Nucleotide Polymorphisms Of DNA Barcoding Sequences In Ganoderma

Species of Ganoderma include several ecologically important and pathogenic fungal species.ITS sequences are comnonly used for taxonomic or phylogenetic studies of fungi,EF1-? and RPB2 genes were shown to have high phylogenetic utility.However,double or multiple nucleotide peaks were often found in the sequencing electropherograms of the three gene-markers,we hypothesized that intragenomic variations of ITS,RPB2 and EF1-? were present and might expand intraspecific variation.In this study,the intragenomic variation of ITS,EF1-? and RPB2 in 36 Ganoderna specimens was detected by using PCR cloning to determine whether intragenomic variation affects accuracy of molecular identification,and at least 30 clones were selected.Further,a desirable gene that could effectively distinguish between Ganoderma species was choosed.In the results of PCR amplification and sequencing,the number of specimens with heterogeneity in ITS gene could be up to 148(29.13%),and sequencing success rate is the lowest(69.96%),EF1-? and RPB2 were relatively higher(87.55%and 88.74%).Analysis of polymorphism of sequences showed that intragenomic variations of ITS,EF1-? and RPB2 genes were present in Ganoderma,this phenomenon was more likely to appear in ITS,and the variation within an individual was 0.18%-2.00%,EF1-? gene had the highest variation within an individual(3.57%),whereas RPB2 showed the lowest intragenomic variation level(0.085%to 0.76%)except for G.orbiforme.Intraspecific variations might be expanded and surpass the variation between species in the tested Ganoderma species,the highest intraspecific variations in ITS,EF1-? and RPB2 genes were 2.9%,4.0%and 3.6%,respectively.Therefore,the use of these sequences for phylogenetic analyses and molecular identification of Ganoderma species might be problematic.In total,RPB2 gene had less specimens with heterogeneity,the highest sequencing success rate,relatively low the level of nucleotide polymorphism and the intraspecific variations,and high variation between species.Therefore,RPB2 could be more suitable as a barcode candidate for Ganoderma.