Show-case project to outline mutation identification in the absence of reference sequences, segregating populations or genetic maps

Genes underlying mutant phenotypes can be isolated by combining marker discovery, genetic mapping and resequencing, but a more straightforward strategy for mapping mutations would be the direct comparison of mutant and wild-type genomes. Applying such an approach, however, is hampered by the need for reference sequences and by mutational loads that confound the unambiguous identification of causal mutations. Here we introduce NIKS (“Needle in the k-stack”), a reference-free algorithm based on comparing k-mers in whole-genome sequencing data for precise discovery of homozygous mutations. We applied NIKS to eight mutants induced in non-reference rice cultivars and to two mutants of the non-model species Arabis alpina. In both species, comparing pooled F2 individuals selected for mutant phenotypes revealed small sets of mutations including the causal changes. Moreover, comparing M3 seedlings of two allelic mutants unambiguously identified the causal gene. Thus, NIKS enables forward genetics in any species amenable to mutagens without requiring segregating populations, genetic maps and reference sequences.