<scp>M</scp>ut<scp>M</scp>ap‐<scp>G</scp>ap: whole‐genome resequencing of mutant <scp>F</scp>2 progeny bulk combined with <i>de novo</i> assembly of gap regions identifies the rice blast resistance gene <scp><i>Pii</i></scp>

Abstract

Next-generation sequencing allows the identification of mutations responsible for mutant phenotypes by whole-genome resequencing and alignment to a reference genome. However, when the resequenced cultivar/line displays significant structural variation from the reference genome, mutations in the genome regions missing from the reference (gaps) cannot be identified by simple alignment. Here we report on a method called 'MutMap-Gap', which involves delineating a candidate region harboring a mutation of interest using the recently reported MutMap method, followed by de novo assembly, alignment, and identification of the mutation within genome gaps. We applied MutMap-Gap to isolate the blast resistant gene Pii from the rice cv Hitomebore using mutant lines that have lost Pii function. MutMap-Gap should prove useful for cloning genes that exhibit significant structural variations such as disease resistance genes of the nucleotide-binding site-leucine rich repeat (NBS-LRR) class.