Quantitative RT-PCR

To verify the results of the DEG analyses, 20 genes with differential expression were randomly selected and measured by qRT-PCR, as described previously (Chao et al. 2014). Leaves at mature and old stages were harvested and total RNA from each was isolated using a Plant RNA kit. Genomic DNA was removed at a volume of 10 µL, containing 2 µL 5 × gDNA Eraser Buffer (TaKaRa, Dalian, China), 1 µL gDNA Eraser (TaKaRa, Dalian, China) and 7 µL total RNA, under the condition of 42 °C for 2 min. The cDNA was synthesized according to the manual of PrimeScript RT reagent Kit (TaKaRa, Dalian, China). The reaction solution consisted of 5 μL gDNA-free RNA, 1 μL PrimeScript RT Enzyme Mix I, 1 μL RT Primer Mix (containing 2 primers, Oligo d(T) and random 6 mers), 4 μL 5 × PrimeScript Buffer 2 and 9 μL RNase Free dH₂O, and the reverse transcript was performed under the conditions of 37 °C for 15 min and then 85 °C for 5 s. Each cDNA was diluted 2 times with dH₂O and 1 μL diluted product was used for qRT-PCR. The qRT-PCR was performed in 96-well blocks with an RT-PCR system (CFX Connect, BIO-RAD, Hercules, California, USA) using an UltraSYBR Mixture (CWbio, Beijing, China) at a volume of 25 µL, which contained 12.5 μL 2 × UltraSYBR Mixture, 1 μL forward primer, 1 μL reverse primer, 1 μL cDNA and 9.5 μL dH₂O. A two-step amplification protocol included initial denaturation at 95 °C for 10 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min; and a melt curve step at the end of the PCR (95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, 60 °C for 15 s). The relative expression was calculated using the 2^−∆∆Ct method. All results are presented as the means of three independent RNA extractions (three biological replicates with three technical replicates) with corresponding standard deviations (SD). Based on the Illumina sequencing results, the red clover UBQ gene (TGAC_v2_mRNA12221) was used as an internal control, and all primers used in the qRT-PCR analyses are reported in Supplementary Table 3.