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Differentially expressed genes that are associated with phytohormone metabolism and signalling. A transcript is considered to be differentially expressed in a library, when it has a mean RPKM value of ≥ 5 at least in one of the two samples (e.g., IMF or FF in IMFvsFF library), with a log2 fold change value ≥│1│and FDR p-value ≤ 0.05. Of these, 61 in the IMFvsFF and MFvsFF comparisons potentially control anther/ pollen development, floral transition and floral organ identity.
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A total of 245 sex determination and floral development associated DEGs were identified. Differentially expressed cannabis transcripts homologous to genes associated with floral development, including anther/pollen development & flower organ identity. A transcript is considered to be MF-specific, when it has a mean RPKM value of ≥ 5 in MF, but zero mean RPKM in both genetically female background FF and IMF samples. Male-specific differentially expressed transcripts that contain BLAST hits against UniProtKB. All of the targeted genes had similar expression patterns in both qPCR and RNA-Seq analyses between the two flower sex types. RNA-Seq data were shown as mean values ± standard errors (n= 2-4) of log2 RPKM. The relative expression (qPCR) in the samples of FF was set arbitrary to 1 (log2(1)= 0). qPCR data were expressed as mean values ± standard errors (n= 2) of log2 fold change. These DEGs include nine transcription factors: APETALA 3 (AP3), Dysfunctional Tapetum1 (DYT1), Agamous-like MADS-box 11 (AGL11), MADS2, WUSCHEL (WUS), MYB35, MYB80, bHLH91and ABORTED MICROSPORES (AMS), and six other genes: Spermidine hydroxycinnamoyl transferase (SHT), Eceriferum 26-like (CR26), MEN-8 (male-specific protein - Men8), Cytochrome P450 703A2 (C70A2), Serine threonine- kinase AFC2 (AFC2) and Mannose glucose-specific lectin (LEC) involved in floral development and sex determination. qPCR validation of selected DEGs in flowers of IMF and MF plants. More differentially expressed transcripts were observed in IMFvsFF and MFvsFF libraries than those in IMFvsMF. White color indicates no differential expression between the plant flower sex types. Dark blue color indicates higher expression in IMF or MF than FF, and dark red color shows more expression in FF compared to IMF or MF. Each square represents a single DEG, and the log2 fold change (log2 FC) was used to generate the color scale varying from -4.5 (more red) to 4.5 (more blue). The metabolic pathway bins: 1- photosynthesis, 2- cellular respiration, 3-carbohydrate metabolism, 4- amino acid metabolism, 5- lipid metabolism, 6- nucleotide metabolism, 7- coenzyme metabolism, 8- polyamine metabolism, 9- secondary metabolism, 10- redox homeostasis, 11- phytohormone action, 12- chromatin organization, 13- cell cycle organization, 14- DNA damage response, 15- RNA biosynthesis, 16- RNA processing, 17- protein biosynthesis, 18- protein modification, 19- protein homeostasis, 20- cytoskeleton organization, 21- cell wall organization, 22- vesicle trafficking, 23- protein translocation, 24- solute transport, 25- nutrient uptake, 26- external stimuli response, and 35- unclassified proteins that can’t be assigned and/ or annotated. Summary of enriched MapMan metabolic pathways from the top 500 DEGs between flower tissues of cannabis sex type comparisons (IMFvsFF, MFvsFF and IMFvsMF). Most of these transcripts were involved in cellular and metabolic processes under cellular component (CC), catalytic activity and binding under molecular function (MF) as well as cell and organelle under biological process (BP). The top 20 GO category distribution for assembled transcripts. The majority of the transcripts/ contigs were clustered into 200-400 bp in length. The size distribution of total assembled transcripts from three cannabis flower sex types.