Septoria and Stagonospora Diseases of Cereals - CIMMYT ...

Thirty-eight arbitrarily chosen
field isolates of S. nodorum collected
from New York in different places
and years were grown on
cellophane discs overlaid on V-8
juice agar. After approximately
seven days, mycelia were scraped
off the cellophane and placed in
microcentrifuge tubes to be
lyophilized.
DNA extraction
Lyophilized mycelia were
ground in liquid nitrogen. The field
isolates were ground directly in
microcentrifuge tubes with a small
amount of white quartz sand (-50 +
70 mesh, Sigma Chemical
Company, St. Louis, MO). DNA
was extracted by a miniprep
procedure (Wirsel et al., 1996).
Amplification of the HMG
and a-boxes by PCR
PCR primers (Sn-HMG1 and
Sn-HMG2) for the HMG box (Table
1) were designed from conserved
regions in the HMG box of the MAT
gene of Cochliobolus heterostrophus,
M. zeae-maydis, and A. alternata,
whereas the primers (Sn-ab1 and
Sn-ab2) for the a-box were designed
from the a-boxes of M. zeae-maydis
and A. alternata. The primers were
synthesized by the Cornell
University DNA Services Facility
and were dissolved (100 µM) in
Table 1. PCR primers used to amplify
fragments of MAT genes in Stagonospora
nodorum.
MAT-1
Sn-ab1 5’ AA(A/G)GCN(C/T)TNAA(C/
T)GCNTT (C/T)GTNGG 3’
Sn-ab2 5’ TC(C/T)TTNCC(A/G/T)AT(C/T)
TG(A/G)TCNCG(A/G/T)AT 3’
MAT-2
Sn-HMG1 5’ AA(A/G)GCNCCN(AC)
GNCCNATGAA 3’
Sn-HMG2 5’ TT(C/T)TT(C/T)TT(C/T)T(CG)
NCCNGG(C/T)TT 3’
sterile distilled water and stored at
–20C. Each PCR reaction mixture
had approximately 20 ng of
genomic DNA in 50 µl reaction
buffer [1X PCR Buffer (Perkin
Elmer, Norwalk, CT), 0.2 mM
dNTPs, 2.5 mM MgCl2, , 2 µM each
primer, and 0.025 U Taq
polymerase]. The samples were
denatured at 95C for 2 min, and
then subjected to 30 cycles of 95C
for 1 min, 50C for 30 sec, and 72C
for 1.5 min. After extension at 72C
for 10 min, the samples were kept
at 4C. 5 µl aliquots of the PCR
products were analyzed on a 2%
agarose gel in TAE buffer.
Cloning, sequencing, and
analysis of PCR products
The MAT sequences from
related fungi predicted that a PCR
product of ~270 bp for the HMG
box and a product of ~230 bp for
the a-boxes would result if the
amplifications were successful.
Products of these sizes were cloned
from the PCR reaction into the
vector pCR2.1, using the guidelines
Mating Type-Specific PCR Primers for Stagonospora nodorum Field Studies 91
of the manufacturer (Invitrogen
Co., San Diego, CA). DNA
sequences were determined at the
Cornell University DNA Services
Facility. Sequences were aligned
with the LaserGene program
MegAlign (DNASTAR Inc.,
Madison, WI), using the clustal
method. A BLAST search was done
using the NCBI/Genbank internet
databases.
Gel blot hybridization
A standard DNA gel blot
hybridization procedure was
followed (Sambrook et al., 1989).
Results
HMG alpha
The primers for the HMG box
were used with genomic DNA of
(+) and (-) S. nodorum strains as
templates. A PCR product
approximately 0.3 kb, matching the
~270 bp predicted, was present in
the (+) (MAT-2) strain, but was not
present in the (-) (MAT-1) strains
(Figure 1). The primers for the abox,
however, amplified the
1 2 3 4 5 6 7 8 9 101112131415161718
Figure 1. PCR products amplified with the HMG and a-box primers. Lanes 2-9 were amplified using
HMG (MAT-2) primers, and lanes 10-17 were amplified with a-box (MAT-1) primers. Lanes 2,10 and
3,11: C. heterostrophus C5 (MAT-1) and C. heterostrophus C4 (MAT-2), respectively; lanes 4,12 and
5,13: plasmid with cloned a-box from SN435PL-98 and plasmid with cloned HMG box from SN436GA-
98, respectively; lanes 6 and 14: SN436GA-98; lanes 7 and 15: SN437GA-98; lanes 8 and 16: SN005NY-
85; lanes 9 and 17: SN197NY-88. A ~0.3-kb band (arrowhead) was amplified in the MAT-2 isolates
only, and a band above 0.2-kb (asterisk) was amplified only in the MAT-1 isolates.