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Figure 1 | BMC Plant Biology

Figure 1

From: TILLING to detect induced mutations in soybean

Figure 1

Schematic of the soybean TILLING process [39]. Seeds are mutagenized and grown to generate the M1. Since the embryo consists of many cells, M1s may be mosaic for mutations induced by the mutagen. M1 plants are allowed to self and a single M2 plant is grown from each M1 line. Tissue and M3 seed are collected from the M2 plants. The concentration of DNAs isolated from the M2 tissue is normalized, and the samples are pooled eight-fold in 96-well plates. IRDye labeled primers are used for amplification of a particular target. Following PCR, samples are denatured and allowed to reanneal such that if a mutation is present, heteroduplexes will form. CJE is used to cleave 3' of the mismatch. Samples are denatured and electrophoresed on polyacrylamide gels using LI-COR 4200 or 4300 machines. Putative mutations are identified by bands appearing in the 700 and 800 channels that add up to the molecular weight of the full length PCR product. Pools are deconvoluted to identify mutant individuals, and the individuals are sequenced. Sample soybean gel section and complete results from the gmclavb primer set screened on the A population are shown.

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