Illustra exostar kit

For confirmation of the specificity of the amplification, all PCR products were cleaned up using Illustra ExoStar Kit (GE Healthcare, UK) and sequenced with both forward and reverse primers using the Big Dye Terminator Cycle Sequencing kit v3.1 (Life Technologies, USA), according to the supplier's protocol. Purification of the sequences from residual dye terminators was performed by Isopropyl alcohol 75%/Ethanol 70% precipitation. Capillary Electrophoresis of the sequences was carried out in an ABI3500 Genetic Analyzer (Life Technologies, USA). Electropherograms were visualized and edited with Sequencher v4.8 software (Gene Codes Corporation, USA). The obtained sequences were aligned with GeneBanks reference sequence.

The spff mutant was backcrossed four times with Micro-Tom WT in order to purify the responsible mutation and finally obtain BC₄F₂ plants (Supplementary Figure S1). Linkage analysis was performed using DNA extracted from F₂, BC₂F₂, BC₃F₂, and BC₄F₂ populations (Supplementary Table S1). Genomic DNA was extracted by DNeasy Miniprep kit (QIAGEN) and amplified by PCR with TaKaRa Ex Taq (TAKARA) and the primer set shown in Supplementary Table S2. The PCR products were purified by the Illustra ExoStar kit (GE Healthcare) and then sent to Eurofins Genomics for sequencing.