CE-SSCP and CE-FLA, simple and high-throughput alternatives for fungal diversity studies

• Published in: Journal of microbiological methods Vol. 72; no. 1; pp. 42 - 53
• Main Authors: Zinger, Lucie, Gury, Jérôme, Alibeu, Olivier, Rioux, Delphine, Gielly, Ludovic, Sage, Lucile, Pompanon, François, Geremia, Roberto A.
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 2008; Elsevier Science; Elsevier
• Subjects: Biodiversity; Biodiversity and Ecology; Biological and medical sciences; CE-FLA; CE-SSCP; DNA Fingerprinting - methods; DNA, Fungal - analysis; DNA, Fungal - isolation & purification; Ecology, environment; Ecosystem; Electrophoresis, Capillary - methods; Environmental Sciences; Fundamental and applied biological sciences. Psychology; Fungi; Fungi - classification; Fungi - genetics; Fungi - isolation & purification; High-throughput; Life Sciences; Microbiology; Polymorphism, Restriction Fragment Length; Polymorphism, Single-Stranded Conformational; Reproducibility of Results; Sensitivity and Specificity; Soil; Soil Microbiology; Fungi; High-throughput; Soil; CE-SSCP; Biodiversity; CE-FLA; High throughput screening; Soils; Single strand conformation polymorphism; registered designation; biodiversity; origin salers; capillary-electrophoresis; soil; soil bacterial; microbial; gradient gel-electrophoresis; ribosomal-rna genes; cheese; t-rflp; keratitis; community structure; fungi; high-throughput; Strand-conformation polymorphism; mycotic
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2007.10.005

Fungal communities are key components of soil, but the study of their ecological significance is limited by a lack of appropriated methods. For instance, the assessment of fungi occurrence and spatio-temporal variation in soil requires the analysis of a large number of samples. The molecular signature methods provide a useful tool to monitor these microbial communities and can be easily adapted to capillary electrophoresis (CE) allowing high-throughput studies. Here we assess the suitability of CE-FLA (Fragment Length Polymorphism, denaturing conditions) and CE-SSCP (Single-Stranded Conformation Polymorphism, native conditions) applied to environmental studies since they require a short molecular marker and no post-PCR treatments. We amplified the ITS1 region from 22 fungal strains isolated from an alpine ecosystem and from total genomic DNA of alpine and infiltration basin soils. The CE-FLA and CE-SSCP separated 17 and 15 peaks respectively from a mixture of 19 strains. For the alpine soil-metagenomic DNA, the FLA displayed more peaks than the SSCP and the converse result was found for infiltration basin sediments. We concluded that CE-FLA and CE-SSCP of ITS1 region provided complementary information. In order to improve CE-SSCP sensitivity, we tested its resolution according to migration temperature and found 32 °C to be optimal. Because of their simplicity, quickness and reproducibility, we found that these two methods were promising for high-throughput studies of soil fungal communities.