Development and amplification of multiple co-dominant genetic markers from single spores of arbuscular mycorrhizal fungi by nested multiplex PCR |
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| Authors: | Stukenbrock Eva H Rosendahl Søren |
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| Affiliation: | 1. Genetic/Genomic Research Center, YARSI Research Institute, Universitas YARSI, Cempaka Putih, Jakarta Pusat, DKI Jakarta, Indonesia;2. Department of Pharmacology, Faculty of Medicine, Universitas YARSI, Cempaka Putih, Jakarta Pusat, DKI Jakarta, Indonesia;3. The Indonesian Pharmacogenomics Working Group, DKI Jakarta, Indonesia;1. Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China;2. The Donghai Sea Collaborative Innovation Center for Industrial Upgrading Mariculture, Ningbo University, Ningbo 315211, China;1. Key Laboratory of Animal Parasitology of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China;2. Shanghai Animal Disease Prevention and Control Center, Shanghai, China;3. Fengxian Center for Animal Disease Prevention and Control of Shanghai, Shanghai, China;1. Programa de Pós-Graduação em Medicina Veterinária (Clínica e Reprodução Animal), Universidade Federal Fluminense, Brazil;2. Departamento de Medicina e Cirurgia Veterinária, Universidade Federal Rural do Rio de Janeiro, Brazil;3. Laboratório de Biologia Estrutural, Instituto Oswaldo Cruz, IOC/Fiocruz, Rio de Janeiro, RJ, Brazil;4. Laboratório de Sanidade Animal, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brazil;5. Departamento de Estatística, Universidade Federal Fluminense, Brazil;6. Laboratório de Anatomia Patológica Veterinária, Universidade Federal Fluminense, Brazil |
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| Abstract: | 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. |
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