Fig. 2

qPCR data describing the fold change in gene expression in cannabis leaves across SD transition. Four biological replicates per assay, from low RFR group. UB used as reference, delta-delta Ct method used to calculate relative expression change. In each assay, the last long day is set as control (1). For all experiments, student’s t-test was utilized for calculating statistical significance of means separation. One star (*) indicates a P-value below 0.05, while two stars (**) indicates a P-value below 0.005. Panel A, CO-like genes: All but one CO-like genes were downregulated promptly by the third short day, which persisted into the seventh short day. COL9 expression remained relatively unchanged, which is characteristic of AtCO. The greatest fold change was exhibited by CsCOL10. Trend line indicates average fold change across all COL genes. Individual expression values across all biological replicates and genes used to calculate fold change, standard error and significance. Overall, the CO-like gene family in cannabis was down-regulated significantly upon SD transition. A one-way ANOVA comparing LLD to SD7 revealed significant differences in gene expression between groups across CO-like genes (F(1, 44) = 14.81, p < 0.001). Post-hoc Tukey’s HSD test indicated significantly lower expression in SD7 compared to LLD (***p < 0.001). Second Y axis quantifies trend line. Panel B, PRR and WNK: PRR37 is heavily downregulated upon SD transition. Contrary to the DESeq results, qPCR results for APRR3 suggest that it was upregulated slightly by the third short day, while WNK4 was upregulated slightly by the seventh day. The qPCR results for WNK9 are consistent with the DESeq. Panel C: Most genes are marginally upregulated while TOE1 is downregulated. Although the fold change increase in expression of DICER is significantly lower in the qPCR assay than the DESeq, we still consider the data to confirm that expression levels are increased by the seventh short day. Four biological replicates per assay, from low RFR group, except in the case of DICER and TOE1 where 8 replicates are used from both the high and low RFR groups.