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Summer Research Fellowship Programme of India's Science Academies

Phylogenetic Analysis of Lasiodiplodia Species Using Dual DNA Barcoding Data

Dr S. Shalini Devi

Assistant Professor, Dept of microbiology, Bhavans Vivekananda College for Science, Humanities and Commerce,
Sainikpuri, Secunderabad 500094

Dr Gnanesh B.N

Ramanujan Fellow Scientist, Molecular Biology lab-1, CSRTI, Mysore 570008

Abstract

Fungi are a heterogeneous group of microbes that include the second largest group of eukaryotic organisms. Proper delineation of closely related fungal species can be achieved by utilizing protein- coding sequences as secondary DNA barcodes like TEF1-α, RPB1, RPB2, β-tubulin and CaM genes. Finer-scale species-level identification of fungi can be achieved by concatenated alignment of the ITS region with one or more protein-coding genes. In the present study, two phytopathogenic fungal isolates from mulberry plants were identified using sequence data of ITS and β-tubulin genes. BIOEDIT software and Clustal W multiple sequence alignment tools were used for initial alignment of ITS and β-tubulin sequences of both test isolates. Further Molecular evolutionary genetic analysis (MEGA X) software was employed for concatenation of ITS and β-tubulin sequence data and phylogenetic analysis. Expasy-translate tool and NCBI –ORF finder tools were used to annotate the protein-coding sequences. The two fungal species were identified as Lasiodiplodia theobromae. The phylogenetic analysis revealed the relationship of these isolates with other Lasiodiplodia spp. Phylogenetic analysis carried out using dual barcode sequences individually and concatenated sequence data revealed the close relation of test isolates and L. theobromae reference sequences with a maximum of similarity with L. theobromae strain 7E86.

Keywords: Bioedit, NCBI, protein coding regions, gene annotation, fungal identification

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