*4.3. Semi-Quantitative PCR and Quantitative Real-Time PCR (qRT-PCR)*

Total RNA of *I. lactea* roots under Cd stress was extracted using Plant RNeasy Mini Kit (Vazyme) and cDNA was generated with PrimeScript® RT kit (Takara, Dalian, China). Oligo 6.0 was used to design primers for semi-quantitative PCR and qRT-PCR (Table 1). Semi-quantitative PCR and qRT-PCR were performed using *IlUBC* as the reference gene [20]. Each reaction mix of semi-quantitative PCR contained 1 μL Taq enzyme, 2 μL 10 × PCR Buffer (Mg2+ Free), 1.5 <sup>μ</sup>L Mg2+ (25 mmol·L<sup>−</sup>1), 1.3 <sup>μ</sup>L dNTP mixture (2.5 mmol·L−<sup>1</sup> each), 6 pmol of each primer and 1 <sup>μ</sup>L cDNA (about 1000 ng·μL−1). PCR was performed under the following cycling conditions: 94 ◦C for 4 min, 25 cycles of 94 ◦C for 30 s, 55◦C for 30 s, 72◦C for 30 s and 72 ◦C for 10 min. PCR products were detected by 1% agarose gel electrophoresis. qRT-PCR was performed using AceQ qPCR SYBR Green Master Mix

(Vazyme, Nanjing, China) and StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The reaction mixture consisted of 2 μL diluted cDNA, 10 μL 2 × SYBR Green Master Mix (Bimake, Houston, TX, USA), 0.4 μL ROX (Dye I), 1 μL of each forward and reverse primer and ddH2O to a total volume of 20 μL. The PCR procedure included an initial denaturation step set at 95 ◦C for 10 min, followed by 40 cycles of 95◦C for 15 s, 60 ◦C for 30 s and 72 ◦C for 30 s. Melting curve analyses of the amplified products were conducted at 60–95 ◦C. Three biological replicates were performed for each treatment and three technical replicates for each reaction. Expression levels were analyzed with the 2−ΔΔCT calculation method.
