Characterization of microsatellites in the fungal plant pathogen, Sclerotinia sclerotiorum

Transcription

1 Characterization of microsatellites in the fungal plant pathogen, Sclerotinia sclerotiorum Caroline Sirjusingh and Linda M. Kohn, Department of Botany, University of Toronto at Mississauga, Ontario, Canada L5L 1C Molecular Ecology Notes Volume 1(4): KEYWORDS: DNA fingerprints, multilocus haplotypes, white mold, Sclerotinia trifoliorum, Sclerotinia minor *Corresponding Author: Caroline Sirjusingh, Department of Botany, University of Toronto, Mississauga, Ontario, Canada L5L 1C6 csirjusi@credit.erin.utoronto.ca FAX: RUNNING TITLE: Microsatellites in Sclerotinia 1

2 Abstract Twenty-five primers produced unambiguous amplification products of 23 microsatellite-containing loci and two microsatellite-like polymorphic loci, with two to ten alleles at each locus in the plant pathogenic fungus, Sclerotinia sclerotiorum. Haplotypes are polymorphic among individuals sharing the same DNA fingerprint and DNA sequence haplotype, facilitating epidemiological monitoring worldwide. Fourteen of these primers also successfully amplified the closely related S. trifoliorum and S. minor Sclerotinia sclerotiorum causes such diseases as white mold and stem rot in agricultural and native plants. Because of significant clonality in most populations of this haploid fungus, DNA fingerprints (Kohn et al 1991; Kohli et al 1995) can be combined with multilocus DNA sequence haplotypes to infer phylogenies that span both contemporary and less recent time scales (Carbone et al 1999; Carbone and Kohn 2001). Microsatellite loci, due to their high mutation rates and multi-allelic nature, provide characters that can be scored for phylogenetic inference. Microsatellite haplotypes will add to the power of fingerprints and multilocus DNA sequence haplotypes. Because of their ease of use, we hope that many plant disease researchers will adopt them, facilitating epidemiological studies worldwide. Tester strains included three species. S. sclerotiorum: Isolates with a common DNA fingerprint included 14 isolates representing fingerprint (clone) 2 sampled from 1989 to 1999, 2

3 and 10 isolates of fingerprint (clone) 39 from 1991 and Isolates with different DNA fingerprints, but sharing a DNA sequence haplotype included 20 isolates from diverse hosts representing 4 haplotypes, each from one of four pathogen populations (Carbone and Kohn 2001). S. trifoliorum and S. minor: one isolate each. A DNA library for S. sclerotiorum containing bp fragments was prepared following Rassmann et al (1991) and Estoup et al (1993). Transformants on membrane filters were hybridized by standard methods (Sambrook et al 1989) with oligonucleotide probes containing the repeat motifs motifs (TC) 10, (TG) 10, (TGTA) 6 TG, CT(ATCT) 6, CT(CCT) 5 and (CAC) 5 CA, at a final concentration of 10ng of oligonucleotide probe per ml of hybridization mix. Oligonucleotides were end-labelled using T4 polynucleotide kinase (GIBCO BRL) and 32 P γ ATP(>3000 Ci/mmol, NEN Life Science Products). All six probes were used together (Estoup and Turgeon 1996; Putative positive clones were re-screened in the same way. Clones containing putative microsatellites were PCR amplified using M13 universal primers (Amersham Pharmacia Biotech). Amplified inserts were sequenced using the same primers used for amplification. Sequencing reactions were prepared for the ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) according to instructions, but using 1/4 of the recommended Terminator Ready Reaction mix for cycle sequencing. Reactions were analyzed on the ABI PRISM 310 Genetic Analyzer (Applied 3

4 Biosystems). Over 70 simple sequence repeat motifs were identified from the S. sclerotiorum library. Amplification primers were designed for 70 simple sequence repeat motifs using GeneWorks (IntelliGenetics Inc.) and synthesized by Sigma Genosys (Sigma-Aldrich Canada Ltd). Forward primers were end-labelled with P33 ATP following standard protocols (Carbone and Kohn 1999 ). PCR amplification mixtures (20 µl) contained 0.5 µm of each primer, 200 µm dntps, 1x PCR Buffer II and 2 mm MgCl 2 (Applied Biosystems), 0.5U of Amplitaq DNA polymerase (Applied Biosystems), and 10 µl of a 0.2 to 0.5 ng/µl genomic DNA solution. Amplifications were performed in a Perkin-Elmer GeneAmp System 9700 Thermocycler programmed for an initial denaturation at 95 C for 8 min, followed by 35 cycles of denaturation at 95 C; primer annealing at 48 to 60 C (Table 1); and extension at 72 C for 60-90s, with a 5 min extension at 72 C on the final cycle. PCR products were separated on a denaturing polyacrylamide gel (Sequagel XR, National Diagnostics) using the Sequencing Gel Electrophoresis System (Model S2, Life Technologies). Twenty-three primer sets produced unambiguous, polymorphic amplicons of the microsatellite-containing loci, as well as two microsatellite-like polymophic loci, in S. sclerotiorum (Table 1). Two primers (13-2, 106-4) yielded amplicons that were polymophic among clone 2 testers. Twenty-four primers produced amplicons that were variable within haplotypes; all 25 were variable among haplotypes. A GenBank search yielded a 79 bp region of 4

5 homology lacking the microsatellite motif for locus 42-4f with Botrytis cinerea, as well as 24 bases of homology including the microsatellite motif for locus 99-4 with human DNA. Acknowledgements This work was supported by NSERC. References Carbone I, Anderson JB, Kohn LM (1999) Patterns of descent in clonal lineages and their multilocus fingerprints are resolved with combined gene genealogies. Evolution, 53, Carbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91, Carbone I, Kohn LM (2001) A microbial population-species interface: nested cladistic and coalescent inference with multilocus data. Molecular Ecology, 10, Estoup A, Solignac M, Harry M, Cornuet JM (1993) Characterization of (GT)n and (CT)n microsatellites in two insect species: Apis mellifera and Bombus terrestris. Nucleic Acids Research, 21, Kohli Y, Brunner LJ, Yoell H et al (1995) Clonal dispersal and spatial mixing in populations of the pathogenic fungus, Sclerotinia sclerotiorum. Molecular Ecology, 4, Kohn LM, Stasovski E, Carbone I, Royer J, Anderson JB (1991) Mycelial incompatibility and molecular markers identify genetic variability in field populations of Sclerotinia sclerotiorum. Phytopathology, 81,

6 Rassmann K, Schlötterer C, Tautz D (1991) Isolation of simple sequence loci for use in polymerase chain-reaction based DNA fingerprinting. Electrophoresis, 12, Sambrook E, Fritsch F, Maniatis T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York. Table 1 Microsatellite and polymorphic loci* for Sclerotinia sclerotiorum Locus Repeat Motif Primer sequence (5' - 3') Ta Size No of Other (Accession Range Alleles Species No) (bp) 41- (TA) 5 and CCGAGCATAATATAATCC No 1:AF (CA) 10 AAGGTTATATTTCCCTCGC 5- (GT) 8 GTAACACCGAAATGACGGC Yes 2:AF GATCACATGTTTATCCCTGGC 6- (TTTTTC) 2 (TTTT GGGGGAAAGGATAAAGAAAAG Yes a 2:AF TG) 2 (TTTTTC) CAGACAGGATTATAAGCTTGGTCAC * 7- (GA) 14 TTTGCGTATTATGGTGGGC No 2:AF ATGGCGCAACTCTCAATAGG 9- (CA) 9 (CT) 9 GCCGATATGGACAATGTACACC No 2:AF TCTTCGCAGCTCGACAAGG 11- (GA) 6 GG(GA) 6 (GG CTTTCCTTTCGTTTGAGGG Yes 2:AF GA) 2 GGCAGGTAATGTTGCTTGG 6

7 12- (CA) 9 CGATAATTTCCCCTCACTTGC No 2:AF GGAAGTCCTGATATCGTTGAGG 13- (GTGGT) 6 TCTACCCAAGCTTCAGTATTCC No 2:AF GAACTGGTTAATTGTCTCGG 5- [(GT) 2 GAT] 3 (GT) CAGACGAATGAGAAGCGAAC No 3:AF GAT(GT) 5 [GAT( TTCAAAACAACGCTCCTGG GT) 4 ] 3 (GAT) 3 7- GT 10 CCTGATATCGTTGAGGTCG No 3:AF ATTTCCCCTCACTTGCTCC 8- CA 12 CACTCGCTTCTCCATCTCC No 3:AF GCTTGATTAGTTGGTTGGCA 17- (TTA) 9 TCATAGTGAGTGCATGATGCC Yes 3:AF CAGGGATGACTTTGGAATGG 20- (GT) 7 GG(GT) 5 GACGCCTTGAAGTTCTCTTCC Yes 3:AF CGAACAAGTATCCTCGTACCG 23- (TG) 10 CTTCTAGAGGACTTGGTTTTGG Yes 4:AF CGGAGGTCATTGGGAGTACG 36- CA 6 (CGCA) 2 CAT 2 GAATCTCTGTCCCACCATCG Yes 4:AF AGCCCATGTTTGGTTGTACG 42- GA 9 GGTCTCATACAGTCTACACACA Yes 4:AF CTCAGAGGATCTGCTGACA 50- CA 7 (TACA) 2 CCCTACAATATCCCATGGAGTC No 4:AF CCTCGTCTATCCGTCCATC 55- TACA 10 GTTTTCGGTTGTGTGCTGG Yes 4:AF GCTCGTTCAAGCTCAGCAAG 7

8 92- (CT) 12 TCGCCTCAGAAGAATGTGC No 4:AF AGCGGGTTACAAGGAGATGG 99- (GTAA) 2 (GCAA)( CTCATTTCATCCCATCTCTCCAATT Yes 4:AF GTAA) 3 CAAGCCTTCCTCAGCC * 106- (CATA) 25 TGCATCTCGATGCTTGAATC Yes a 4:AF CCTGCAGGGAGAAACATCAC 110- (TATG) 9 ATCCCTAACATCCCTAACGC Yes 4:AF GGAGAATTGAAGAATTGAATGC 114- (AGAT) 14 (AAGC) 4 GCTCCTGTATACCATGTCTTG Yes 4:AF GGACTTTCGGACATGATGAT 117- (TAC) 6 C(TAC) 3 TCAAGTACAGCATTTGC Yes 4:AF TTCCAGTCATTACCTACTAC 119- (GTAT) 6 and GTAACAAGAGACCAAAATTCGG No 4:AF (TACA) 5 TGAACGAGCTGTCATTCCC a Amplifies S. sclerotiorum and S. minor, not S. trifoliorum. 8