Septoria and Stagonospora Diseases of Cereals - CIMMYT ...

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Session 3B — N.E.A. Murphy, R. Loughman, R. Wilson, E.S. Lagudah, R. Appels, and M.G.K. Jones
families to identify homozygous F2
plants. The DNA of eight
homozygous resistant F2 plants
and 14 homozygous susceptible F2
plants was selected for the analysis.
Equal amounts of DNA from
the homozygous resistant F2 plants
were bulked together as was the
DNA from the homozygous
susceptible F2 plants. Each bulked
sample and the DNA from each
parent were divided into two
aliquots. One aliquot was left
untreated. The second aliquot was
treated to enrich the proportion of
low copy sequence DNA by
removing the repetitive DNA
sequences using hydroxyapatite
after selective renaturation. The
technique followed Eastwood et al.
(1994). The double-stranded DNA
was removed from the samples
after a Cot value of 120 had been
achieved (Smith and Flavell, 1975).
The DNA was re-suspended in
0.1xTE buffer and diluted to a final
concentration of 5ng/ml of DNA.
RAPD reactions were carried
out on a Perkin-Elmer PE9600. The
random primers were produced by
Operon Technologies (Alameda,
Ca.). The RAPD products were
electrophoresed on polyacrylamide
gels, and the gels were stained
using ethidium bromide with gel
images recorded digitally using a
Bio-Rad Gel Doc 1000 system. For
each primer nine reactions were
carried out: a negative control,
sterile distilled water, was used
instead of template DNA, the next
four reactions used the total
genomic DNA as template, and the
final four reactions used the nonrepetitive
DNA as template.
If a polymorphism was
identified, the polymorphic band
was stabbed with a sterile syringe
needle. The needle was washed in
sterile distilled water which was
used as template for a further
RAPD reaction using the same
primer and reaction conditions that
had generated the polymorphism.
The amplified fragment was ligated
into the plasmid pGEM-T. The
plasmids were transformed into E.
coli cells by heat shock. The
following day colonies were
screened using PCR primers, and
permanent cultures were
established from insert-containing
clones.
The cloned polymorphic band
was used as an RFLP probe to
verify and determine the linkage of
the potential marker to the
resistance gene. Total genomic
DNA of the parental accessions was
digested with a number of
restriction enzymes, singly and in
combination, to find a restriction
enzyme that would produce a
polymorphism with the marker
between the resistant and the
susceptible parent. The marker
band was prepared using an alkali-
SDS minprep, double digested with
NcoI and SacI, separated on an
agarose gel and purified using
agarase. The purified band was
labeled using random nonamer
primers with 32P as dCTP. The
membranes were hybridized
overnight, washed the following
morning, and exposed to
autoradiograph film for up to one
week.
When a suitable restriction
enzyme had been selected, the
DNA from the selected seven
homozygous resistant F2 plants
and seven homozygous susceptible
F2 plants was digested,
electrophoresed and blotted using
an alkali capillary blot. The
membranes were autoradiographed
for up to two weeks.
Results
Sixty random primers were
used to screen the DNA samples.
Two polymorphic bands were
identified by comparing the
banding pattern of the resistant
parent and the resistant bulk with
the susceptible parent and the
susceptible bulk. The first was
amplified using primer OPJ6 and
was approximately 770bp long
(OPJ6770 ). The second polymorphic
band was amplified using primer
OPJ18 and the fragment was
approximately 350bp (OPJ18350 ).
Both bands were ligated into
pGEM-T.
For the first marker, OPJ6770 , no
restriction enzyme nor combination
of enzymes tested was able to
produce a polymorphism between
the resistant and the susceptible
parent. Consequently the marker
could not be tested for its linkage to
resistance. The second marker,
OPJ18350 , produced a
polymorphism between the
resistant and susceptible parent
when the DNA was double
restricted with HindIII and
followed by MseI. The
polymorphism was present as an
extra band in the resistant parent.
All of the F2 plants tested were
correctly classified as resistant or
susceptible when the marker was
used.