Identification Species of Powdery Mildew Pathogens by Direct Sequencing
I. Amplification Using PMITS Primers
A. Single-Round PCR
Sample preparation
Amplification using the PMITS1/2 primer pair works well on fresh tissue and has
reportedly worked well on herbarium specimens (Cunnington et al, 2003). Remove 100
mg of fresh or dried plant tissue containing heavy mycelial growth and sporulation and
extract genomic DNA using the DNEasy Plant Mini Kit. Add tissue to a sterile beadbeater tube containing two 5-mm diameter beads. Freeze in liquid nitrogen, beat tube
twice for 30 sec at 2500 rpm, add Buffer AP1 and RNase A, incubate at 65°C as
indicated in kit instructions, and follow remaining kit instructions. Elute in 100 µl Buffer
AE.
PCR Reaction Mixture (per 50 µl reaction)
Nanopure water
5 µl
MgCl2, 25 mM
2 µl
Titanium Taq 10X buffer
5 µl
dNTPs, 1.0 mM
10 µl
Primer PMITS1, 0.8 µM
10 µl
Primer PMITS1, 0.8 µM
10 µl
Titanium Taq
1 µl
Smart Cycler Additive Reagent
5 µl
Sample
2.0µl of DNA extract (or controls as noted below)
Primers
Prepare to a concentration of 0.8 µM (0.8 pmol/µl) in Nanopure water:
PMITS1 5’-TCGGACTGGCCYAGGGAGA-3’
PMITS2 5’-TCACTCGCCGTTACTGAGGT-3’
Controls
Negative control:
Positive control:
2 µl Nanopure water
Genomic DNA from a known powdery mildew extract
Cycling Protocol
Name of protocol (on GeneAmp thermocycler): pmits.primers
Ramp rate=MAX
1. 94ºC for 10 min
2. 35 cycles of: 94ºC for 1 min
63ºC for 1 min
72ºC for 2 min
3. 72ºC for 10 min
Duration of protocol: not determined
Expected Results
Expected amplicon from PMITS primers is 700-800 bp in size (Cunnington et al, 2003).
Sequencing Amplicon
If a product of the correct size is amplified, gel-purifying may be necessary since
fragments of other sizes may also amplify. Mix 42 µl of reaction mixture with 8 µl of
loading buffer and dispense into three lanes joined together in a 1.5% agarose gel. Excise
band and clean it using the QiaQuick gel extraction kit or equivalent, paying special
attention to instructions in the kit which relate to direct sequencing. Elute in 30-50 µl
Buffer EB. Sequence using PMITS1 and PMITS2.
B. Nested PCR (for difficult targets)
For targets that do not amplify well with the above reaction mix, a second, nested PCR is
recommended (Cunnington et al, 2003), as follows.
PCR Reaction Mixture (per 50 µl reaction)
Nanopure water
7.3 µl
MgCl2, 25 mM
2 µl
Titanium Taq 10X buffer
5 µl
dNTPs, 1.0 mM
10 µl
Primer PMITS1, 0.8 µM
20 µl
ITS4, 5 µM
3.2 µl
Titanium Taq
1 µl
Bovine serum albumin, 10 mg/ml* 1 µl
*Sigma Cat. No. A-7030, filter-sterilized.
Samples
Run one tube with 0.5 µl of a 1:100 dilution of the reaction mixture from the SingleRound PCR Reaction (above), and run another with an undiluted 0.5 µl.
Cycling Protocol
Name of protocol (on GeneAmp thermocycler): pmits.nested
Ramp rate=MAX
4. 94ºC for 10 min
5. 35 cycles of: 94ºC for 1 min
60ºC for 1 min
72ºC for 2 min
6. 72ºC for 10 min
Sequencing
Gel-purify product and sequence using PMITS1 and ITS4.
II. Amplification Using Primers of Takamatsu et al. (2001)
This protocol has not been evaluated in our laboratory, but the paper by Takamatsu et al
(2001) suggests it also has promise. This may be useful for targets that fail to amplify
with the primers of Cunnington et al (2003). For details, see paper.
Primers
ITS1 5’-TCCGTAGGTGAACCTGCGG-3’
PM5 5’-TTGCTTTGGCGGGCCGGG-3’
PM4 5’-CCGGCCCGCCAAAGCAAC-3’
PM6 5’-GYCRCYCTGTCGCGAG-3’
Controls
Negative control:
Positive control:
2 µl Nanopure water
Use universal primer set ITS1/ITS4
References
Cunnington, J. H., Takamatsu, S., Lawrie, A. D., and Pascoe, I. G. 2003. Molecular
identification of anamorphic powdery mildews (Erysiphales). Australasian Plant
Pathology 32:421-428.
Takamatsu, S., and Kano, Y. 2001. PCR primers useful for sequencing of rDNA of the
powdery mildew fungi. Mycoscience 42:135-139.
Document Control: February 17, 2016