The first true leaves of L. bicolor plants were collected at stages A and B and stored at –80°C (Yuan et al., 2015) and their total RNA was extracted. Template cDNA was obtained by reverse transcription using ReverTra Ace qPCR RT kit (TOYOBO, Japan). With reference to the Iso-seq transcriptome (unpublished) of L. bicolor, primers (LbSAD2-S and LbSAD2-A) of Lb125774 (LbSAD2) were designed to clone the full-length coding sequence using Primer Premier 5.0 (Supplementary Table 1).

Alignment was performed using DNAman and DNAstar to compare nucleic acid and protein sequences. After NCBI BLASTp using LbSAD2, 33 SAD2 proteins were chosen for a phylogenetic tree construction using the neighbor-joining method with MEGA5.11 and ClustalX, with statistical support for nodes obtained from at least 1,000 trials. The ProtParam tool in the ExPASy online software was used to predict the physical and chemical properties of the protein based on its primary structure2. The Prot-Scala tool in ExPASy was used to analyze the hydrophilicity and hydrophobicity of the amino acid sequence3. The signal peptide was detected with Signal4.14, the GOR4 tool in ExPASy was used to predict the secondary structure of the protein5, SMART software was used to predict the conserved domain of the protein6, and SWISS-MODEL in ExPASy was used to predict the tertiary structure7.

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