Gene expression analysis by RT-qPCR
Expression analysis was performed as described in Samarth et al. (2019). RNA (1 μg) was used to prepare cDNA using a Superscript Reverse Transcriptase III kit (Invitrogen Inc.). The prepared cDNA was diluted 5-fold with double ionised water and subsequently used in the quantitative PCR (qPCR). The reaction mix contained 7.5 μL of Kappa SYBR mix, 4.5 μL of water, 1 μL each of the forward and reverse primers and 1 μL of cDNA to be used for the gene expression studies. The following PCR program was used to amplify the cDNA during the RT-qPCR reaction: 95 °C for 10 min hold, followed by 50 cycles of 95 °C for 10 s, 60 °C for 15 s and 72 °C for 20 s. The primer sequences used are listed in Table S2. In addition to CpFT1, CpFT2, CpFT3, CpFT4, CpFT5 and CpTFL1 , homologues of other floral-promoting genes known to regulate the expression of FT- like genes (Song et al., 2013), includingCpGI , CpHd1 , CpEhd3 , CpMADS1 , CpTPS1, CpMADS50 and a temperature regulator of flowering in temperate grasses (CpVRN1 ) were also included in the expression studies. Despite much effort, the expression of CpFT1 could not be detected in the leaf samples.