Strategy for targeted knockout of <i>PpASCL</i> and genotyping of the resulting stable transformants by PCR.

<p>(a) Schematic diagram of insertion of the linear knockout construct into the <i>PpASCL</i> locus via double homologous recombination. 35S-P, CaMV 35S promoter; <i>nptII</i>, 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 <i>PpASCL</i> (Primers 1 and 2, which bind to genomic DNA sequences located outside the locus-specific regions used for homologous recombination) or to the <i>nptII</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146817#pone.0146817.s005" target="_blank">S1 Table</a>. (c) PCR products using locus-specific primers 1 and 2 with DNA from untransformed control and each of three stable putative <i>PpASCL</i> knockout lines: <i>ascl-1</i>, <i>-2</i> and <i>-3</i>. (d) PCR products, indicative of 5′ and 3′ recombination between the knockout vector and homologous DNA in the <i>PpASCL</i> 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.