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. 2018 May 24;9:699. doi: 10.3389/fpls.2018.00699

FIGURE 4.

FIGURE 4

The UvSTL2 gene and deletion mutants. (A) The UvSLT2 gene and gRNA spacers. The position and direction of gRNA spacers and primers used to generate and screen Uvslt2 deletion mutants are marked with arrows. The on-target scores of spacers SLT01, SLT02, and SLT03 are labeled in the bracket. (B) PCR assays for the deletion of UvSTL2 (upper panel) and presence of the geneticin-resistance gene (lower panel) in 16 transformants generated with pCas9-tRp-gRNA-SLT01 (spacer SLT01). The 571-bp UvSTL2 fragment was only amplified in the wild-type (CK) and transformant 1, 3, 5, 6, 12, and 15. M: 1-kb DNA ladder marker. (C) PCR assays to verify gene replacement events in 16 putative Uvslt2 deletion mutants. The 2013-bp upstream and 2081-bp downstream recombination products were amplified with primer pairs SLT27F(S7F)/G855R and SLT8R(S8R)/G856F, respectively. Amplification with P1 was used as the negative control. M: 1-kb DNA ladder marker.