Bole cfx96 system

Total RNA was extracted from evenly mixed samples (1 g, the sample is a mixture of each separately treated sample) using a Fast Pure Plant Total RNA Isolation Kit (RC401, RC401, Vazyme, Nanjing, China) and reverse transcribed to generate the first-strand cDNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (R323-01, Vazyme, Nanjing, China) according to the manufacturer’s instructions. Quantitative real-time PCR (RT-qPCR) consisted of three biological and three technical replicates and was carried out using the Bole CFX96 system (Bio-Rad, Hercules, CA, USA) and Ultra SYBR mixture (CW2601M, CWBIO, Beijing, China,). The calculation method for RT–qPCR was 2^−ΔΔCt with FaACTIN as the internal control. The specific primers used are listed in Table S1.

RNA was extracted via a kit (TransGen Biotech, Beijing, China), as described by Xu et al^{19 (link)}. The qRT-PCR analyses were carried out on the Bole CFX96 system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommended protocols. Each sample was analyzed in triplicate, with MdActin used as the internal reference gene for each quantification. The Ct values were read under default conditions, and the 2^−ΔΔCT method was used for data analysis^{60 (link)}.

Total RNA was extracted from evenly mixed samples (1 g, the sample is a mixture of each separately treated sample) using a Fast Pure Plant Total RNA Isolation Kit (RC401, Vazyme, Nanjing, China) and reverse transcribed to generate the first strand cDNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (R323-01, Vazyme, Nanjing, China) according to the manufacturer’s instructions. Quantitative real-time PCR (RT-qPCR) consisted of three biological and three technical replicates and was carried out using the Bole CFX96 system (Bio-Rad, Hercules, CA, USA) and Ultra SYBR mixture (CW2601M, CWBIO, Beijing, China,). The calculation method for RT–qPCR was 2^−ΔΔCt with FaACTIN as the internal control. The specific primers used are listed in Table S6.