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. 2016 Jan 11;11(1):e0146817. doi: 10.1371/journal.pone.0146817

Fig 2. Strategy for targeted knockout of PpASCL and genotyping of the resulting stable transformants by PCR.

Fig 2

(a) Schematic diagram of insertion of the linear knockout construct into the PpASCL locus via double homologous recombination. 35S-P, CaMV 35S promoter; nptII, neomycin phosphotransferase II gene; 35S-T, CaMV 35S transcription termination signal. (b) Schematic diagram of recombined gene locus after successful insertion. Expected PCR product sizes based on sequence information are shown. Single-headed arrows denote the locations of primers specific to PpASCL (Primers 1 and 2, which bind to genomic DNA sequences located outside the locus-specific regions used for homologous recombination) or to the nptII resistance cassette (Primers 3 and 4) used in the PCR analyses. Primer 1, ASCL-gDNA-F; 2, ASCL-gDNA-R; 3, pTN182-5ʹ-R; 4, pTN182-3ʹ-F. Primer sequences are provided in S1 Table. (c) PCR products using locus-specific primers 1 and 2 with DNA from untransformed control and each of three stable putative PpASCL knockout lines: ascl-1, -2 and -3. (d) PCR products, indicative of 5′ and 3′ recombination between the knockout vector and homologous DNA in the PpASCL locus, using primers 1 plus 3 (5ʹ recombination) and primers 2 plus 4 (3ʹ recombination). Amplified DNA products were resolved electrophoretically on 1.2% agarose gels and visualized by ethidium bromide fluorescence.