Sequence-based identification of T-DNA insertion mutations in Arabidopsis: actin mutants act2-1 and act4-1

A method is presented to facilitate the isolation of mutations in plant genes, which requires knowledge of the target gene or protein sequence, and is independent of mutant phenotype. The polymerase chain reaction was used to amplify the junctions between a T-DNA insert and the gene of interest from pools of mutant plant lines. The approach was used to identify mutations in Arabidopsis thaliana actin genes. The Arabidopsis genome encodes 10 actins in six ancient subclasses each with distinct expression patterns. Primers in the T-DNA border and highly degenerate actin primers, designed from conserved amino acid motifs, were used to prime the amplification. The PCR products were transferred to filters and probed for actin at low stringency. Thus, mutations in all 10 actin genes were screened for simultaneously. Mutations in the vegetative constitutive actin gene, ACT2, and the pollen-specific actin gene, ACT4, were identified in a population of 5300 lines containing approximately 1.5 T-DNA insertions per line. The screen was sensitive enough that actin insertion alleles were easily distinguished among pools of 100 plant lines. PCR techniques were used which accelerated the purification of mutant lines, and segregation, physical mapping, and sequencing of the act2-1 and act4-1 mutations. This strategy should be generally useful in screening mutant libraries made with a variety of plant insertion elements for mutations in any known sequence.