Phenotypic Analysis

Depending on the goals of the investigator, the desired phenotype can be a disruption of function to aid in basic research of a largely uncharacterized gene, or it can be to achieve a specific goal, such as improved agronomic or fruit quality traits. In either case, background mutations are the largest hurdle for any investigator in acquiring a clean phenotype.

Fig. 14. Example of a 96-lane TILLING assay used for detection of mutations in 6-fold pools of tomato DNA. A gel image from the IRD 700 channel (left) and IRD 800 channel (right) with size ladders flanking the left image are shown. The dark bands at the top of the gels represent full-length (759 bp) uncut PCR products (arrow). CELI cleaved heteroduplexes indicating the presence of a mutation are visible as dark bands (circled) shorter than the full-length product. The size of the cleavage products in the IRD 700 and IRD 800 channels corresponding to the same mutation add up to the full-length PCR product. Insets: enlargements of CEL I cut heteroduplexes. The PARSESNP output showing the gene model exons (boxes) and introns (lines) is shown to the left of the IRD 700 image. The brackets adjacent to the gene model indicate areas with homology to other proteins in the database. Triangles indicate the position of the mutations. A marker is added to every twelfth lane to improve the accuracy of the lane calls (small circles)

Fig. 14. Example of a 96-lane TILLING assay used for detection of mutations in 6-fold pools of tomato DNA. A gel image from the IRD 700 channel (left) and IRD 800 channel (right) with size ladders flanking the left image are shown. The dark bands at the top of the gels represent full-length (759 bp) uncut PCR products (arrow). CELI cleaved heteroduplexes indicating the presence of a mutation are visible as dark bands (circled) shorter than the full-length product. The size of the cleavage products in the IRD 700 and IRD 800 channels corresponding to the same mutation add up to the full-length PCR product. Insets: enlargements of CEL I cut heteroduplexes. The PARSESNP output showing the gene model exons (boxes) and introns (lines) is shown to the left of the IRD 700 image. The brackets adjacent to the gene model indicate areas with homology to other proteins in the database. Triangles indicate the position of the mutations. A marker is added to every twelfth lane to improve the accuracy of the lane calls (small circles)

A population with a high mutation frequency is desired for efficient TILLING, but the resulting mutation loads in M3 plants can greatly impede phenotypic analysis. Each M3 plant can have hundreds or thousands of background mutations. Any of these background mutations can interfere with plant growth and reproduction, making it difficult to obtain tissue for analysis and in some cases, the background mutations can interfere specifically with the anticipated phenotype of the mutated gene. M3 siblings that are wt for the mutation of interest but that contain a similar complement of background mutations as the homozygous plant are therefore essential controls in early phenotyping efforts. In cases where the M2 plant is homozygous, no sibling controls will exist in the M3 generation and the first controlled look at phenotype will take place after crossing to the unmu-tagenized parent line. The genotyping of progeny is greatly facilitated by the ability to use the identified mutation as its own molecular marker in a breeding program. Additional backcrosses will eliminate background mutations for advanced phenotypic analysis. For commercial crop development, mutations identified by TILLING can be introgressed into preferred germplasm or combined with other mutations to stack traits.

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