enzyme name is followed, in parentheses, by the number of contigs homologous to the gene families that encode this enzyme. MEP, 2-C-Methyl-D-erythritol 4-phosphate; MEV, Mevalonate; G3P, Glyceraldehyde 3-phosphate; DXP, 1-Deoxy-D-xylulose 5-phosphate; CDP-ME, 4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol; MECDP, 2-C-Methyl-D-erythritol-2,4-cyclodiphosphate; HMBPP, (E)-4-Hydroxy-3-methylbut-2-en-1-yl diphosphate; IPP, Isopentenyl diphosphate; DMAPP, Dimethylallyl diphosphate; GPP, Geranyl diphosphate; FPP, Farnesyl diphosphate; HMG-CoA, (S)-3-Hydroxy-3-methylglutaryl-CoA; DXS, 1-Deoxy-D-xylulose-5-phosphate synthase; DXR, 1-Deoxy-D-xylulose-5-phosphate reductoisomerase; ISPD, 2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase; ISPE, 4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase; ISPF, 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase; ISPG, 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase; ISPH, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IPPI, Isopentenyl diphosphate isomerase; AAT, Acetyl-CoA acetyltransferase; HMGS, HMG-CoA synthase; HMGR, HMG-CoA reductase; MVK, Mevalonate kinase; PMK, Phosphomevalonate kinase; DPM-DC, Diphosphomevalonate decarboxylase; GPPS, GPP synthase or FPP synthase; and TPS, Terpene synthase

Fig. 9

A phylogenetic tree of terpene synthase (TPS). Deduced amino-acid sequences of chrysanthemum (Cluster ID of our data) and those of similar protein family members (Genbank accession number in Additional file 5: Table S5) previously identified in higher plants were compared, and a phylogenetic tree was constructed using the neighbor-joining method. For the phylogenetic analysis, the chrysanthemum contig that was the most homologous to the Arabidopsis ortholog at the amino-acid level was used as a representative of the clusters in this study