**Session VI -- Plant Breeding, Germplasm Utilization and Cereal
Genomics -- Poster VI-8**

**Genic Microsatellite Markers from Expressed Sequence Tags (ESTs) of**

**Developing Oat Seed**

Peter Eckstein, Aaron Beattie, Brian Rossnagel, and Graham Scoles

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

Microsatellite markers (simple sequence repeat (SSR)) have been widely
available for the genetic analysis of agronomically important cereal
crops such as wheat, barley, and rice, and have formed the backbone of
genetic studies in these crops due to their high levels of polymorphism
and ease of use. Despite the benefits of microsatellite markers, few are
available for use in oat genomics, and even fewer have been placed on
genetic maps, a pre-requisite for their efficient association with
agronomically important traits. The cross-applicability to oat of SSR
markers developed from wheat, barley, and sorghum has generally been
found to be poor. Though the levels of polymorphism are adequate (if not
high), the polymorphisms are often dominant in nature resulting from
primer-template mis-match, as opposed to the co-dominant, multi-allelic
polymorphisms that are generated at true microsatellite loci and from
which SSR markers gain their analytical power.

Current efforts to increase the number of oat-specific SSR markers, such
as the development of microsatellite-enriched libraries from cultivars
Kanota and Ogle, and the in-silico analysis of publicly available oat
EST sequences for simple sequence repeats, are yielding results. We have
generated an EST sequence database in cooperation with the Natural
Products Genomics Resource (NAPGEN) initiative of PBI-NRC. Approximately
15,000 ESTs were isolated and sequenced (single pass) yielding 6149
unigenes (1685 contigs plus 4464 singletons). This database was mined
for SSR regions using the Web-based "SSR Primer Discovery" tool , and
primers flanking the SSRs were designed through the integrated Primer 3
program (set at default values). The analysis identified 260 EST
sequences (4.2%) that contained perfect and/or imperfect SSRs of n 6 for
di-, n 5 for tri-, n 4 for tetra, and n 4 for penta-nucleotide repeat
motifs. Sixty-eight percent of all repeats were tri-nucleotide motifs,
18.5% were dinucleotide motifs, 13% were tetra-nucleotide motifs and
penta-nucleotide motifs was limited to one. Approximately one third of
the SSR containing sequences were BLASTX annotated to genes of known
function, one third of sequences of unknown function, and one third of
sequences were unique. Fifty SSR primer pairs were chosen on the basis
of repeat lengths ≥ 20 nucleotides (irrespective of motif length) for
initial analysis on the cultivar Kanota. Ninety percent of primers pairs
mediated the amplification of one to three DNA fragments, while 10%
amplified multi-band and/or weak banding patterns. Forty-five SSR
markers were subsequently analysed against nine oat cultivars (Kanota,
Ogle, TAM O-301, Marion, Terra, Mn841801-1, Noble-2, CDC SolFi, HiFi) of
five mapping populations. Data will be presented on marker quality,
polymorphism potential, and mapping potential.

Based on early indications of marker quality and levels of polymorphism
among nine oat cultivars for the initial 50 SSR primer pairs, and on
studies similar to ours, there is good potential for the development of
additional SSR markers from the remaining pool of 210 primer pairs.