**Report of Informal Discussion on Oat Molecular Marker Development at
Plant and Animal Genomics XV Symposium, San Diego, California, Jan.
13-17, 2007**

Topics discussed and written reports submitted include:

**Oat DArT progress report** \-- Andrzej Kilian (Diversity Arrays
Technology) made a presentation and led discussion on the exciting
progress and next steps in the process. Nick Tinker (Agric. and
Agri-Food Canada, Ottawa) coordinator of the project, together with
Andrzej, prepared a summary and status report updated through March 2007
(see below).

**Oat genomic SSR library** \-- Joe Anderson (USDA-ARS, West Lafayette,
IN) prepared a written summary of his progress report (see below).

**Barley and wheat genomic SSR markers in oat** \-- Eric Jackson
(USDA-ARS, Aberdeen, ID). See abstract of poster P140 referenced below.

**Wheat SSRs in oat** \-- Report from North Carolina. Although not able
to attend, Paul Murphy (North Carolina State Univ.) and Jeanette Lyerly
(USDA-ARS, Raleigh) sent in a written report to share at the meeting
(see below).

**Oat EST-SSRs** \-- Rayco Becker\'s (Germany) results were summarized
by Howard Rines. Published article in *Plant Breeding* now available
online.

**Oat BAC library** \-- Howard Rines shared an estimate he had been
requested to obtain from the Arizona Genomics Institute (see below).

**GrainGenes, Avena Avenue** \-- Dave Matthews (USDA-ARS, Ithaca, NY)
reiterated that the Avena Avenue web site
(http://wheat.pw.usda.gov/GG2/oat.shtml) developed for the oat community
contains information and linkages to data and reports on oat SSR and
other markers, QTLs, marker maps, germplasm data bases, regional
nurseries, and meetings and other reports. Information of almost any
kind (such as this report) thought to be of interest to the oat
community can be posted by contacting Dave (matthews@wheat.pw.usda.gov).

**Announcement**: 2008 International Oat Conference, June 28-July 2,
2008, Minneapolis, Minnesota. Contact: Deon Stuthman,
<stuth001@umn.edu>.

**Possible \"Oat Genomics\" Workshop at Plant and Animal Genome XVI,
January 2008**. Eric Jackson and Nick Tinker will submit a proposal to
the PAG organizing committee.

**Oat posters at PAG:** Abstracts available at
http://www.intl-pag.org/15/abstracts/

#### P140 Surveying and mapping of genomic-derived SSRs from barley and wheat improves oat linkages -- Gongshe Hu, Eric Jackson, J. Michael Bonman

#### P341 Development of markers associated with traits of agronomic importance in oats -- Catherine Howarth, Tim Langdon, Alexander Cowan, John Valentine

#### P342 An oat (*Avena sativa* L.) cDNA library representing early seed development \-- Aaron D. Beattie, Peter E. Eckstein, Tom Zatorski, Graham J. Scoles, Brian G. Rossnagel

#### P343 Mapping qualitative and quantitative oat crown rust resistance in Ogle and TAMO 301 using precise phenotyping approaches -- Eric W. Jackson, Don E. Obert, Gongshe Hu, Monica Menz, Anne Sturbaum, John M. Bonman

#### P737 Using the Affymetrix wheat microarray as an oat expression platform -- Joseph Anderson, Elizabeth Buescher

#### 

#### Reports:

**Summary and status of oat DArT project**

March 2007 - Nick Tinker

-       A consortium to fund and develop a typing array for oat DArT

analysis was initiated in early 2006. 

-       Members were solicited from the international oat research

community based on potential interest in using the technology. 

-       Members from 13 different institutions agreed to:

o       fund portions of the total cost

o       provide DNA from germplasm representative of diverse areas of

world oat production

o       participate in planning and discussion

o       Develop a publication of results

-       A panel of 170 diverse oat varieties was assembled, plus a small

set of wild relatives, and the Kanota x Ogle mapping population.

-       Initially, 12,000 random clones (=2 discovery arrays) were to be

developed from this material for the purpose of screening differential

clone presence in DArT arrays.  The number of clones has now been

increased to 18,000 due to slightly lower polymorphism than was found in

wheat and barley.

-       From these clones, we expect to find 1000 to 1200 that give

reliable marker polymorphisms.  These will be assembled into a final

typing array.

-       The clones on the typing array will be sequenced for future

conversion to individual PCR marker assays.

-       Polymorphism screening from the initial panel and mapping

population will be collected and analyzed as part of the project.

Current status:

-       Results from 1 array (6000 clones) have been completed and

analyzed.  This provided approximately 400 clones that were polymorphic

on the germplasm panel. 

-       At this point, the consortium will initiate preparation of a

manuscript describing results, and in selecting and sequencing clones

for the typing array.

-       A typing array is expected to be available to members of the

consortium by mid summer.  This will be available to the broader

community as soon as the work is published.

**Progress of Oat genomic SSR identification** \-- Joe Anderson

We have made enriched libraries of 8 different SSR motifs from both
Kanota and Ogle as they are the parents so the most widely used mapping
population. The libraries were constructed by digesting genomic DNA with
3 blunt cutting restriction enzymes and one enzyme that leaves "sticky"
ends. DNA ranging from 400 bp to 1000bp was size selection from agarose
gels and linkers were ligated to the purified DNA. These digested/size
selected/linker DNAs were individually enriched for a particular SSR
motif using a SSR oligo-biotin/strepavidin-magnetic bead affinity
chromatography followed by a PCR amplification step. DNAs were pooled by
plant line and SSR motif and cloned into a PCR cloning vector for a
total of 16 enriched libraries. The SSR motifs are AGG, AC, GT, GA, CAG,
CCG, GAA, and AAC).

From these libraries 3,072 clones per motif per oat line were randomly
robotically picked and arrayed into 384 well microtiter plates for a
total of 49,152 clones. These clones were arrayed onto macroarrays for a
final selection step which has been completed. This array selection
indicated that approximately 40% of the clones hybridized to the SSR
motif used at the probe. This 40% enrichment suggests that we have
approximately 1,228 putative SSR clones/motif/oat line. These putative
SSR clones were rearrayed into 384 well plates and a subset are now in
the process of being sequenced. Initially we are doing one pass
sequencing of one 384-well plate per motif per oat line for a total of
5,568 clones. These sequences will be analyzed for the presence of the
repeat, number of repeats etc. Primer pairs will be designed from those
sequences which meet certain criteria, such as number of repeats and
position of the repeat in the sequence. This part should be done by the
end of February, 2007

My lab will test a number of these to generate some statistics such as
the percent that amplify from Kanota and Ogle and a panel of oat lines,
the percent that are polymorphic etc. (In a future-mail I will propose a
panel that I would like the community to examine to see if it meets our
needs) At this point there have been a number of labs which have
indicated an interest in helping out with the most challenging part -
identifying useful primer sets that can be mapped in then relevant
populations. Therefore, my plan is to coordinate with the oat community
on developing and mapping a significant number of SSR markers.

**Update on SSRs in oat at NCSU and the USDA Genotyping Lab, January
2007**

From Paul Murphy and Jeanette Lyerly

In the spring of 2006 the Small Grains Group at NCSU, in conjunction
with the USDA Eastern Regional Small Grains Genotyping Center, began
screening genomic wheat SSR primers in lines of oat, with the goal of
expanding the pool of available SSRs. We optimized a PCR protocol for
wheat primer amplification in oat using primers with the M13-universal
fluorescent label, and began an initial primer screening.

To date, 530 primers have been screened from seven primer groups (xcfa,
xcfd, xbarc, xgdm, xgwm, xwmc, and xpsp) on six oat lines (Kanota, Ogle,
Wintok, Norline, Fulghum, and Rodgers) and a wheat control. Of these
530, 153 (28%) were identified as primers that amplified something in
most of the oat lines tested.

As of December 2006, 119 of those 153 selected primers had been repeated
with an expanded set of oat samples (six above with TAM and two *A.
sterilis* accessions) and two wheat controls. Of those, 20 are
amplifying fragments of a consistent size in most oat lines. The
difficulty for these is that thus far, very few are polymorphic.
Thirty-one additional primers are promising, but need further study. In
several cases these 31 primers amplify more than one or two fragments
with some variation in intensity, making it harder to distinguish
consistent SSRs that may be present. Though these are more difficult to
score, if this could be sorted out then these present the greatest
potential for polymorphism from the first round of screening. Together,
this is about 9% of the original 530 tested, which is lower than the
range of reported in the literature of 10-30% for transferability of SSR
primers across species.

For our next phase, we are testing some of the primers identified thus
far in a population as a final confirmation of the presence of the SSR.
This approach was very helpful previously when we checked several tall
fescue SSR primers for amplification in oat. Additionally, there are
hundreds of wheat SSR primers in the NCSU/USDA collection that have not
been screened at all in oat that could be tested. We would only need to
confirm that we are not overlapping with other labs by testing large
numbers of the same primers.

**Report on estimated cost of an oat BAC library**

From a letter from Dave Kurdna, Arizona Genomics Institute, University
of Arizona, to Howard Rines

Costs based on a "per clone basis of 30 cents per clone of average size
130 kb. For example: 4x coverage of *Avena sativa*, genome size 12,500
Mbp = \$115,200

BAC end sequencing is done at a cost of \$2.25/BAC clone