A protocol for validation of doubled haploid plants by enzymatic mismatch cleavage

Abstract

Doubled haploidy is an important tool for plant breeders. It provides a rapid means of developing recombinant populations consisting of individuals that are homozygous and therefore genetically fixed. Homozygosity is also important in plant mutation breeding where many induced mutations are predicted to be recessive and mutant alleles need to be in a homozygous state before new traits are expressed. While production of doubled haploids has been described for many plant species, efficient means to validate that produced materials are indeed homozygous are needed. Polymorphism discovery methods utilizing enzymatic mismatch cleavage are ideally suited for validation of doubled haploid plants. We describe here a low-cost protocol that utilizes self-extracted single-strand-specific nucleases, standard PCR reactions and agarose gel electrophoresis that can be applied to most plant species.