MEN_322.fm Page 563 Tuesday, November 12, 2002 11:16 PM
Molecular Ecology Notes (2002) 2, 563 –565
doi: 10.1046/j.1471-8278 .2002.00322.x
PRIMER NOTE
Blackwell Science, Ltd
Isolation of twelve microsatellite loci, using an enrichment
protocol, in the phytopathogenic fungus Puccinia striiformis
f.sp. tritici
J . E N J A L B E R T ,* X . D U A N ,† T . G I R A U D ,‡ D . V A U T R I N ,§ C . D E V A L L A V I E I L L E - P O P E * and
M. SOLIGNAC§
*Unité de Pathologie Végétale et Epidémiologie, INRA, 78850 Thiverval Grignon, France, †Institute of Plant Protection, Chinese
Academy of Agricultural Sciences, Beijing, 100094, China, ‡Ecologie, Systématique et Evolution, Bâtiment 362, Université Paris-Sud,
91405 Orsay cedex, France, §Populations, Génétique et Evolution, CNRS, avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France
Abstract
We report the characterization of 12 microsatellite markers in the biotrophic fungus Puccinia
striiformis f.sp. tritici, responsible for yellow rust on wheat. An enrichment protocol was
used to isolate microsatellite loci, and polymorphism was explored with 96 isolates from
natural populations collected from several French and Chinese locations. Eight primers
(67%) showed cross-amplification when tested with eight isolates of P. triticina.
Keywords: basidiomycota, clonal biotrophic fungus, enriched library, Puccinia striiformis f.sp. tritici,
Yellow rust
Received 11 June 2002; revision received 15 August 2002; accepted 15 August 2002
Puccinia striiformis f.sp. tritici is a fungus (basidiomycota)
responsible for wheat yellow rust (stripe rust), one of the
most damaging diseases world-wide on wheat (Triticum
aestivum). Spread by dikaryotic urediniospores, this
biotrophic fungus shows no evidence of sexual cycle.
Despite its clonal reproductive behaviour, yellow rust
presents a high ability to break down the specific resistance
genes introduced in wheat cultivars (Bayles et al. 2000).
Recent studies using AFLP markers (Steele et al. 2001;
Hovmøller et al. 2002) demonstrated that there was very
low level of polymorphism in yellow rust populations and
this situation is related to the reproductive system of the
species.
Because of the dikaryotic structure and the low molecular diversity of P. striiformis f.sp. tritici, codominant and
highly polymorphic molecular markers are greatly needed
for the study of dynamics of yellow rust populations and
the population genetics, especially to detect the cryptic
sexuality. This prompted a search for microsatellite loci in
P. striiformis.
After an unsuccessful direct screening of a genomic
bank, from which one microsatellite was isolated over
2200 clones tested, a microsatellite enriched-library of
*Correspondence: Jérôme Enjalbert. Fax: 33 1 30 81 53 06; E-mail:
[email protected]
© 2002 Blackwell Science Ltd
P. striiformis was built according to a modified protocol
by 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 mixture of 20 French isolates of
P. striiformis using the method of Justesen et al. (2002)
modified by grinding spores by vortexing a 2-mL tube
containing 200 µL of buffer and seven glass beads, 4 mm
in diameter, for 2 min 30 s. Recombinant colonies were
screened by hybridization of dioxigenine-labelled oligoprobes [(TG)10 and (AAG10)]. A total of 1700 clones were
screened and 79 gave positive hybridization signal. Inserts
were of an appropriate size, i.e. mainly between 300 and
650 bp. All clones were sequenced. 86% of the clones contained microsatellite loci and most of them were short
(from 5 to 7 repeats). PCR primers were designed for 28 loci
containing di- or tri-nucleotide microsatellites repeated
from six to 13 times, using the computer program oligo™
(National Bioscience).
Polymerase chain reaction (PCR) amplifications were
performed using a iCycler (Biorad) thermal cycler with 35
cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s.
Each reaction (10 L) contained 1 L of 10X reaction buffer
(1X = 50 mm KCl, 0.1% Triton X-100, 10 mm Tris-HCl,
pH 9.0), 75 m of dCTP, dGTP, dTTP and dATP, 0.2 g/L
BSA, 1.5 mm MgCl2, 2.5 pmol of each primer, 0.25 U of Taq
MEN_322.fm Page 564 Tuesday, November 12, 2002 11:16 PM
564 P R I M E R N O T E
Table 1 Description of Puccinia striiformis f.sp. tritici pathotypes used to evaluate polymorphism of microsatellite loci: pathotype code, host,
number of isolates and geographical origin
Pathotype
Host
N° of isolates
Origin
6E16
32E0
45E140
109E141
169E136
232E137
237E141
Unknown*
Unknown*
Durum and Bread Wheat
Bread Wheat
Bread Wheat
Bread Wheat
Bread Wheat
Bread Wheat
Bread Wheat
Bread Wheat
Bread Wheat
7
7
13
7
7
14
9
7
25
Southern France
Southern France
Northern France
Northern France
Northern France
Northern France
Northern France
Sichuan, China
Gansu, China
*Not tested on European differential cultivars.
Table 2 Repeat motif, primer sequences, size, amplification conditions, number and size of alleles of the 12 microsatellite loci isolated from
Puccinia striiformis
Locus
Repeat motif
Primer sequences (5′–3′)
RJ3
(TGG)8
GCA
GAT
GTG
GCT
ATC
TCA
CAG
CGG
ATC
CAC
TGG
ACA
CTG
GAT
AGA
CCT
TTC
CAG
CCC
ATC
TTG
CTC
CGT
ATG
RJ4
(TGG)8
RJ12
(AC)7
RJ13
(TTG)7
RJ15
(TG)7
RJ17
(GTT)5 + (GTT)7
RJ18
(TGT)5
RJ20
(CAG)4
RJ21
(GTT)6
RJ22
(CA)8
RJ24
(GTT)5+9
RJ27
(TC)10
GCA
GAA
GGT
AAT
ATT
CAC
GTT
ACC
GAG
TGG
TGA
GCA
CCC
GAA
AGA
CCG
CTG
TTC
TTC
AAG
CTG
AAG
CCC
AGT
CTG
TCA
TGG
CCA
CCG
TGA
CGT
CAG
CAC
ACA
GTG
ACA
ATG
GTG
TCG
ATT
GAT
TCA
GTC
AAG
AGT
CCC
GAC
TAG
GCA
GGA
GCT
TTC
ATT
TCC
TGT
TCC
GTC
GAC
GAT
AAC
CTC
GGT
ACG
GGC
TGA
CTC
TGT
ATT
AGT
ATC
TAA
TTT
GGT
TGG
GGA
CAC
TCT
CAA
GGT
ACC
CAA
GAC
GAG
TCA
TTC
GCT
CAC
TTA
ATT
GGA
CAT
CCT
TTG
CTC
TCT
AGA
GG
CTC
GTC
GCA
TTC
TAG
GAG
CAA
ATC
GGT
CTG
CCC
GTC
GCT
CCG
GGC
CGT
CCC
CCG
GGG
CGG
CAA
GGT
TCA
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 base pairs.
Of the 28 microsatellites tested, 24 successfully amplified
fragments of appropriate size. The double-banded patterns
of amplification observed for most of the primers in addition to the classical stuttering indicated intra clonal heterozygosity caused by the divergence of the two nuclei of this
dikaryotic species. The polymorphism of the 24 microsatellites was evaluated over 96 yellow rust isolates, composed
Size (bp)
Ta (°C)
No. alleles (size)
208
52
198
52
273
52
238
52
246
52
278
52
358
52
294
52
170
52
363
52
286
52
229
52
2
(208–210)
2
(203–206)
2
(271–273)
2
(234–238)
2
246–310
2
(278–281)
2
(358–355)
3
(294-297-303)
2
(170–176)
3
(363-364-365)
5
(268-277-278-286–292)
4
(227-229-233-237)
C
CC
TCA CC
ATC AG
TGG
C
G
TG
G
ATC
G
CG
AG
TGA G
TGA G
CC
CC
GGT CGA C
of 64 French samples and 32 Chinese samples (Table 1). 12
microsatellites were polymorphic (Table 2) and enabled us
to distinguish 6E16, a Mediterranean pathotype specific of
southern France, from all the other European pathotypes
and to separate the Chinese isolates into two other classes.
The primers were tested for cross-amplification over 15
pathotypes (one isolate per pathotype) of Puccinia triticina,
a rust species well differentiated from P. striiformis at both
molecular and morphological levels. Eight primers (RJ3,
RJ4, RJ5, RJ17, RJ20, RJ22, RJ24 and RJ27) of 12 gave positive amplification, none being polymorphic.
© 2002 Blackwell Science Ltd, Molecular Ecology Notes, 2, 563 – 565
MEN_322.fm Page 565 Tuesday, November 12, 2002 11:16 PM
P R I M E R N O T E 565
In conclusion, the isolated microsatellites revealed the
low polymorphism feature in the species of P. striiformis.
These markers will be useful to describe the structure of
this clonal species at continental scale.
Acknowledgements
We thank 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. We are grateful to Shelin Jin, Institute of Plant Protection, Gansu Academy of
Agricultural Sciences, Lanzhou, China, and Ge Yao, Institute of Plant
Protection, Sichuan Academy of Agricultural Sciences, Chengdu,
China, for providing us the Chinese samples of yellow rust.
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© 2002 Blackwell Science Ltd, Molecular Ecology Notes, 2, 563–565
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