Transcriptome sequencing and whole genome expression profiling of chrysanthemum under dehydration stress

Abstract

Background: Chrysanthemum is one of the most important ornamental crops in the world and drought stress seriously limits its production and distribution. In order to generate a functional genomics resource and obtain a deeper understanding of the molecular mechanisms regarding chrysanthemum responses to dehydration stress, we performed large-scale transcriptome sequencing of chrysanthemum plants under dehydration stress using the Illumina sequencing technology.

Results: Two cDNA libraries constructed from mRNAs of control and dehydration-treated seedlings were sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 98,180 unique transcripts which were further extensively annotated by comparing their sequencing to different protein databases. Biochemical pathways were predicted from these transcript sequences. Furthermore, we performed gene expression profiling analysis upon dehydration treatment in chrysanthemum and identified 8,558 dehydration-responsive unique transcripts, including 307 transcription factors and 229 protein kinases and many well-known stress responsive genes. Gene ontology (GO) term enrichment and biochemical pathway analyses showed that dehydration stress caused changes in hormone response, secondary and amino acid metabolism, and light and photoperiod response. These findings suggest that drought tolerance of chrysanthemum plants may be related to the regulation of hormone biosynthesis and signaling, reduction of oxidative damage, stabilization of cell proteins and structures, and maintenance of energy and carbon supply.

Conclusions: Our transcriptome sequences can provide a valuable resource for chrysanthemum breeding and research and novel insights into chrysanthemum responses to dehydration stress and offer candidate genes or markers that can be used to guide future studies attempting to breed drought tolerant chrysanthemum cultivars.

Figure 1

Length distribution of chrysanthemum unique transcripts.

Figure 2

Functional classification of chrysanthemum unique transcripts. (A), Within the category of biological process. (B), Within the category of molecular function.

Figure 3

Number of unique transcripts annotated as transcription factor and protein kinase in chrysanthemum transcriptome sequences. (A), Transcription factor. (B), Protein kinase.

Figure 4

Verification of RNA-seq results by qRT-PCR. Eighteen unique transcripts with significantly altered expression pattern in response to dehydration were selected from transcription factors, signal components, and biochemical pathways. (A), Comparison of expression level of unique transcripts between RNA-seq and qRT-PCR. Primers for qRT-PCR are listed in Additional file 4. (B), Scatter diagram of log ratios (Log₂ FC) of unique transcripts. qRT-PCR data were normalized using the ‘housekeeping’ gene CmUBI.

Figure 5

Number of unique transcripts annotated as transcription factor and protein kinase in response to dehydration stress. (A), Transcription factor. (B), Protein kinase.

Figure 6

Proline biosynthetic pathway in chrysanthemum under dehydration. P5CS, pyrroline-5-carboxylate synthetase; P5CR: pyrroline-5-carboxylate reductase; ProDH, proline dehydrogenase; OAT, ornithine aminotransferase. Genes in red and blue mean up- and down-regulated by dehydration, respectively. Numbers in brackets represent numbers of unique transcript regulated by dehydration stress.

Figure 7

ABA biosynthetic pathway in chrysanthemum under dehydration. NSY: neoxanthin synthase; NCED: 9-cis-epoxycarotenoid dioxygenase. Genes in red mean up-regulated by dehydration. Numbers in brackets represent numbers of unique transcript regulated by dehydration stress.

Figure 8

Chrysanthemum plants under dehydration and well-watered conditions.