Fig. 2.
Expression of TaIPK1 homoeologs in seeds and illustration of gRNA expression Cassette. (A) Expression analysis of three TaIPK1 homoeologs at two different developmental stages of seed i.e. 14 and 21 days after anthesis (DAA). 2AL, 2BL, 2DL represent expression of TaIPK1 homoeologs on the long arm of chromosome 2 of the A, B and D genomes. qRT-PCR assays were performed using SYBR green and Ct values were normalized against TaActin as an endogenous control. Blue bars represent expression of TaIPK1 homoeologs at 14 DAA stage and gray bars represent the expression at 21 DAA stage. (B) Wheat Ta.IPK1.A gene model and target sequences are shown. The Ta.IPK1.A sequences for sgRNA1Ta.IPK1.A (Target 1) and sgRNA2Ta.IPK1.A (Target 2) are shown below the target regions with the PAM highlighted in magenta colour. Restriction endonucleases (BtgZ1 andBsa1) recognition sequences are underlined. Primers used for mutation detection are shown in both panels. (C) Schematic of binary plasmid vector delivered to wheat is depicted. The construct sgRNATa.IPK1.A houses a Kanamycin resistant cassette flanked by the Gateway recombination sequence attL1 and attL2 is mobilized to the binary vector pOs-Cas9 through the Gateway recombination. The expression cassette consists of a pair of sgRNAs (sgRNA1Ta.IPK1.A and sgRNA2Ta.IPK1.A) followed by endonuclease Cas9 driven by Ubiquitin promoter from Rice.