The HEM Lines: A New Library of Homozygous Arabidopsis thaliana EMS Mutants and its Potential to Detect Meiotic Phenotypes

Abstract

Genetic screens have been crucial for deciphering many important biological processes, including meiosis. In Arabidopsis thaliana, previous forward screens have likely identified almost all the meiotic genes that when mutated lead to a pronounced decrease in fertility. However, the increasing number of genes identified in reverse genetics studies that play crucial roles in meiosis, but do not exhibit strong phenotypes when mutated, suggests that there are still many genes with meiotic function waiting to be discovered. In this study, we produced 897 A. thaliana homozygous mutant lines using Ethyl Methyl Sulfonate (EMS) mutagenesis followed by either single seed descent or haploid doubling. Whole genome sequencing of a subset of lines showed an average of 696 homozygous mutations per line, 195 of which (28%) modify a protein sequence. To test the power of this library, we carried out a forward screen looking for meiotic defects by observing chromosomes at metaphase I of male meiosis. Among the 649 lines analyzed, we identified 43 lines with meiotic defects. Of these, 21 lines had an obvious candidate causal mutation, namely a STOP or splicing site mutation in a gene previously shown to play a role in meiosis (ATM, MLH3, MLH1, MER3, HEI10, FLIP, ASY4, FLIP, PRD2, REC8, FANCL, and PSS1). Interestingly, this was the first time that six of these genes were identified in a forward screen in Arabidopsis (MLH3, MLH1, SGO1, PSS1, FANCL, and ASY4). These results illustrate the potential of this mutant population for screening for any qualitative or quantitative phenotype. Thus, this new mutant library is a powerful tool for functional genomics in A. thaliana. The HEM (Homozygote EMS Mutants) lines are available at the Versailles Arabidopsis stock center.

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“…The intention of this reverse genetics approach is to test the function of specific genes by analyzing the consequences of their disruption on the process of interest. Recently, for example, an EMS screen in Arabidopsis thaliana was applied to generate a mutant collection in order to identify genes that lead to meiotic defects (Capilla-Perez et al., 2018). Mutant collections are very useful resources for functional genomics and basic research in Arabidopsis thaliana .…”

A restorer‐of‐fertility‐like pentatricopeptide repeat protein promotes cytoplasmic male sterility in Arabidopsis thaliana

“…EMS mutagenesis was applied to these seeds as described by Capilla‐Perez et al . (2018), but with 0.2% (v/v) EMS instead of 0.3%.…”

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“…Other examples using this approach, also called bulked segregant analysis by whole-genome re-sequencing, are the identification of mutations restoring the photorespiratory defect of er-ant1 [ 57 ] or anthocyanin accumulation of tt19 [ 58 ]. A direct whole genome sequencing approach has recently proven successful to identify causal mutations of allelic M3 lines by comparison to the parental line in mutants with meiotic defects [ 30 , 31 , 32 ]. According to Jander et al [ 59 ], 50,000 M1 lines need to be tested to have 95% chance to find mutation in any G:C in the genome.…”

“…Indeed, the low cost of sequencing allows to sequence a large number of mutants. Sequencing a large number of mutants with a similar phenotype can be used to retrieve the mutated gene by finding allelic mutations in the same gene [ 29 , 30 , 31 , 32 ]. The goal of this study is to categorize the isolated mutants for potential allelism thereby facilitating downstream analysis of the whole genome sequencing data.…”