Arabidopsis Map-Based Cloning in the Post-Genome Era

Abstract

Map-based cloning is an iterative approach that identifies the underlying genetic cause of a mutant phenotype. The major strength of this approach is the ability to tap into a nearly unlimited resource of natural and induced genetic variation without prior assumptions or knowledge of specific genes. One begins with an interesting mutant and allows plant biology to reveal what gene or genes are involved. Three major advances in the past 2 years have made map-based cloning in Arabidopsis fairly routine: sequencing of the Arabidopsis genome, the availability of more than 50,000 markers in the Cereon Arabidopsis Polymorphism Collection, and improvements in the methods used for detecting DNA polymorphisms. Here, we describe the Cereon Collection and show how it can be used in a generic approach to mutation mapping in Arabidopsis. We present the map-based cloning of the VTC2 gene as a specific example of this approach.

A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

“…In the case of a monogenic recessive trait, F 2 mutants are the individuals of choice. These 1,317 mutant F 2 segregants were subsequently genotyped for insertion/deletion (InDel) polymorphisms of the Cereon InDel collection (18). In the subsequent steps, gradually decreasing subsets of recombinants were genotyped with pairs of InDel markers flanking the genomic area containing the PMM gene until no recombinants were left.…”

“…Therefore, to avoid the time-consuming step of self-fertilizing the relevant wild type recombinants and growing the F 3 progeny to determine their genotype, we only used the mutant F 2 individuals for subsequent analysis. In total, 1,317 F 2 mutants (13.3%) were identified, harvested, and submitted to linkage analyses using InDel markers developed from the Cereon InDel collection (18). Initially, two InDel polymorphisms (CER452389 and CER449256) flanking the previously defined 673-kb interval were identified and used to screen the F 2 mutant population.…”

ArabidopsisPEN3/PDR8, an ATP Binding Cassette Transporter, Contributes to Nonhost Resistance to Inappropriate Pathogens That Enter by Direct Penetration

“…Key individuals were retested for their pen3 phenotype in the F3 generation. New markers within the mapping interval were generated using the CEREON database of Col-0 and Ler polymorphisms (Jander et al, 2002) and the Whitehead Institute online primer design software, Primer3 (Rozen and Skaletsky, 2000; http://frodo.wi. mit.edu/cgi-bin/primer3/primer3_www.cgi).…”

Self-Incompatibility in the Brassicaceae

“…In Arabidopsis , insertional mutagenic techniques using T-DNA or transposons have become popular tools for functional genomics. However, insertional mutagenesis often leads to complete gene knockouts, making it diffi cult to associate nuanced phenotypes with essential genes (Jander et al 2002 ). Similarly, radiation mutagenesis, for example, fast neutrons, often induces large genomic deletions that affect multiple genes (Li et al 2001 ).…”