Practice towards DNA barcoding of the nectriaceous fungi

Abstract

Fungi of Nectriaceae are economically important and of high species diversity. For the purpose of accurate and rapid species identification, ITS, 28S rDNA, β-tubulin gene and EF-1α gene were selected as the candidate DNA barcode markers to investigate their feasibility in identification of 28 well-circumscribed species belonging to 9 genera of the nectriaceous fungi. A total of 216 sequences of the candidate genes were analyzed. Intra- and inter-specific variations and success rate of PCR amplification and sequencing were considered as important criteria to estimate the candidate genes. The partial β-tubulin gene met the requirements for an ideal DNA barcode and functions well for correct species delimitation. No overlapping between the intra- and inter-specific pairwise distances was found. The smallest inter-specific distance of β-tubulin gene was 3.45%, while the largest intra-specific distance was 2.77%; which appeared to possess the appropriate intra- and inter-specific variations. Twenty-eight clusters were recognized in accordance with the 28 morphological species tested. In addition, it had a high PCR and sequencing success rate. As to the other candidates, EF-1α gene showed fairly good sequence variations among species, but the PCR and sequencing success rate reached only 75.3%. ITS had a high PCR and sequencing success rate (93.5%) and recognizes 92.9% of the total number of species, nevertheless, overlapping occurred between the intra- and inter-specific distances, which may lead to incorrect species identification. 28S rDNA is most conservative compared with any other candidate markers and able to recognize merely 60.7% of the total species. We propose β-tubulin gene as the possible barcode for the nectriaceous fungi.