Tomato SlIAA9 Gene Mutation Detection

Genomic DNA was isolated from tomato leaves using Nucleospin Plant II (TaKaRa, Japan), and used for amplification of target sequences by PCR using PrimeSTAR GXL DNA Polymerase (TaKaRa, Japan). An AccI site is located on the SlIAA9 target sequence, and PCR-RFLP was used for detection of the mutation. In PCR-RFLP, PCR fragments were digested with AccI (NEB, Japan) and analyzed by agarose gel electrophoresis. For Sanger sequencing analysis, PCR fragments purified from agarose gels were cloned by the Seamless ligation cloning extract (SLiCE) method (Motohashi, 2015 (link)) into cloning vector pNEB193 (NEB, Japan). All primers used for PCR are listed in Supplementary Table S1.