MSA - Ecology/Pathology
Acevedo, Manuel , Cantrell, Sharon A. , Perez-Jimenez, Jose R. .
Accountability of fungal molecular signatures from microbial mats in TRFLP profiles.
Diversity studies conducted in Puerto Rico using traditional techniques has documented a total of 36 species of known fungi from hypersaline microbial mats. Some species are common across layers, sites and seasons, such as Cladosporium cladosporioides, C. dominicanum, C. fusiforme, C. sphaerospermum, and Hortaea werneckii. Others, such as species of Aspergillus, Penicillium and Preussia, are found during the rainy season and the oxic layer of the mats. Also, we have been documented the fungal diversity using TRFLP and clonal sequencing. TRFLP can illustrate the community structure based on profiles of phylotypes after the digestion of the amplicons with a restriction enzyme. But the technique will not provide identity for the phylotypes. The goal of this study is to evaluate TRFLP profiles generated from pure cultures, in silico digestions and environmental samples. ITS-amplicons (~10 ng) were digested with HaeIII and analyzed with GenMapper on an ABI 3130 automatic sequencer. The ITS region was sequenced for all isolated cultures and were cut in silico to obtain the expected peak pattern and fragment sizes. Results from TRFLP analysis showed that isolates from the same species give the same TRF pattern, also different genera showed different patterns indicating that peaks are unique for the species analyzed. In silico digested sequences showed profiles similar to the environmental samples. Both TRFLP profiles and in silico digestions showed multiple peaks, which can represent a problem at the moment of estimating richness of TRFLP profiles from environmental samples. Preliminary results from digestions at different times (30, 60, 90 and 120 min) showed that at 30 min samples are under-digested and at 120 mins samples are over-digested. Profiles from 60 and 90 mins are identical. Future work will optimize the digestion protocol to achieve a good accountability of fungal molecular signatures obtain in TRFLP profiles from environmental samples.
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1 - Universidad del Turabo, Department of Biology, School of Science and Technology , P.O. Box 3030, Gurabo, PR, 00778, Puerto Rico
2 - Universidad del Turabo, Interdisciplinary Research Institute, P. O. Box 3030, Gurabo, PR, 00778, Puerto Rico
Presentation Type: Poster:Posters for Topics
Location: Event Tent/Cliff Lodge
Date: Tuesday, July 28th, 2009
Time: 5:30 PM