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.