*2.4. Di*ff*erential Gene Expression Analysis between "Variegatum" and "Green Pittosporum" under Cold Condition*

The differential gene expression analysis was performed on all expressed genes by comparing their expression levels between "Variegatum" and "Green Pittosporum". As shown in Figure 6A, a total of 309 differentially expressed genes (DEG) were obtained, including 156 up-regulated and 153 down-regulated genes in the variegated leaves. To validate our differential expressed gene result, we selected five up-regulated genes and five down-regulated genes (Table S3) and performed qRT-PCR using the cDNAs from leaves of the two cultivars. The qRT-PCR results were strongly correlated with the RNA-seq data (R<sup>2</sup> = 0.89, Figure S1). This result confirms well the high reliability of the RNA-seq data obtained in the present study.

We performed gene ontology (GO) enrichment analysis of these DEGs based on three ontologies: biological process, cellular component and molecular functions. In the biological process components, metabolic and cellular process was found to be the most dominant group (Figure 6B). Within the cellular components, cell and cell part represented the most dominant functional groups. Meanwhile, the catalytic activity and binding were the most abundant functional groups among the molecular functions, showing that enzymes and transcription factors encoding genes may play key roles in the differential cold response. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the DEGs showed that the biosynthesis of unsaturated fatty acids, sesquiterpenoid and triterpenoid biosynthesis, fatty acid metabolism, phenylalanine metabolism and protein processing in endoplasmic

reticulum were the main pathways contributed by the DEGs (Figure 6C). The diversity of these molecular pathways highlights the complex mechanism of the improved cold response in relation with leaf variegation in *P. tobira*.

**Figure 6.** Differentially expressed genes (DEG) analysis between "Green Pittosporum" and "Variegatum". (**A**) Volcano plot depicting the up-, down- and no- regulated genes between the two cultivars. (**B**) Gene ontology enrichment analysis of the DEGs. (**C**) KEGG enrichment analysis of the DEGs. \* represent the significantly enriched pathways.
