Genotyping Leaf Tissue for LanFTc1 Alleles

Young leaf tissue from three biological replicates per plant was sampled. Frozen (−80°C) plant tissue (50 mg) was homogenized using TissueLyser II (Qiagen, Hilden, Germany) and two stainless steel beads (ø 5 mm) in 2 ml tubes (Eppendorf, Hamburg, Germany). DNA was isolated using DNeasy Plant Mini Kit (Qiagen). PCR was performed using GoTaq Long PCR Master Mix (Promega, Mannheim, Germany) and published LanFTc1_INDEL2 primers (Taylor et al., 2019 (link)), provided here for reference in Supplementary Table 2. PCR conditions were as follows: initial denaturation (94°C for 2 min), then 35 cycles composed of denaturation (94°C for 30 s), annealing (62°C for 30 s), and elongation (72°C for 5 min), followed by the final extension (72°C for 10 min). Products were resolved by agarose gel electrophoresis and SYBR Safe DNA staining (Invitrogen, Carlsbad, CA, United States) and visualized on UV-transilluminator (Uvitec, Thermo Fisher Scientific, Waltham, MA, United States). Wild P27255 allele (ku, without deletion) was encoded as “A,” Palestinian allele (Pal, 1208 bp deletion) as “B,” 83A:476 allele (Ku, 1423 bp deletion) as “C,” and Krasnolistny allele (Jul, 5162 bp deletion) as “D.”