Tilling by Sequencing for Genome-Wide Mutation Discovery and Functional Genomics in Camelina sativa

TILLING (Targeting Induced Local Lesions in Genomes) is a non-transgenic reverse genetic approach that allows genome-wide discovery of induced point mutations in EMS-mutagenized populations. The development of genotyping-by-sequencing (GBS) to rapidly detect nucleotide variation at the whole genome level, in highly multiplexed sets of discrete individuals simultaneously has provided a transformative genetic profiling technique. We have successfully established a GBS-based approach for high-throughput screening for mutations in an EMS-mutagenized population of Camelina sativa. To assay the mutation rate and efficacy of the GBS approach, ~400 M₂ lines were grown, and genomic DNA and seeds were collected from ~200 individuals. Two separate DNA pools of 96 individuals, for a total of 192, were constructed by mixing equal amounts of genomic DNA. GBS analysis of these individuals utilized two methylation sensitive restriction enzymes (PstI and MspI), one with a six-base and the second with four-base recognition site, to effect genome reduction. GBS libraries were sequenced on Illumina HiSeq 2000 platform. A customized bioinformatics pipeline identified 6,057 unique candidate EMS SNPs of which 2,594 were exonic and the remaining aligned to non-exonic regions. The number of mutations in each line varied significantly potentially due to varying EMS penetration or lack of mutagenization in seeds. High proportions of SNPs were EMS induced as ~90% of the recovered mutations were C-to-T and G-to-A transitions, a hallmark feature of EMS mutagenesis. A detailed functional analysis and phenotypic characterization of recovered mutations will be presented. Additionally, potential applications of this effective approach of mutation discovery to other polyploid crops, such as Brassicas and wheat will be discussed.