*4.4. RNA Extraction, cDNA Library Construction, and Transcriptome Sequencing*

The complete leaves from the cultivars "Green Pittosporum" and "Variegatum" were collected in replicates from three different plants under cold conditions at November 15th (Temperature = 10 ◦C), immediately frozen in liquid nitrogen and stored at −80 ◦C until further use. Total RNAs were extracted using Spin Column Plant total RNA Purification Kit following the manufacturer's protocol (Sangon Biotech, Shanghai, China). Purity of the extracted RNAs was assessed on 1% agarose gels followed by NanoPhotometer spectrophotometer (IMPLEN, Los Angeles, CA, USA). We quantified the RNA using Qubit RNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, Carlsbad, CA, USA). RNA integrity was checked using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).

Libraries preparation, and sequencing on Illumina HiSeq 4000 platform (Illumina Inc., San Diego, CA, USA) were performed as described by Zhuang et al. [78].

## *4.5. De novo Assembly, Functional Annotation, Classification and Metabolic Pathway Analysis*

Raw transcriptome data were submitted to NCBI SRA, freely accessible at www.ncbi.nlm.nih. gov/bioproject/PRJNA553027. The clean reads were retrieved after trimming adapter sequences, removal of low quality (containing > 50% bases with a Phred quality score < 15) and reads with unknown nucleotides (more than 1% ambiguous residues N) using the FastQC tool (http://www. bioinformatics.babraham.ac.uk/projects/fastqc/). The high-quality reads from all the six libraries were de novo assembled into transcripts using Trinity (Version r20140717) [79] by employing paired-end method. Next, the transcripts were realigned to construct unigenes. The assembled unigenes were then annotated by searching against various databases such as Kyoto Encyclopedia of Genes and Genomes (KEGG) [80], Gene Ontology (GO) [81], Clusters of Orthologous Groups (COG) [82], Pfam [83], Swissprot [84], egNOG [85], NR [86], euKaryotic Orthologous Groups (KOG) [87] using BLAST [88] with a threshold of E-value <1.0 E−<sup>5</sup> .

The software KOBAS2.0 [89] was employed to get the unigene KEGG orthology; the analogs of the unigene amino acid sequences were searched against the Pfam database [83] using HMMER tool [90] with a threshold of E-value < 1.0 E−10. The sequenced reads were compared with the unigene library using Bowtie [91], and the level of expression was estimated in combination with RSEM [92]. The gene expression level was determined according to the fragments per kilobase of exon per million fragments mapped (FPKM).
