Development and amplification of multiple co-dominant genetic markers from single spores of arbuscular mycorrhizal fungi by nested multiplex PCR.
Multiple co-dominant genetic markers from single spores of the arbuscular mycorrhizal (AM) fungi Glomus mosseae, Glomus caledonium, and Glomus geosporum were amplified by nested multiplex PCR using a combination of primers for simultaneous amplification of five loci in one PCR. Subsequently, each marker was amplified separately in nested PCR using specific primers. Polymorphic loci within the three putative single copy genes GmFOX2, GmTOR2, and GmGIN1 were characterized by sequencing and single strand conformation polymorphisms (SSCP). Primers specific for the LSU rDNA D2 region were included in the multiplex PCR to ensure correct identification of the Glomus spp. spores. Single AM fungal spores were characterized as multilocus genotypes by combining alleles of each amplified locus. Only one copy of each putative single copy gene could be amplified from each spore, indicating that spores are homokaryotic. All isolates of G. mosseae had unique genotypes. The amplification of multiple co-dominant genetic markers from single spores by the nested multiplex PCR approach provides an important tool for future studies of AM fungi population genetics and evolution.
Stukenbrock
Eva H
EH
Department of Microbiology, Institute of Biology, University of Copenhagen, Øster Farimagsgade 2D, DK-1353 Copenhagen K, Denmark. eva.stuckenbrock@ipw.agrl.ethz.ch
Rosendahl
Søren
S
D000483:Alleles; D018797:Cell Cycle Proteins / Q000235:genetics; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D005656:Fungal Proteins / Q000235:genetics; D005799:Genes, Dominant; D005800:Genes, Fungal; D005819:Genetic Markers; D005838:Genotype; D008969:Molecular Sequence Data; D016533:Mycological Typing Techniques; D038821:Mycorrhizae / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA; D013172:Spores, Fungal / Q000235:genetics*
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004275:DNA, Ribosomal; C400127:FOX2 protein, Glomus mosseae; D005656:Fungal Proteins; D005819:Genetic Markers; D019869:Phosphatidylinositol 3-Kinases