Whirly Genes Expression Analysis

Real-time PCR was performed to detect the expression pattern of Whirly genes according to a previous study (Liu et al., 2022 (link)). The total RNA was isolated using RNApure Plant Kit (CWBIO), and the first-strand cDNA was synthesized from 1 μg of total RNA using Prescript III RT ProMix (CISTRO). The real-time PCR was performed using gene-specific primers (Supplementary Table S1) with 2× Ultra SYBR Green qPCR Mix (CISTRO), and the TaActin gene was selected as a reference control. The real-time PCR cycling parameters were 95°C for 30 s, followed by 45 cycles at 95°C for 5 s and 60°C for 30 s, with a melting curve analysis. All reactions were performed on three technical and biological replicates. The relative expression levels of target genes were calculated using the 2^−△△CT method (Livak and Schmittgen, 2001 (link)).