Cloning of an Albino Mutation of Arabidopsis thaliana 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.

Functional Genomics for Plant Breeding 3.0

“…Rodriguez-Alcocer et al [3] report on an EMS-induced mutation in Arabidopsis thaliana leading to albinism and lethality at the seedling stage. By identifying the underlying genetic mutation and its effects on the splicing of At2g04030 transcripts, this study provides insights into the deregulation of genes encoding plastid-localized proteins, advancing our understanding of chloroplast-localized proteins.…”