Cloning of an Albino Mutation of <i>Arabidopsis thaliana</i> Using Mapping-by-Sequencing

Abstract

We report the molecular characterization of an ethyl methanesulfonate (EMS)-induced mutation that causes albinism and lethality at the seedling stage in Arabidopsis thaliana. We identified the mutation using a mapping-by-sequencing approach that uses Fisher’s exact tests to detect changes in allele frequencies among the seedlings of an F2 mapping population, which had been pooled according to their phenotypes (wild-type or mutant). After purifying genomic DNA from the plants of both pools, the two samples were sequenced using the Illumina HiSeq 2500 next-generation sequencing platform. The bioinformatic analysis allowed us to identify a point mutation that damages a conserved residue at the acceptor site of an intron of the At2g04030 gene, which encodes the chloroplast-localized AtHsp90.5 protein, a member of the HSP90 family of heat shock proteins. Our RNA-seq analysis demonstrates that the new allele alters the splicing of At2g04030 transcripts in multiple ways, leading to massive deregulation of genes encoding plastid-localized proteins. A search for protein–protein interactions using the yeast two-hybrid method allowed us to identify two members of the GrpE superfamily as potential interactors of AtHsp90.5, as has previously been reported for green algae.

Keywords

• mapping-by-sequencing
• Fisher’s exact test
• chloroplast
• HSP90 proteins
• heat shock proteins
• <i>Arabidopsis thaliana</i>