Pre-processed RNA-Seq paired reads for each flower sex type (3 flower bud samples for FF, 4 flower bud samples for IMF and 2 flower bud samples for MF) were mapped to the final combined assembled transcriptome using the CLC Genomics Workbench. The expression levels of mapped paired reads were normalized as reads per kilobase per million mapped reads (RPKM) with the CLC RNA-Seq analysis tool. The normalized reads were then used for the differential expression analyses in three groups, including “IMF versus FF”, “IMF versus MF”, and “MF versus FF” using the same software. Differentially expressed genes (DEGs) were identified based on criteria set as an absolute log2 fold change ≥ 1, and a false discovery rate (FDR) p value ≤ 0.05. Differentially expressed genes were further characterized based on enriched metabolic pathways with MapMan (Usadel et al. 2009). The Mercator4 vs2.0 platform (Schwacke et al. 2019) was employed to annotate the DEG sequences with default settings to generate the mapping file that was used as an input in the pathway enrichment analysis in MapMan.

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