Transcriptomic Analyses of Camellia oleifera 'Huaxin' Leaf Reveal Candidate Genes Related to Long-Term Cold Stress

Abstract

'Huaxin' is a new high-yielding timber cultivar of Camellia oleifera of high economic value, and has been widely cultivated in the red soil hilly region of Hunan Province of the People´s Republic of China in recent years. However, its quality and production are severely affected by low temperatures during flowering. To find genes related to cold tolerance and further explore new candidategenes for chilling-tolerance, Illumina NGS (Next Generation Sequencing) technology was used to perform transcriptomic analyses of C. oleifera 'Huaxin' leaves under long-term cold stress. Nine cDNA libraries were sequenced, and 58.31 Gb high-quality clean reads were obtained with an average of 5.92 Gb reads for each sample. A total of 191,150 transcripts were obtained after assembly. Among them, 100,703 unigenes were generated, and 44,610 unigenes were annotated. In total, 1564 differentially expressed genes (DEGs) were identified both in the A_B and A_C gene sets. In the current study, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed, andrevealed a group of cold-responsive genes related to hormone regulation, photosynthesis, membrane systems, and osmoregulation; these genes encoded many key proteins in plant biological processes, such as serine/threonine-protein kinase (STPK), transcription factors (TFs), fatty acid desaturase (FAD), lipid-transfer proteins (LTPs), soluble sugars synthetases, and flavonoid biosynthetic enzymes. Some physiological indicators of C. oleifera 'Huaxin' were determined under three temperature conditions, and the results were consistent with the molecular sequencing. In addition, the expression levels of 12 DEGs were verified using quantitative real-time polymerase chain reaction (qRT-PCR). In summary, the results of DEGs analysis together with qRT-PCR tests contribute to the understanding of cold tolerance and further exploring new candidate genes for chilling-tolerance in molecular breeding programs of C. oleifera 'Huaxin'.

Keywords: Camellia oleifera; DEGs; long-term cold stress; transcriptome sequence.

Figure 1

Effect of different temperaturetreatments on phenotypic changes, chlorophyll contents, net photosynthetic rate (P_n), electron transfer rate (ETR), soluble sugar, starch content, and relative water contentin Camelliaoleifera‘Huanxin’ leaves. (a) 25 °C; (b) 6 °C; (c) Low environmental temperature; (d) Chlorophyll content; (e) Net photosynthetic rate; (f) Electron transfer rate; (g) Soluble sugar content; (h) Starch content; (i) Relative water content. Data represent the mean ± SE (n = 3). Lowercase letters indicate significant differences among different temperature treatments at p ≤ 0.05 according to Duncan’s multiple range test (DMRT).

Figure 2

Functional annotations of the assembled transcriptome. (a) NR annotated species distribution map similar to the Camellia oleifera ‘Huaxin’ transcriptome; (b) GO classification of the annotated unigenes; (c) KOG function classification of the consensus sequence.

Figure 3

The graphical representation of differentially expressed genes (DEGs) of Camellia oleifera ‘Huaxin’ in response to low-temperature stress. (a) Number of up/down-regulated DEGs in the A_B, A_C, and B_C differential expression gene sets; (b) Venn diagram showing all DEGs numbers among the three gene sets; (c) Venn diagram showing up-regulated genes DEGs numbers among the three gene sets; (d) Venn diagram showing down-regulated DEGs numbers among the three gene sets.

Figure 4

Analyses of the transcription factors (TFs) involved in Camellia oleifera ‘Huaxin’ response to low-temperature stress. (a) Distribution of transcription factor families in the A_B gene set; (b) Distribution of transcription factor families in the A_C gene set; (c) Expression profiles of 18 differentially expressed TFs among different samples. The heat map was generated from the log10 (FPKM values). Changes in expression level are represented by a change in color; green indicates a lower expression level, whereas red indicates a higher expression level. FPKM: fragments per kilobase of transcript per million mapped reads

Figure 5

The differentially expressed genes in the hormone signaling pathways in response to low temperature in Camellia oleifera ‘Huaxin’. (a) The green and red boxes show the mapped differentially expressed genes in the plant hormone signaling pathways, as modified from a Kyoto Encyclopedia of Genes and Genomes map (KO 04075), and the green boxes indicate down-regulated genes, whereas red boxes indicate up-regulated genes. (b) The expression patterns are shown as a heat map. Changes in expression level are indicated by a change in color, green indicates a lower expression level, whereas red indicates a higher expression level.

Figure 6

Differentially expressed genes (DEGs) related to photosynthesis pathways in response to low temperature in Camellia oleifera ‘Huaxin’. (a) and (b) Green and red boxes show the mapped DEGs in the ‘Photosynthesis’ and ‘Photosynthesis—antenna proteins’ pathways, as modified from the KEGG maps (KO 00195 and KO 00196). (c) A comparison of the gene expression patterns. Changes in expression level are indicated by a change in color, green indicates a lower expression level, whereas red indicates a higher expression level.

Figure 7

The comparison of the expression levels of 12 DEGs identified related to cold acclimation between RNA-Seq and qRT-PCR analyses from three samples treated at different temperatures.