Utilizing next generation sequencing to identify cell wall mutants

Christopher K Dugard, Purdue University

Abstract

Through a combination of traditional marker-based mapping and the utilization of next-generation sequencing, I found that low cell-wall Ara mutant mur5 encodes a defective allele of Reversibly Glycosylated Polypeptide 2 (RGP2) - a mutase implicated in the interconversion of UDP-Arap and UDP-Araf. Identity of the mutant was confirmed by rescue of the wild type phenotype by genetic transformation of mur5 and two associated SALK alleles with wild-type RGP2 placed under the control of the 35S promoter. Following expression analysis of the RGP family, I found a lack of expression of RGP2 in the mur5 background as well as increased expression of RGP1. Conversely, in overexpressing lines of RGP2, expression of RGP1 decreases, which is evidence of cross talk within the RGP family. This mapping strategy was also used for the low Ara mur6, which revealed a mapping interval containing chromatin remodeling factor, AUG3. The T-DNA knockout of this mutant is homozygous lethal. An allelism test and genetic complementation experiments are proposed to determined if AUG3 is a strong candidate. I then determined whether whole-genome DNA sequencing alone could be employed to narrow a list of candidate genes from single backcrossed lines of mutants derived by enrichment of ethyl methanesulfonate (EMS)-induced polymorphisms. Single lines of a ferulate-5-hydroxylase mutant, fah1, and chromatin remodeling factor mutant, pkl, gave defined intervals of enriched EMS-type polymorphisms on single chromosomes that included the known mutated genes. Subsequently, the deep sequencing of single recombinant lines of low cell-wall Ara mur6 and mur7, low cell-wall Rha mur8, and re-mutagenized lines of pkl screened for enhancing or suppressing phenotypes revealed similar intervals with enriched EMS-type mutations that contained small numbers of non-synonymous candidate genes of these unknown mutants. A minimum of three backcrosses of the original mutant against wild type appeared to be required for the identification, but five backcrosses narrowed the interval of EMS-type mutations substantially, thus narrowing the list of candidate genes. I propose whole-genome sequencing of a sufficiently back-crossed line to be a suitable method to identify mutants, which obviates the need for a mapping population. It is anticipated that three backcrosses would be sufficient if multiple independent lines were selected, thereby amplifying the relevant EMS-interval containing the mutated gene of interest.

Degree

M.S.

Advisors

Carpita, Purdue University.

Subject Area

Molecular biology|Genetics|Biochemistry

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