Analysis of Fungal Diversity in the Composting by Sequencing of Cloned PCR-Amplified 18S rDNA and Denaturing Gradient Gel Electrophoresis

Wan Li; Xiu Hong Xu; Jia Lei Xiao; Bi Xian Zhang; Hong Tao Li; Yong Cai Lai

doi:10.4028/www.scientific.net/AMR.356-360.1747

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Analysis of Fungal Diversity in the Composting by Sequencing of Cloned PCR-Amplified 18S rDNA and Denaturing Gradient Gel Electrophoresis

Abstract:

Like bacteria, fungi play an important role in the composting process as major decomposers of organic substances. As only a small fraction of the fungi present in composting can be cultured because conventional microbiological techniques limited information on the composition of fungal communities in composting. Molecular methods are expected to give a more realistic view of species richness and distribution. For this purpose, we selected universal PCR primer set that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences. DNA was extracted from composting samples, and 18S rDNA genes were amplified by EF4/Fung5 (0.6kb) and EF4/NS₂-GC (0.4kb). DGGE analysis of the fungal community in the composting of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. Clear banding patterns were obtained with amplified production. 13 different bands excised from the DGGE gel were sequenced and compared with genbank. Sequencing showed that some could not be cultured; some were efficient cellulose-degrading strains. The results showed that diversity and composition of the fungal community in the composting can be analyzed by the combination of 18S rDNA PCR amplification and DGGE.