Heteroplasmy In The Mitochondrial Genome Of The Nematode-trapping Hyphomycetes Arthrobotrys Oligospora And Comparative Mitogenomic Analyses With Other Related Species

DNA barcoding has recently become increasingly popular for species delimitation,identification and systematics.Cytochrome oxidase I(COI),as an animal DNA barcode,has been successfully employed in the species identification.But its feasibility as molecular marker for fungal identification is problematic.Nematode-trapping hyphomycetes belong to a modern lineage of the broad predatory fungi.There is a great interest in using these fungi as model samples in adaptative evolution researches and as biological control agents against parasitic nematodes.It was found that there were many SNPs(single nucleotide polymorphism)in the COI sequence of the same strain,which was far more beyond the variation range of this gene in the similar fungal groups during the early molecular phylogenetic analysis of nematode-trapping hyphomycetes and its related groups.SNPsIn order to explore whether the emergence of SNPs is the presence of mitochondrial heterogeneity or the recombination of mitochondrial genes in vitro PCR reactions,we conducted single spore isolation of the model nematode-trapping fungus Arthrobotrys oligospora strain YMF1.03037 and passed them on to posterity to six generations.And the degree and range of SNPs changes of each generation of strain was investigated.At the same time,we assembled and annotated the mitochondrial genomes of five related fungal strains,and comparative mitochondrial genomic analyses were conducted with seven additional related strains.The results are as follows:(1)70 single spore isolations were obtained through six generations.The selected isolations were amplified by nested PCR to obtain the target COI sequence and 124 available sequences were obtained.Then,nucleotide and amino acid similarity and phylogenetic analysis were performed for the 124 sequences.We found that the SNPs of YMF1.03037 exist inter-and intra-generations,even different PCRs within single single-spore strains.Though the SNPs don’t diminish from generation to another,but major molecular variance was found among different single-spore strains,and molecular variance within the same generations is less than those between generations.Most private alleles are found in F1 and F6,which had not been found in the parental generation,indicating that there might be potential other genotypes that we haven’t explored.During subsequent amplification,new genotypes that were not previouslyamplified may appear.Recombination was found between different SNPs in COI gene,indicating there is mitochondrial heteroplasmy in this haploid hypomycete.(2)To ensure the reliability of this experiment and detect the relationships intraand inter-single spore lineages,we did five generations of single spores isolation from strain YMF1.03037 original plate and each single spore was passed down to five progenies.We found that SNPs still exist,such as C454.1,C454.2,C454,3 and C454.4,and there are still SNPs between the COI amplicons of the same single spore strain under different PCR reactions.Lineage A45 has accumulated the most private alleles.(3)We amplified the COI exon sequences from 7 other A.oligospora strains of YMF1.02787,YMF1.02896,YMF1.03104,YMF1.03142,YMF1.03162,YMF1.01883 and YMF1.03038 by nested PCR,and found that SNPs also existed in these strains.However,we did not find SNPs among the nuclear genes ITS,RPB2,TEF,mat1-2,and the designed primers amplification of two fragments of mitochondrial COX3,COB,and the last exon of COI,that is,SNPs were found only in the specific COI segments we studied in the entire mitochondrial genome.(4)Mitochondrial genomes of five strains A.oligospora YMF1.02765,YMF1.02775,YMF1.03037,YMF1.01838 and YMF1.03216 were assembled and annotated.In addition to the previously annotated strains A.oligospora YMF1.01883 and Dac.haptotyla,7 mitochondrial genomes including both intra-and inter-species strains were analyses jointly,the number of protein coding genes(PCGs),GC content,AT skew,and GC skew varied among these mitogenomes.The increased number and total size of introns were the main contributors to the size differences among the mitogenomes.