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Molecular Plant Breeding 2012, Vol.3, No.8, 80
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with CR resistance in wheat and barley. If the
homoeologous allelic relationship is true between the
barley 3H CR QTL and the wheat 3B CR Locus, it
could be relatively easier to characterise the
functional expression of the candidate genes underlie
the homologous locus and to isolate this major gene
for CR resistance in barley than in wheat, considering
the difference in chromosome sets and genome size
between the two crops.
3 Materials and Methods
3.1 Wheat and barley populations used in this study
A wheat doubled haploid (DH) population consisting
of 153 lines derived from a cross between two bread
wheat varieties Batavia and Ernie was used for
mapping QTL conferring CR resistance and for
comparative mapping; a linkage map was constructed
for this population by using 104 SSR and 852 DArT
markers (Li et al., 2010a). Ernie is an American soft
red winter wheat variety with high-level resistance to
CR, and Batavia is an Australian variety highly
susceptible to this disease. Four additional populations
were developed for validating the effect of the 3B
major QTL derived from the resistant variety Ernie
(Li et al., 2010a). The four validation populations
include
67 F4/F5 lines from a cross of 27868/Ernie,
120 F4/F5 lines from a cross of 13832/Ernie, 55 DH
lines from a cross of Kennedy/Ernie, and 120 F2/F3
lines from a cross of 10903/Ernie.
A barley DH population of 92 lines was used to
identify QTL conferring CR resistance and for
comparative mapping. The DH population was derived
from TX9425 (a Chinese landrace resistant to CR)
and Franklin (an Australian variety highly susceptible
to CR). A linkage map for this population was
constructed using 412 DArT, 80 AFLP and 28
microsatellite markers (Li et al., 2008; 2010b). Three
additional populations were developed for validating
the effect of the 3H QTL derived from TX9425. The
three validation populations used in this study include
74 DH lines from a cross of TX9425/Naso Nijo, 120
F3/F4 lines from a cross of TX9425/Gairdner, and 119
F3/F4 lines from a cross of TX9425/DYSYH.
3.2 Marker genotyping
A total of 34 barley primers and 28 Wheat SSR
primers (S-Table 1) from the related genomic regions
associated with CR resistance in wheat and barley
were screened for polymorphism between parents of
the wheat and barley DH populations. Fourteen barley
SSR markers showed polymorphism between TX9425
and Franklin, and 11 wheat SSR markers are
polymorphic between Ernie and Batavia. These SSRs
were amplified using fluorescent dUTPs (Molecular
Probes, Eugene, Oregon, USA). Amplification reactions
were performed in a total volume of 12.5 μL
containing 1× Buffer, 1.5 mM of MgCl
2
, 0.2 mM of
dNTPs, 0.2 μM of unlabeled primer, 0.6 μM
fluorescent dUTPs, 0.5 U of Taq polymerase and 20
ng of template DNA. SSR name and amplification
conditions were those published in the Genetics
supplemental data site: http:/www.genetics.org/cgi/
content/full/156/4/1997/DC1. Gel electrophoresis was
performed on an automated Gel scanner (Gel-Scan
2000, Corbett Research). Samples were electrophoresed
on an 18-cm long 4% polyacrylamide gel containing
7M urea. Allele sizes were calculated by comparison
with a 350 (TAMRA) size standard.
3.3 CR screening
A highly aggressive isolate of
Fusarium pseudogram-
inearum
(Fp3096) collected from Northern New
South Wales, Australia (Akinsanmi et al., 2004) was
used in this study. The procedures used for inoculum
preparation were based on that described by Mitter et
al (2006). Specifically, plates of 1/4 strength PDA
(potato dextrose agar) inoculated with Fp3096 were
incubated for a week at room temperature. The
mycelium was scraped and plates were incubated for a
further week under a combination of cool white and
black (UVA) fluorescent light with a 12-h photoperiod.
Macroconidia were harvested, suspended in sterile
distilled water after straining through layers of
cheesecloth and spore concentration was adjusted to
1×10
6
spores/mL. Tween 20 was added (0.1% v/v) to
the spore suspension prior to use.
CR assay was based on procedures described by Li et
al (2008). Seeds were germinated in petridishes on
3-layers of filter paper saturated with water. Newly
germinated seedlings were immersed in the conidial
suspension (1×10
6
m/L) for 1 min. Three treated
seedlings were sown in a 5 cm×5 cm square punnet