CE-SSCP and CE-FLA, simple and high-throughput alternatives for fungal diversity studies.
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 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.
Zinger
Lucie
L
Laboratoire d'Ecologie Alpine UMR 5553 UJF/CNRS, Grenoble Cedex 9, France.
Gury
Jérôme
J
Alibeu
Olivier
O
Rioux
Delphine
D
Gielly
Ludovic
L
Sage
Lucile
L
Pompanon
François
F
Geremia
Roberto A
RA
D044822:Biodiversity; D016172:DNA Fingerprinting / Q000379:methods; D004271:DNA, Fungal / Q000032:analysis / Q000302:isolation & purification; D017753:Ecosystem; D019075:Electrophoresis, Capillary / Q000379:methods*; D005658:Fungi / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012988:Soil Microbiology
D004271:DNA, Fungal