Identification of genes required for cold acclimation using next generation sequencing of sensitive to freezing mutants

Abstract

Wild type Arabidopsis plants are able to survive temperatures below 0°C, providing they have been subjected to low positive temperatures for several days prior to freezing. This brief period of low temperature is known as cold acclimation and results in the activation of a number of pathways that bring about physical and biochemical changes necessary to withstand freezing stress. However, when the sensitive to freezing (sfr) mutants are subjected to temperatures that would promote acclimation in wild type plants, they fail to become more freezing-tolerant. This suggests that the mutation lies in a gene intrinsic to the acclimation process.

The sensitive to freezing mutants were discovered as part of an ethyl methanesulfonate (EMS) chemical mutagenesis screen, which induces single nucleotide polymorphisms (SNPs) into the genome. As a result, for each mutant the deficiency in freezing tolerance is caused by a SNP in an unknown gene. sfr4, sfr5, sfr8 and sfr9 were the focus of this investigation. Mapping intervals had been previously determined for each of the four sfr mutants using classical recombination-based techniques, giving a specific region of the genome in which to search for the SNP responsible for freezing sensitivity.

Full Illumina genome sequences were produced for sfr4, sfr5, sfr8 and sfr9, and these were mapped against the TAIR 10 Arabidopsis genome. Using the Integrative Genomics Viewer software, the mapping interval was scanned and all SNPs of interest were recorded. Additionally, a command line-based method of identifying SNPs was also applied to the sequencing data by collaborators at the University of Liverpool. SNPs found by both methods were verified to exist in the DNA of the mutant they corresponded to via direct DNA sequencing. This provided a number of candidate genes for each sfr mutant line. T-DNA insertional mutants were obtained for each of the genes in question, and these were genotyped via PCR. Homozygotes were tested for freezing tolerance, comparing them to the respective sfr mutant to the knockouts/knockdowns in each potential gene candidate.

Additional phenotypic data was collected for each of the sfr mutants, as they had not received much work prior to the investigation. At the end of this investigation, several candidates remained as potential causes of freezing tolerance for sfr5, sfr8 and sfr9; however no candidates were isolated for sfr4.