Detection of crop off-types is of interest for
multiple uses, including the assessment of uniformity
for new plant variety applications during distinctness,
uniformity and stability (DUS) testing for the awarding
of plant breeders’ rights (PBR). Here, we investigate
whether genetic markers, in this case Kompetitive
Allele-Specific PCR (KASP), can be used for the identification
off-types for phenotypes assessed for DUS in
the inbreeding cereal crop, barley (Hordeum vulgare).
To demonstrate proof of principle, KASP markers diagnostic
for phenotypic expression of nine DUS phenotypes,
and DNA from two barley varieties (‘Pelican’ and
‘Felicie’) carrying contrasting alleles at each marker
were used. We found that for the majority of markers,
it was possible to robustly call alleles down to template
DNA concentrations of 2 ng, but not ≤ 0.2 ng. When
used in mixtures of DNA consisting of ‘Felicie’ DNA
spiked with different concentrations of ‘Pelican’ DNA,
robust allele calling was possible in DNA mixtures
down to 18 ng:2 ng. Collectively, this demonstrates that
where diagnostic markers are available, molecular identification
of a single off-type for a given DUS trait
within a bulk of ten individuals should be possible. We
validated this assumption, with all of the diagnostic
genetic markers investigated found to robustly detect
DUS off-types at a frequency of 10% in DNA extracted
from tissue collected from pools of 10 individuals. Ultimately,
this work demonstrates that, where diagnostic
polymorphisms are known for DUS traits, KASP
markers should be able to robustly detect off-types or
cross-contamination within DNA samples from a diploid
inbred species down to 10%. While just two varieties
that contrasted for the eight DUS targeted were
investigated in this study, as the markers used are diagnostic
for their relevant phenotype (or a proportion of
the variation observed for that phenotype), in theory the
approach should be valid for any variety studied—
although the introduction of novel alleles via spontaneous
mutation or more exotic germplasm pools may
mean that marker sets would need to be periodically
added to or updated. However, we nevertheless demonstrate
the principle that, for a subset of DUS traits,
molecular markers can now be robustly used as a tool
towards determining all three components of the DUS
testing process in barley. These results are relevant for
the assessment of varietal uniformity by crop breeders,
crop testing authorities and germplasm maintenance, as
well as highlighting the potential use of bulk samples
rather than individual plant samples for assessment of
distinctness by molecular methods
