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Total genomic DNA of all isolates of Alternaria was isolated by CTAB method and molecular characterization by ITS sequence. The genetic identification of different isolates of Alternaria was confirmed by the DNA sequence analysis, a rapid mean of identification. Sequence similarities were determined by molecular method using DNA and ITS region. DNA analysis not only enables the species identification but also permits phylogenetic analysis. Internally transcribed spacer (ITS) region including 5.8S rRNA coding region in ribosomal DNA is one of the favorite targets for this purpose. The ITS region was amplified using the polymerase chain reaction (PCR) and the universal primers ITSl and ITS4. The focus of present study was on the genetic diversity analysis of the ITS regions of rRNA gene complex of local isolates of Alternaria in Pakistan. The genomic DNA of these isolates KI1, KI4, KI5, KI6, KI, KI8, KI9, and KI10 was amplified using primers ITS1 and ITS4 designed at the end, and start of conserved 18S and 28S region and between ITS1 and ITS4 respectively. By the comparison of isolates of all species have maximum genetic diversity only KI7 and KI8 have maximum homology with each other that showed that both are may be originated from same ancestor. Their phylogenetic relationships in GenBank were analyzed. Morphological and molecular data obtained might be useful in determining the taxonomy and diversity of Alternaria species.