*2.3. Transcriptome Sequencing in Leaves of "Variegatum" and "Green Pittosporum" and Functional Annotation of Unigenes*

To get an insight into the molecular pathways and genes conferring the strong response to low temperature in "Variegatum", we synthesized six cDNA libraries from leaves collected from "Variegatum" and "Green Pittosporum" plants under cold conditions (10 ◦C) and generated *de novo* RNA-sequencing data for the first time in *P. tobira*.

The RNA-seq yielded a total of 40.88 Gb clean data with 92.78% of bases scoring Q30 and above (Table 1). The assembly was performed using the Trinity software and a total of 112,875 unigenes were obtained with N50 length about 1,017 bp. The assembly integrity was high and specific statistics are shown in Table 2. A total of 51,718 unique genes were functionally annotated based on various databases (Table 3, Table S1). The clean data of each sample was serialized with the assembled unigene libraries and the mapping result statistics are presented in Table 4. Of these genes, 19,677 genes were expressed with the number of fragments per kilobase of exon per million fragments mapped (FPKM) values ranging from 0.04 to 22578.37 (Table S2, Figure 5A).

Hierarchical clustering of the samples based on FPKM showed that all the biological replicates clustered together, suggesting a high reliability of our RNA-sequencing data (Figure 5B). Moreover, a clear separation of the two leaf sample types was observed, implying that a large number of genes may be differentially expressed between the two cultivars to explain the relative stronger response to cold stress in "Variegatum".

**Figure 5.** Overview of the transcriptome sequencing. (**A**) Gene expression profile in the 6 libraries. T01-T03 represent the three replicates libraries of the cultivar "Green Pittosporum" and T04-T06 represent the three replicates libraries of the cultivar "Variegatum". (**B**) Heatmap clustering showing correlation among *P. tobira* different samples based on global expression profiles. Numbers in the heatmap represent the Pearson correlation value.




**Table 2.** Statistics of the assembly results.

**Table 3.** Functional annotation statistics of the unigenes.


**Table 4.** Statistics of the mapping of sequencing data with assembly results.

