Molecular Markers for the Oat Stem Rust Resistance Gene Pg16.

1
Molecular Markers for the Oat Stem Rust
Resistance Gene Pg16. Peter
Eckstein1, Tom Fetch2, Donna Hay1, Tom Zatorski1,
Brian Rossnagel1, Graham Scoles1
1 Department of Plant Sciences/Crop
Development Centre, University of Saskatchewan,
Saskatoon, SK, CANADA, S7N 5A8
2 Cereal Research Centre, Agriculture and
Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB,
CANADA, R3T 2M9
Results
Introduction

• The ratio of homozygous resistant to homozygous
  susceptible lines deviated from the expected
  (11) with a predominance of susceptibles and a
  greater than expected number of segregating lines
  in both crosses (Tables 1 and 2).
• Five 10-mer primers (OPB20, UBC110, UBC172,
  UBC297, UBC331) amplified six polymorphic
  fragments (Figure 1). All polymorphic fragments
  amplified from DNA of the resistant parent ()
  and failed to amplify from the susceptible parent
  (-), and all co-segregated in W99162 x Ronald.
• All polymorphic fragments showed 100 linkage
  (cis) with the disease reaction of lines involved
  in marker discovery that were homozygous for
  resistance/susceptibility (Table 1).

Puccinia graminis Pers. f. sp. avenae Eriks. E.
Henn. is the causal agent of stem rust which
occurs commonly in Manitoba and eastern
Saskatchewan. Infection level depends largely on
the amount, race structure, and time of arrival
of inoculum carried on southerly winds from the
USA. Several genes conferring resistance have
been identified of which Pg16 is effective
against many races of Puccinia graminis f. sp.
avenae, including race NA67. This gene was
stably introgressed into hexaploid oat from Avena
barbata Pott. collection D203 by Brown et al
(1986), and is the same source of Pg16 resistance
used in current breeding. Individual stem rust
resistance genes are often difficult to screen
for since their effects may be masked by the
presence of others. The ability to screen using
molecular markers-assisted selection (MMAS) is
therefore desirable.
Table 1. Polymorphic fragment sizes for five
random primers and the Pg16-1100 SCAR marker, and
their segregation ( present, - absent) among
progeny of W99162 x Ronald. Homo.R and Homo.S
lines homozygous for each disease reaction, Seg
lines segregating for the disease reaction.

• All polymorphic fragments were isolated from
  W99162 and sequenced. None were similar to each
  other.
• The 1100bp OPB20 fragment was chosen for
  conversion to a PCR-based MMAS friendly marker.
  Primers complementary to the ends of the fragment
  produced a robust single band from DNA of
  resistant genotypes, while failing to amplify
  fragments from susceptible genotypes (Figure 2).

Materials, Methods
Plant Materials W99162 (resistant) x Ronald, 162
F4 derived F5 RILs. Ninety-three lines involved
in marker discovery, 69 additional lines used for
linkage confirmation. OT399 x OT2030
(resistant), 58 F5 RILs used for linkage
confirmation.

• Linkage between SCAR marker Pg16-1100 and
  resistance was identical to that of the RAPD
  fragments (Table 1).
• Linkage of SCAR marker Pg16-1100 with disease
  resistance in additional lines from W99162 x
  Ronald, and OT399 x OT2030 was absolute (Table 2).

Marker Discovery RAPD analysis of
bulked-segregant DNA ( resistant and susceptible
DNA bulks, each from eight constituent lines).

Phenotyping Twelve plants per RIL were seeded in
5 fibre pots and grown in a greenhouse at 22 /-
2C, 14 hour photoperiod. Seven day old
seedlings were inoculated with a 5 mg/ml
urediniospore suspension of P. graminis f. sp.
avenae race NA67, using a rust inoculator and air
pump. Inoculated plants were placed in a dew
cabinet set at 19C with near 100 humidity and
dark for 16 hours, then returned to the
greenhouse. Disease severity was rated at 13
days post-inoculation, on a 0-4 scale where 0-2
is indicative of low infection type (IT)
(resistance) and 3-4 of susceptibility (Gordon,
1933).
Discussion and Summary

• Five RAPD markers co-segregated perfectly with
  Pg16 type stem rust resistance.
• A robust PCR based marker for Pg16 has been
  developed for use in MMAS.
• Segregation ratios for the disease reaction in
  both crosses did not support single gene disease
  resistance. However, perfect linkage with a
  number of markers, and the fact that all of the
  cis linked fragment amplified only from lines
  carrying Pg16, suggests a single resistance gene
  located on a segment of unique DNA introgressed
  (inserted) from A. barbata. Slightly impeded
  transmission of the gene has been previously
  noted by Brown et al (1986).
• Segregation ratios for disease reaction in
  OT399 x OT2030 may have been further skewed
  through previous selection for a number of
  agronomic traits.
• Attempts to locate the gene to an oat genetic
  map have failed since none of the polymorphic
  fragments were amplified in either the
  Kanota/Ogle or the Marion/Terra mapping
  populations.
• Attempts to design a co-dominant marker, or a
  marker for the alternate allele, have failed
  since the six polymorphic fragments failed to
  amplify from DNA other than from resistant lines
  (W99162, OT2030).

PCR Primers Random 10-mer primers originated
from the University of British Columbia (UBC) and
Operon Corporation (O series). SCAR primer
sequences 5 - GGACCCTTACATCAC 5 -
GGACCCTTACTCTGG
PCR Conditions for Pg16-1100 20 mM Tris-HCl,
50 mM KCl, 2 mM MgCl2, 400 µM dNTP, 200 nM each
primer, 1 unit Taq polymerase, 50 ng template
DNA. Cycling 94ºC (3 min), followed by 35
cycles of 94ºC (30 sec), 57ºC (45 sec), 72ºC (60
sec).
Acknowledgements We would like to thank our
industry partners for their continued support of
oat research Pepsi-Quaker Tropicana
Gatorade Canada Can-Oat Milling
Cargill Inc.
References Brown, P.D., Forsberg, R.A.,
McKenzie, R.I.H., and Martens, J.W. 1986. The
use of disomic alien addition lines in the
transfer of oat stem rust resistance to hexaploid
oats. In Lawes, D.A. and Thomas, H. (eds.).
Proc. 2nd International Oat Conference,
Dordrecht, Netherlands. pp 16-20.