CE-SSCP and CE-FLA, simple and high-throughput alternatives for fungal diversity studies
Résumé
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 ITSI 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 ITS I region provided complementary information. In order to improve CE-SSCP sensitivity. we tested its resolution according to migration temperature and found 32 degrees 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. (c) 2007 Elsevier B.V All rights reserved.