Molecular Ecology Notes (2003)
doi: 10.1046/j.1471-8286 .2003.00350.x
PRIMER NOTE
Blackwell Science, Ltd
Isolation of 12 microsatellite loci, using an enrichment
protocol, in the phytopathogenic fungus Puccinia triticina
X . D U A N ,* J . E N J A L B E R T ,† D . V A U T R I N ,‡ M . S O L I G N A C ‡ and T . G I R A U D §
*Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100094, China, †Unité de Pathologie Végétale et
Epidémiologie, INRA, 78850 Thiverval Grignon, France, ‡Populations, Génétique et Evolution, CNRS, avenue de la Terrasse, 91198
Gif-sur-Yvette cedex, France, §Ecologie, Systématique et Evolution, Bâtiment 362, Université Paris-Sud, 91405 Orsay cedex, France
Abstract
We report the characterization of 12 polymorphic microsatellite markers in the biotrophic
fungus Puccinia triticina, the causal agent of leaf rust on wheat. An enrichment protocol
was used to isolate microsatellite loci and polymorphism was explored with 15 European
isolates. Significant level of cross-amplification (44% of the loci) was found in P. striiformis.
Keywords: basidiomycota, biotrophic fungus, dikaryote, leaf rust, microsatellite enriched library,
Puccinia triticina
Received 3 September 2002; revision received 3 October 2002; accepted 3 October 2002
Puccinia triticina Eriks. (P. recondita f.sp. tritici: Anikster et al.
1997) is a Basidiomycota responsible for wheat leaf rust, a
common and damaging disease of wheat (Triticum aestivum).
Mainly spread by asexual dikaryotic urediniospores, this
biotrophic fungus is however, able to complete a sexual
cycle on the aecidious host species Thalictrum speciosissimum
in Mediterranean regions and in Central America.
The development of leaf rust populations relies on its
fast uredial cycle, allowing bursts of epidemics when climatic
conditions are favourable. Despite clonal multiplication of
spores, P. triticina populations exhibit a high level of diversity,
both at virulence and molecular levels (Kolmer & Liu 2000).
The importance of sexual reproduction for the maintenance
of polymorphism or for the adaptive dynamic of the species remains unknown, and its determination required
molecular markers. As P. triticina is a dikaryotic organism,
codominant markers were needed, which prompted a search
for microsatellite loci.
A microsatellite enriched-library of P. triticina was built
according to Dutech et al. (2000) and Giraud et al. (2002),
using biotin-labelled microsatellite oligoprobes [(TC) 10 (TG)10
(AAT)10 and (AAG)10] and streptavidin-coated magnetic
beads. Total genomic DNA was extracted from a mix of
spores sampled in Grignon (France) in 1999. 10 mg of spores
were ground by vortexing a 2-mL tube containing 200 µL
of buffer (25 g/L D-Sorbitol, 10 g/L N-Lauroylsarcosine,
Correspondence: Jérôme Enjalbert. Fax: 33 1 30 81 53 06. E-mail:
[email protected]
© 2003 Blackwell Publishing Ltd
0.8 m NaCl, 20 mm EDTA, 0.1 m Tris, PH 8) and seven glass
beads, 4 mm in diameter, for 2 min 30 s. After adding
300 µL of buffer and 10 µL proteinase K (10 mg/mL), the
suspension was incubated 1.5 h at 65 °C, then mixed with
0.5 mL chloroform, and centrifuged at 4 °C for 20 min at
20 000 g. DNA was precipitated from the supernatent with
one volume of isopropanol (−20 °C), centrifuged for 20 min
(20 000 g), and DNA pellet washed with 70% ethanol, air
dried and dissolved overnight at 4 °C in 20 µL TE-buffer
(10 mm Tris pH 8, 1 mm EDTA) + 1 µL RNase (10 mg/mL).
After digestion of genomic DNA and transformation,
recombinant colonies were screened by hybridization of
dioxigenine-labelled oligoprobes [(TG)10 (TC)10 and (AAT)10].
A total of 1500 clones were screened and 133 gave positive
hybridization signal (8.9%). Inserts were of an appropriate
size for sequencing, i.e. mainly between 300 and 650 bp. All
positive clones were sequenced, and we found microsatellites in 110 of them (83%). Most of the microsatellites were
however, too small (from 5 to 7 repeats) to be expected to
be polymorphic. Polymerase chain reaction (PCR) primers
were designed for 36 loci, containing di-, tri or tetra-nucleotide
microsatellites repeated from six to 19 times, using the
computer program oligo™ (National Bioscience).
PCR amplifications were performed using a iCycler
(Biorad) thermal cycler with 35 cycles of 94 °C for 30 s, 52 °C
for 30 s, and 72 °C for 30 s. Each reaction (10 µL) contained
1 µL of 10X reaction buffer (1X = 50 mm KCl, 0.1% Triton X100, 10 mm Tris-HCl, pH 9.0), 75 µm of dCTP, dGTP, dTTP
and dATP, 0.2 g/L BSA, 1.5 mm MgCl2, 2.5 pmol of each
66 P R I M E R N O T E
Table 1 Description of leaf rust isolates used in evaluation of the polymorphism of microsatellite loci: isolate code, avirulence and virulence
genes, date of collection and geographical origin. Avirulence/virulence formulas were established by Henriette Goyeau, Unité de
Pathologie Végétale et Epidémiologie, INRA, 78850 Thiverval Grignon
Isolate
Avirulence genes
Virulence genes
Date
Country
B8907-3B4
B9201-2C3
B9384 -1C1
B9387 -1A1
B9405 -2B
B9407-1CA3
B9414 -1CA2
B9506-2B
B950506-A
B9833-B
B9834-E
B77SaBa
B347
B950019-A
B950365
1,2a,2b,3,3 ka,9,10,15,17,17b,19,23,24,26
1,2a,2b,3,3 bg,3 ka,9,10,15,17,19,20,23,24
1,2a,2b,9,10,19,23,24
1,2a,2b,9,13,15,16,17,17b,19,20,23,24,26
1,2a,2b,9,10,19,23,24
1,2a,2b,3 ka,9,10,13,15,17,17b,19,23,24,26
2a,2b,3 ka,9,10,13,15,17,17b,19,20,23,24,26,17b
1,2a,2b,3,3 bg,3 ka,9,10,15,17,19,20,23,24
2a,2b,2c,3,3 bg,3 ka,9,13,14a,16,19,23,24,26
1,2a,2b,9,19,20,23,24
1,2a,2b,3,3 bg,3 ka,9,15,17,17b,19,20,23,24,26
9,10,14a,16,19,23,24
9,14a,17b,19,23,24,26
1,2a,2b,3,3 bg,3 ka,9,13,14a,15,16,17,19,23,24,26
1,2a,2b,9,13,15,16,17,17b,19,20,23,24,26
2c,3 bg,13,14a,16,20
2c,13,14a,16,17b,26
2c,3,3 bg,3 ka,13,14a,15,16,17,17b,20,26
2c,3,3 bg,3 ka,10,14a
2c,3,3 bg,3 ka,13,14a,15,16,17,17b,20,26
2c,3,3 bg,14a,16,20
1,2c,3,3 bg,14a,16
2c,13,14a,16,17b,26
1,10,15,17,17b,20
2c,3,3 bg,3 ka,10,13,14a,15,16,17,17b,26
2c,10,13,14a,16
1,2a,2b,2c,3,3 bg,3 ka,13,15,17,17b,20,26
1,2a,2b,2c,3,3 bg,3 ka,10,13,15,16,17,20
2c,10,17b,20
2c,3,3 bg,3 ka,10,14a
1989
1992
1993
1993
1994
1994
1994
1995
1995
1998
1998
France
France
France
France
France
France
France
France
France
France
France
Czechoslovakia
Czechoslovakia
Switzerland
Germany
1995
1995
Table 2 Repeat motif, primer sequence, size, amplification conditions (Ta: annealing temperature), number and size in base pair (bp) of
alleles, expected (HE ) and observed (HO ) heterozygosity of the 15 microsatellite loci isolated from Puccinia triticina. Last column describes
the polymorphism found in P. striiformis
Locus EMBL
Repeat
Motif
RB1
AJ508879 (GT)5
RB4
AJ508880 (GT)8
RB8
AJ508881 (TGG)7
RB10
AJ508882 (GT)7+4+4
RB11
AJ508883 (CA)17
RB12
AJ508884 (AG)5+3
RB16
AJ508885 (TGG)7 + (GT)4
RB17
AJ508886 (TGC)5 + (TGG)6
RB25
AJ508887 (AT)4 + (GT)7
RB26
AJ508888 (CT)8+6
RB27
AJ508889 (CA)4+3
RB28
AJ508890 (TGG)5
RB29
AJ508891 (CA)15
RB31
AJ508892 (GT)5 +
(GGGTGTGT)3
AJ508893 (AC)9 +
(TA)5 + (AG)5
RB35
Primer sequences (5′–3′)
TTGTCGTTCTGGAATGATGC
TGCCCACAACCCTCCTC
CAGTATTGTGGTGGTTGGATG
ACTCAAGAATAATGGGGAACAC
CGCCGTTCCCATCGTTC
TAAAACACTCCACCCACGCC
TAAGATTGGTGGTATGTGGTGGA
TTGTCTTTCATCTCATCCAGCC
AGCAGTGAGCAGCAGCGTC
ACTACTGTGAGTGTCGGCTTGG
CCACAAGCAACCACATACCACC
TGGTCCATGAAGAAGTCTCTGAAC
CATTTGAGCCACGGTTGACTG
AGACATGGTCGCAGCCACTG
CTTCGGTAGGATTTCGAGCG
CAGCTCCAAATCCTTTGCC
ATGTCTGTAGTCGGCAGGGC
GCCTCTGCGGGATCGGT
TCGTCCTGCCTACCTCTGAC
AAAGTGCATGATCTGCATGTG
CTATCGAGTCCAGAACCGAAC
CAAGCCAAGACCTGAGCTATC
CATCTGGCTGGTGAGGTCGC
GAAGCCCGCCGAGCAGC
CTCACCAAACATCAAGCACC
GAGCCTAGCATCAGCATCC
CTTGTCGTTCTGGAATGATGC
CCACAAACAACCACATACCAC
ACTGCGATATCCAGTACACACAC
TGATGGGCTCGCAGTGG
Size
(bp)
Ta
No. Alleles
(°C) (size: bp)
HE
126
52
0.49 0.77 Not amplifying
244
52
147
52
218
52
204
52
288
52
277
52
92
52
228
52
346
52
170
52
318
52
118
52
269
52
246
52
2
(134-126)
2
(244-230)
3
(147-141-138)
1
HO
P. striiformis amplif
Ta, No/size of alleles, He
0.23 0.27 Not amplifying
0.65 0.71 Not amplifying
52 °C, 2 alleles: 216
& 220 bp, HE = 0.12
0.58 0.79 Not amplifying
0
0
4
(208-206-204-178)
2
0.49 0.73 Not amplifying
(298-288)
1
0
0
52 °C, 2 alleles: 156
& 161 bp, HE = 0.12
3
0.65 0.14 Not amplifying
(92-91-89)
2
0.24 0.14 Not amplifying
(228-226)
3
0.52 0.53 Not amplifying
(346-342-340)
1
0
0
52 °C, 2 alleles: 169
& 170 bp, HE = 0.12
3
0.56 0.87 Not amplifying
(318-316-315)
9
0.83 1.00 Not amplifying
(118-180)
2
0.49 0.73 Not amplifying
(277-269)
3
0.64 0.87 52 °C, 2 alleles: 224 &
(248-246-244)
228 bp, He = 0.12
© 2003 Blackwell Publishing Ltd, Molecular Ecology Notes, 3, 65–67
P R I M E R N O T E 67
primer, 0.25 U of Taq DNA polymerase (Promega), and
approximately 10 ng of sample DNA. PCR products were
analysed in 6% polyacrylamide gels and visualized by silver
staining (Chalhoub et al. 1997). Alleles were scored by length
in bp, using as size standard a 25pb DNA ladder (Promega).
Of the 36 microsatellites tested, 31 successfully amplified
fragments of appropriate size. Their polymorphism was
evaluated on a sample of 15 leaf rust isolates, composed of
11 French isolates and four isolates of other European
origins (Table 1). Only 12 microsatellite loci were polymorphic (Table 2). This low rate of polymorphic markers (33%),
as well as the low number of alleles (2– 4), can be at least
partly explained by the low number of repeats of the cloned
microsatellites. If most of the loci have allele sizes corresponding to increments of the repeat unit, RB17 and RB28
present 1 bp allele differences (Table 2): insertion-deletion
events are also involved in the polymorphism detected.
For RB31, the two alleles detected appear to be increments
of the 8pb repeat instead of the 2pb. The majority of the
isolates presented two bands at each polymorphic locus,
indicating a high heterozygosity of the dikaryotic uredinospores. The three isolates from Czechoslovakia and Switzerland were discriminated from the other isolates by four
microsatellites (RB8, RB12, RB28 and RB31). Even if the
very small sampling size used here is expected to create
linkage disequilibrium at random, there was no evidence
of strong linkage disequilibrium between loci for the other
micorsatellites.
The 36 primer pairs were also tested for cross amplification
on 16 P. striiformis f.sp. tritici pathotypes (1 isolate per pathotype) of French and Chinese origins. Sixteen microsatellites
© 2003 Blackwell Publishing Ltd, Molecular Ecology Notes, 3, 65–67
gave positive amplifications, four of them being polymorphic (RB10, RB16, RB27 and RB35).
Despite the limited number of isolates studied here, the
polymorphism found for the 12 microsatellites demonstrate
that these markers will be highly useful for studying sexuality and geneflow in leaf rust populations.
Acknowledgements
We thank Henriette Goyeau for providing us the leaf rust isolates,
Laurent Schibler and Sead Taourit for sequencing facilities in
INRA of Jouy en Josas, and CETIOM institution for hosting the
genotyping activities in their laboratory in Grignon.
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