Flow citometry - Arabidopsis Protocols

Abstract

EMS mutant analysis is a routine experiment to identify new players in a specifi c biological process or signaling pathway using forward genetics. It begins with the generation of mutants by treating Arabidopsis seeds with EMS. A m utant with a p henotype of i nterest ( mpi ) is obtained by screening plants of the M2 generation under a specifi c condition. Once the phenotype of the mpi is confi rmed in the next generation, map-based cloning is performed to locate the mpi mutation. During the map-based cloning, mpi plants ( Arabidopsis Columbia-0 (Col-0) ecotype background) are fi rst crossed with Arabidopsis Landsberg erecta (L er ) ecotype, and the presence or absence of the phenotype in the F1 hybrids indicates whether the mpi is recessive or dominant. F2 plants with phenotypes similar to the mpi , if the mpi is recessive, or those without the phenotype, if the mpi is dominant, are used as the mapping population. As few as 24 such plants are selected for rough mapping. After fi nding one marker (MA) linked to the mpi locus or mutant phenotype, more markers near MA are tested to identify recombinants. The recombinants indicate the interval in which the mpi is located. Additional recombinants and molecular markers are then required to narrow down the interval. This is an iterative process of narrowing down the mapping interval until no further recombinants or molecular markers are available. The genes in the mapping interval are then sequenced to look for the mutation. In the last step, the wild-type or mutated gene is cloned to generate binary constructs. Complementation or recapitulation provides the most convincing evidence in determin- ing the mutation that causes the phenotype of the mpi . Here, we describe the procedures for generating mutants with EMS and analyzing EMS mutations by map-based cloning. Key