*2.2. Phylogenetic Tree, Digital Expression, and Motif Analysis*

The Mega (version 7.0) program was used to create the maximum likelihood phylogenetic tree [19]. The conserved motif in the TUBBY gene was predicted using the online MEME server (latest Version 4.12.0) (http://meme-suite.org/tools/meme) (accessed on 8 March 2022). In response to biotic and abiotic stress, the gene expression levels were determined at various stages in all available tissue. The RNA-seq data were retrieved in transcripts per million (TPM) from the expVIP wheat Expression Browser (http://www.wheat-expression.com/) (accessed on 8 March 2022) [20,21]. The abiotic stress was comprised of temperature stress ranging from 20 to 40 ◦C, and biotic stresses were comprised of abscisic acid (ABA), gibberellic acid (GA), and a combination of *Fusarium graminearum* (FG), ABA, and GA. The ratio of the expression value under treatment to the control was calculated to determine the regulation patterns of a given gene subjected to stress. Ratios greater than or less than 1.0 under a given treatment indicated that the stress treatment had altered gene expression levels. In contrast, a ratio equal to 1.0 showed that

the treatment did not affect gene expression levels [20]. The heatmap was created using the Heml 1.0 software tool (http://hemi.biocuckoo.org/faq.php) (accessed on 8 March 2022).
