Figure 3

TILLING using ENDO1. (a) Universal primers Tilling strategy. Targets were amplified by PCR with gene specific primers (black arrows 1), followed by a nested-PCR with internal gene specific (black parts of arrows 2) primers containing a universal M13 tail (blue parts of arrows 2), in combination with IR DYE-labeled universal M13 primers (blue arrows 3); the red and green dots indicate the position of IRD700 and IRD800-labelling, respectively. The sequence of the primers are indicated in Table 1 (b) Example of a RBR Tilling screen on 8-fold pooled pea DNA. The gel images represent the superposed image from the IRD700 and IRD800 channels. The sizes of the cleavage products (circled) from the 2 dyes-labeled DNA strands (red or green) add up to the size of the full-length PCR products (top of the gel). PCR artifacts are distinguishable from true mutants by yellow points (red and green added) as they appear of the same size in both channels. The size of the cleavage product (sizing ladder in the first and last lanes) indicates approximately where the SNP is located in the fragment. (c) Graphic representation of mutations identified in two regions of RBR gene. This drawing was made using the PARSESNP program [54], which maps the mutation on a gene model to illustrate the distribution of mutations. The purple and black triangles represent silent and missense mutations, respectively. The black lanes indicate the two tilled amplicons.