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. 2013 Apr 24:14:279.
doi: 10.1186/1471-2164-14-279.

Transcriptome analysis of Cymbidium sinense and its application to the identification of genes associated with floral development

Jianxia Zhang  1 , Kunlin Wu, Songjun Zeng, Jaime A Teixeira da Silva, Xiaolan Zhao, Chang-En Tian, Haoqiang Xia, Jun Duan
Affiliations

Transcriptome analysis of Cymbidium sinense and its application to the identification of genes associated with floral development

Jianxia Zhang et al. BMC Genomics. .

Abstract

Background: Cymbidium sinense belongs to the Orchidaceae, which is one of the most abundant angiosperm families. C. sinense, a high-grade traditional potted flower, is most prevalent in China and some Southeast Asian countries. The control of flowering time is a major bottleneck in the industrialized development of C. sinense. Little is known about the mechanisms responsible for floral development in this orchid. Moreover, genome references for entire transcriptome sequences do not currently exist for C. sinense. Thus, transcriptome and expression profiling data for this species are needed as an important resource to identify genes and to better understand the biological mechanisms of floral development in C. sinense.

Results: In this study, de novo transcriptome assembly and gene expression analysis using Illumina sequencing technology were performed. Transcriptome analysis assembles gene-related information related to vegetative and reproductive growth of C. sinense. Illumina sequencing generated 54,248,006 high quality reads that were assembled into 83,580 unigenes with an average sequence length of 612 base pairs, including 13,315 clusters and 70,265 singletons. A total of 41,687 (49.88%) unique sequences were annotated, 23,092 of which were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Gene Ontology (GO) analysis of the annotated unigenes revealed that the majority of sequenced genes were associated with metabolic and cellular processes, cell and cell parts, catalytic activity and binding. Furthermore, 120 flowering-associated unigenes, 73 MADS-box unigenes and 28 CONSTANS-LIKE (COL) unigenes were identified from our collection. In addition, three digital gene expression (DGE) libraries were constructed for the vegetative phase (VP), floral differentiation phase (FDP) and reproductive phase (RP). The specific expression of many genes in the three development phases was also identified. 32 genes among three sub-libraries with high differential expression were selected as candidates connected with flower development.

Conclusion: RNA-seq and DGE profiling data provided comprehensive gene expression information at the transcriptional level that could facilitate our understanding of the molecular mechanisms of floral development at three development phases of C. sinense. This data could be used as an important resource for investigating the genetics of the flowering pathway and various biological mechanisms in this orchid.

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Figures

Figure 1
Figure 1
The length distribution of assembled C. sinense contigs.
Figure 2
Figure 2
The length distribution of assembled C. sinense unigenes.
Figure 3
Figure 3
COG function classification of C. sinense unigenes.
Figure 4
Figure 4
GO classification of C. sinense unigenes.
Figure 5
Figure 5
Metabolic pathway of the circadian rhythm for unigenes by KEGG annotation.
Figure 6
Figure 6
Phylogenetic analysis of the CONSTANS or CONSTANS-like proteins from different plant species. The tree is displayed as a phylogram in which branch lengths are proportional to distance. Bootstrap values for 1,000 replicates were used to assess the robustness of the trees. The proteins are as follows: AtCO (accession no. NP_197088), AtCOL1 (accession no. NP_197089), AtCOL2 (accession no. NP_186887), AtCOL3 (accession no. Q9SK53), AtCOL4 (accession no. Q940T9.2), AtCOL5 (accession no. Q9FHH8), AtCOL6 (accession no. Q8LG76), AtCOL7 (accession no. Q9C9A9), AtCOL8 (accession no. Q9M9B3), AtCOL9 (accession no. NP_001118599), AtCOL10 (accession no. Q9LUA9), AtCOL11 (accession no. O23379), AtCOL12 (accession no. Q9LJ44), AtCOL13 (accession no. O82256), AtCOL14 (accession no. O22800), AtCOL15 (accession no. Q9C7E8), AtCOL16 (accession no. Q8RWD0), OsHd1 (accession no. ABB17664), PhalCOL (accession no. FJ469986), CsCO (accession no. GU168786).
Figure 7
Figure 7
Numbers of DGE unigenes in each comparison.
Figure 8
Figure 8
Differential expression genes related with flower development in C. sinense DGE. Each column represents an experimental sample (e.g. VP, FDP and RP) and each row represents a gene. Expression differences are shown in different colors. Red means high expression and green means low expression.
Figure 9
Figure 9
The qRT-PCR analysis of gene expression in VP (blue bars), FDP (red bars) and RP (green bars). The y-axis indicates fold change in expression among the samples VP, FDP, and RP using the results from RT-qPCR. The Lg(Relative Quantitation) of 16 genes in the VP was calibrated as zero.
Figure 10
Figure 10
Organs from C. sinense used to prepare DGE libraries for Illumine sequencing. (A) A young C. sinense plant in the vegetative phase (VP); (B) A mature C. sinense plant with floral bud (fb) in the floral differentiation phase (FDP); (C) A mature C. sinense plant with inflorescence (i) in the reproductive phase (RP).
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