Fig. 4
From: Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes
Pathway to gene discovery using Can-Seq in a forward genetic screen. The Can-Seq strategy shown in blue can be used in the M2 generation to identify mutants that carry a recessive candidate mutation in a gene known to contribute to the trait of interest. By exclusion, novel mutants carrying a causative mutation in an unknown gene can also be identified. For these novel mutants, BC1F2 mapping populations can be produced, and whole genome or exome sequencing of bulked BC1F2 mutant plants can be used to determine the chromosomal vicinity of the unknown gene contributing to the trait of interest. Reverse genetics on candidate genes in the chromosomal vicinity or complementation tests by crossing multiple alleles can then be used to reveal the identity of the new gene. Additionally, mutants identified by Can-Seq to carry new missense mutations in known genes can be confirmed by using complementation tests, and then potentially used to characterize the biochemical function of the protein encoded by the gene (dotted arrow and dotted box)