Quantitative Real-Time PCR Analysis

Quantitative real-time PCR was determined using the mature method [35 (link)]. The PCR instrument and reagent were a StepOnePlus real-time PCR system (Applied Biosystems) and a SYBR Green Master Mix (Bimake, Houston, TX, USA), respectively. Total RNA was extracted with Plant Rneasy Mini Kit (Qiagen, Hilden, Germany). The first cDNA strand was synthesized by A PrimeScript^® RT kit (TaKaRa, Dalian, China). Genscript online design software (https://www.genscript.com/tools/pcr-primers-designer, accessed on 16 March 2021) was used to design the primer pairs (Table S3). A clustered heat map was drawn using Tbtools software. The reference gene was Actin (HhACT) [14 (link)]. Three independent experiments were conducted. Relative transcript abundances were analyzed using the 2^−ΔΔCT method [36 (link)].