Design of degenerate primers for PCR amplification of genomic DNA encoding eIF4G in Verticillium dahliae using local alignment search

Articles in Press

Document Type : Original Research

Author
Abbas Jolodar
Department of Basic Sciences, Biochemistry and Molecular Biology Section, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Islamic Republic of Iran.
Abstract
Verticillium dahliae is a soilborne fungal pathogen that causes a vascular wilt disease in numerous economically significant crops and ornamentals worldwide. In this study, a partial genomic DNA fragment encoding eukaryotic translation initiation factor 4 gamma was amplified from V. dahliae using degenerate primers design based on the local alignment search method. To identify homologous proteins in closely related species, the NCBI GenBank database was queried using the HomoloGene program and a target keyword. The aligned part of the selected sequences revealed two conserve motifs, which facilitated the design of a pair of degenerate primers. Using Touchdown PCR, a 503-bp genomic DNA from V. dahliae was successfully amplified and sequenced, designated as VdeIF4G. This fragment contained two open reading frames (ORFs), encoding a total of 135 amino acids, separated by a 97-nucleotide intron. The intron-exon junction adhered to the GU-AG rule. At the nucleotide level, taxonomic analysis of VdeIF4G revealed three matches within the genus Verticillium, covering 81% of overlapping regions with sequence similarity ranging from 97.99 to 100%. Phylogenetic analysis at the protein level showed that VdeIF4G was identical to V. dahliae but exhibited divergence from V. nonalfalfae and V. alfalfae with the lowest genetic distance between these sequences being 1.8%. Furthermore, the presence of small and polar amino acids, along with a glycine-rich domain suggests potential involvement in host interactions and may contribute to fungal virulence. Degenerate primers designed using a local alignment search method effectively amplified homologous sequences of the corresponding eIF4G domain-containing protein from V. dahliae.

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Journal of Agricultural Science and Technology

Articles in Press, Accepted Manuscript
Available Online from 07 January 2026