**Session VI -- Plant Breeding, Germplasm Utilization and Cereal
Genomics -- Poster VI-9**

**DArTs without the DArT Board: The Application of Individual DArT
Markers to Marker-assisted Selection**.

Peter Eckstein, Brian Rossnagel, Graham Scoles

Department of Plant Sciences/Crop Development Centre, University of
Saskatchewan, Saskatoon, SK, Canada, S7N 5A8, (306) 966-8349,
peter.eckstein@usask.ca.

The recently developed DArT marker array for oat is a promising tool for
genotyping, molecular mapping, and marker/trait association studies.
While the DArT array can quickly and concurrently analyse thousands of
loci at minimal costs per data point, the platform currently
"over-delivers" for the MMAS needs of many breeding programs. Few
associations have been established between DArT markers and
agronomically important traits. For traits that have previously been
marked and mapped, association between the mapped loci and the newly
placed DArT markers need to be confirmed.

DNA sequences for oat DArT probes have been derived and may be an
interesting resource for the potential development of robust, PCR-based,
locus-specific markers such as SNPs. These SNP markers could then be
interrogated on a variety of platforms that may, at least in the short
term, be more amenable to marker-assisted selection in oat. With a view
to the use/conversion of individual map-based DArT probes in barley, we
have conducted a study of selected barley DArT markers for their
potential application outside of the DArT platform, by conversion to
PCR-based allele-specific markers. The study targeted a leaf scald
resistance locus on chromosome 6HS whose location was determined through
very loose linkage with a poorly amplified RAPD fragment. Attempts to
identify quality markers with tighter linkage included the analysis of
RFLP, SSR, and SNP markers placed near the target on various barley
genetic linkage maps. These were unsuccessful primarily due to a lack of
polymorphism near the locus in the test populations. Having exhausted
most map-based (and publicly available) SSR (48), RFLP (9), and SNP (1)
marker candidates, the DNA sequences for 14 bPb-series DArT probes
located near the locus on the DArT consensus map, and permission for
their analysis, were kindly provided by Dr. Andrzej Kilian of DArT Pty.
DNA sequences ranged in length from 212 to \> 850 nucleotides. Primers
complementary to the ends of the sequences were designed to amplify the
full DArT probe fragments. The study considered the number of amplified
fragment(s) and size(s), levels of polymorphism between the parents of
the study populations, linkage to the trait, and conservation of marker
order. Ten primer pairs amplified single fragments of expected size,
while three primer pairs amplified two to three fragments. Only one
primer pair amplified poorly/multiple fragments. Four of 13 primer pairs
amplified polymorphic banding patterns from DNA of resistant and
susceptible genotypes upon initial amplification (three dominant, one
co-dominant). Sequence analysis of amplification products from an
additional 6 primer pairs revealed sequence variation between DNA of
resistant and susceptible genotypes that would allow for the design of
additional SNP markers. Five polymorphic markers chosen for linkage
analysis in the population were tightly linked to the resistance locus,
three of which co-segregated, and their order was conserved to that of
the consensus DArT map. Results from barley regarding DArT marker
polymorphism rates and locus specificity, demonstrate the potential for
use of individual DArT markers "off" the platform.