Isolation of eight polymorphic microsatellite loci, using an

Molecular Ecology Notes (2002) 2, 121–123
PRIMER NOTE
Blackwell Science, Ltd
Isolation of eight polymorphic microsatellite loci, using
an enrichment protocol, in the phytopathogenic fungus
Fusarium culmorum
T . G I R A U D ,* E . F O U R N I E R ,† D . V A U T R I N ,‡ M . S O L I G N A C ,‡ E . V E R C K E N ,* B . B A K A N § and
Y. BRYGOO†
*ESV, Bâtiment 362, Université Paris-Sud, 91405 Orsay cedex, France, †PMDV, INRA, Route de Saint Cyr 78026, Versailles, France,
‡PGE, CNRS, avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France, §LMTC, INRA Rue de la Géraudière, BP 71 627, 44 316
Nantes cedex 3, France
Abstract
We report the development of eight microsatellite markers in the haploid filamentous fungus
Fusarium culmorum, a pathogen of numerous cereal crops. An enrichment protocol was used
to isolate microsatellite loci, and polymorphism was explored with isolates of Fusarium culmorum and F. graminearum from natural populations collected from several French locations.
Keywords: enriched library, filamentous fungus, Fusarium graminearum, Giberella spp.
Received 14 October 2001; revision accepted 16 November 2001
The genus Fusarium (anamorphs Giberella, Ascomycete)
contains numerous phytopathogenic species, F. culmorum
and F. graminearum being particularly important pathogens of cereal crops in many areas of the world. The fungus
causes head and seedling blight of small grains such
as wheat and barley, ear and stalk rot of corn, and
stem rot of carnation (Nelson et al. 1975; Cook 1981;
Kommedahl & Windels 1981). These diseases cause yield
reduction in many crops, and many Fusarium species also
produce trichothecenes, which are highly toxic to both
plants and animals, including humans (Desjardin et al.
1993). Molecular markers are needed to study the relationships between the different species of the Fusarium complex, and to identify trichothecenes-producing isolates.
The molecular markers that have been used to date either
were not polymorphic enough or exhibited problems of
paralogous sequences (O’Donnell & Cigelnik 1997; Aoki &
O’Donnell 1999). This prompted a search for microsatellite
loci in F. culmorum.
A microsatellite enriched-library of F. culmorum was
built according to (Dutech et al. 2000) using biotin-labelled
microsatellite oligoprobes [(TG)10 and (AAG10)] and
streptavidin-coated magnetic beads. Minor modifications
were as follows: (i) total genomic DNA was extracted from
Correspondence: T. Giraud. Fax: + 33-1-6915 73 53; E-mail:
[email protected]
© 2002 Blackwell Science Ltd
the strain L2 of F. culmorum (isolated in 1996 from wheat
in France) using the method of (Möller et al. 1992); (ii)
the DNA fragments generated by RsaI digestion of the
total genomic DNA were not selected for their sizes
before ligation to the adaptators; (iii) the polymerase
chain reaction (PCR) fragments obtained after enrichment were cloned using TOPO TA Cloning Kit (Invitrogen
K450641); and (iv) recombinant colonies were transferred onto charged Nylon membranes (Hybond N+,
Amersham-Pharmacia) and screened by hybridization of dioxigenine-labelled oligoprobes [(TG)10 and
(AAG10)]. A total of 800 clones were screened and 132
gave a positive response. Despite the lack of a size-selecting
step, inserts were of an appropriate size, i.e. mainly between
300–700 bp. Of the 33 clones that were sequenced all
contained microsatellite loci. Approximately half of
the clones contained microsatellite motifs with too low a
number of repeats, i.e. less than eight repeats. PCR primers
were designed for eight loci, using the computer program oligo™ (Macintosh version 4.0, National Bioscience).
Each locus was screened for variation and crossamplification using a panel of 29 Fusarium spp. isolates
(Table 1). PCR amplifications were performed using a
Biometra 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 µL)
contained 1 µL of 10× reaction buffer (50 mm KCl, 0.1%
Triton X-100, 10 mm Tris-HCl, pH 9.0), 75 µm of dCTP,
122 P R I M E R N O T E
Table 1 Description of isolates used to evaluate polymorphism of microsatellite loci: name, species, host plant and geographical origin
Isolate
Species
Host Plant
French Department
A2
A4
A5
B3
B5
C1
C2
C4
C6
C8
E1
E2
F1
G1
H1
K2
O1
O2
P1
R1
Q2
Fg1
Fg2
Fg3a
Fg3c
Fg4
Fg5
Fg6
Fg7
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. culmorum
F. graminearum
F. graminearum
F. graminearum
F. graminearum
F. graminearum
F. graminearum
F. graminearum
F. graminearum
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Durum wheat
Durum wheat
Durum wheat
Durum wheat
Durum wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Durum wheat
Wheat
Wheat
Calvados
Calvados
Calvados
Loire Atlantique
Loire Atlantique
Marne
Marne
Marne
Marne
Marne
Seine Maritime
Seine Maritime
Somme
Gard
Gers
Loir-et-Cher
Haute-Garonne
Haute-Garonne
Gers
Vendée
Loir-et-Cher
Eure-et-Loir
Loire Atlantique
Loir-et-Cher
Loir-et-Cher
Loire Atlantique
Gers
Drôme
Drôme
Table 2 Repeat motif, GenBank accession number, primer sequences, amplification conditions, size and number of alleles (N = 21
individuals of Fusarium culmorum, N = 8 individuals of F. graminearum) of the eight microsatellite loci isolated in F. culmorum
Number of alleles
Locus
Repeat
motif
GenBank
accession no.
F1
(TG)8
AF444196
F3
(CA)11
AF444197
F4
(GT)11
AF444198
F6
(AC)15
AF444199
F7
(GT)7
AF444200
F9
(AC)13
AF444202
F10
(AAG)28
AF444204
F11
(GT)9
AF444203
Primer sequences (5′− 3′)
GAC
CTT
CAT
TTG
CTT
GCT
TAT
CTT
TGA
GAG
CGA
AAC
AAG
GAC
CAG
CAG
AAG
GAT
ATT
AAT
TTT
TTC
TTC
GGT
CAA
TGG
GCT
ACC
CGC
TGC
TCT
GTT
CAA
AGC
CAA
GAT
CCC
CCT
GTG
CCC
GCA
AGT
AAT
AAA
CAA
CGA
TGG
GGC
GCG
ACG
CCG
AAG
GGC
GCT
CAA
TGG
AGC
TTC
GGT
ACG
CAG
AAC
TCG
ACG
ATA
GAC
ACC
GGC
TCC
CGA
GGA
ATA
GAT
GAT
GGC
GCT
AGA
ACC
CTC
CTT
GGA
CGA
CAC
GAC
ATT
TCG
CTT
TCG
AGG
ATC
AGG
CAT
TGA
GAA
ATC
CTT
dGTP, dTTP, 6 µm of dATP, 0.02 µL of 33P dATP, 0.2 µg/L
BSA, 1.5 mm MgCl2, 2.5 pmol of each primer, 0.25 U of Taq
DNA polymerase (Promega), and approximately 10 ng
of sample DNA. PCR products were analysed in 6%
AA
CG
AA
GG
TT
GG
GG
AA
AA
GC
AT
CG
CGA
A
AG
AA
Size
(bp)
Ta
(°C)
181
51
5
2
4
201
53
13
11
2
129
53
2
1
1
128
51
7
6
1
225
52
5
2
3
188
50
9
8
1
179
55
9
9
No amplification
324
52
5
5
1
Total
F. culmorum
F. graminearum
polyacrylamide gels and visualized by autoradiography.
Alleles were scored by length in bp.
The eight loci successfully amplified fragments of appropriate size in F. culmorum and F. graminearum (Table 2). Only
© 2002 Blackwell Science Ltd, Molecular Ecology Notes, 2, 121–123
P R I M E R N O T E 123
the locus F10 failed to cross-amplify in F. graminearum. The
other loci seem to differentiate the two species, as few
alleles were shared between F. graminearum and F. culmorum.
Despite the low number of individuals genotyped in
each of the two species, the microsatellite loci characterized here proved to be highly polymorphic, and this
opens opportunities to study the Fusarium species complex.
Acknowledgements
We thank Cyril Dutech for invaluable help in applying the protocol,
Bernadette Faivre for help in sequencing, and Jacqui Shykoff for
editing the English text. This work was supported by the French
Bureau des Ressources Génétiques, and by a post-doctoral grant
of the French Société de Secoures des Amis des Sciences to T. G.
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© 2002 Blackwell Science Ltd, Molecular Ecology Notes, 2, 121–123
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