Screen for nucleotide substitutions in selected gene fragments. A. Gene model and amplification schemes of the cucumber phytoene desaturase-3 gene, Female ACC synthase, ramosus-3 and ramosus-4 homologous genes. The approximate positions of the amplicons screened by TILLING are indicated, each delimited by two pairs of nested primers (arrows indicate the internal amplicons). The mutations that were verified by sequencing (from families 53, 254, 48, 540, 928) are indicated; in family 53, two independent point mutations were recovered in two of the genes, respectively. B. Chromatogram of the C to T mutation (read by a reverse primer as G to A), discovered in the PDS-3 gene, in family 53. Top: wild type sequence in cultivar Poinsett76. Below: plant 53–13 is homozygous for the mutation, plant 53–20 is heterozygous.