4.2. cDNA Isolation, Sequence Analysis, and Phylogenetic Tree Analysis of MdFLC

To isolate MdFLC1 cDNA, the apple EST sequences were searched using DDBJ and GDR. DDBJ was also used to search homologous genes in other plants. The cetyltrimethylammonium bromide (CTAB) method [27] and a TaKaRa RNA PCR kit (AMV) (Takara-Bio, Kusatsu, Japan) were used for RNA extraction and reverse transcription, respectively. Cloning of MdFLC3 cDNA was performed by PCR with degenerate primers MdMADSF and MdMADSR (Table S1, Supplementary Materials) based on highly conserved sequences in the MADS-box protein using PROSITE (https://prosite.expasy.org). The restriction sites of EcoRI and BamHI were added to the primers in advance. PCR was performed with cDNA from leaves in the juvenile and adult phases. PCR products were electrophoresed on agarose gel, and amplified fragments of the expected size were collected using TaKaRa RECOCHIP (TaKaRa). pUC18 was digested with EcoRI and BamHI, and the PCR fragments were ligated into this vector and then transformed into Escherichia coli. The plasmid was purified for sequence analysis. Sequence Alignment by ClustalW (http://align.genome.jp) was used to prepare the sequence alignments and phylogenetic trees.