Quantification of Gene Expression by Real-Time PCR.

Total RNA was extracted from leaf primordium samples of Callitriche species using the FastGene RNA Premium Kit (Nippon Genetics). Each sample comprised >10 leaf primordia of approximately the same length. The extracted RNA was then used for reverse transcription with the PrimeScript RT Reagent Kit (TaKaRa Bio) to synthesize cDNA. Real-time PCR was performed using Thunderbird SYBR qPCR Mix (Toyobo) on a Roche LightCycler 480 II PCR platform. We used the SAND1 gene, which shows stable expression in multiple plant species (58, 59), as an internal control. We also confirmed that CpSAND1 was stably expressed in all tissues and conditions in our RNA-Seq data (31) (SI Appendix, Fig. S3A). We quantified the expression of SPCH and MUTE orthologs in the three biological replicates described above using the comparative CT method (Fig. 4). The results from three technical replicates were averaged and are shown in Fig. 4. The primers used for real-time PCR are listed in SI Appendix, Table S2.

For C. palustris, we designed primers based on the sequence of one of the two putative paralogs of SPCH and MUTE. However, considering the high sequence similarity and highly similar expression patterns of the two paralogs (SI Appendix, Fig. S3), we did not design primers that amplified each paralog specifically. We assumed that our primers amplified both paralog and thus labeled the results as “CpSPCH”, for example.