The Intron Analysis Of Mitochondrial Genome In Representative Species Of Basidiomycetes And The Study Of Phlebopus Portentosus Mitochondrial Genome

Mitochondrion is an important organelle with double membrane and plays a crucial role in respiratory action and energy generation within the fungal cell. Despite the stable structure and content of fungal mitochondrial genome, various genomic polymorphisms accumulated in the long evolutionary history, which play an important role in fungi classification, identification and phylogenetics. Meanwhile, the differences in the genome size ranging from 10Kb to over 100 Kb are observed among fungal mitochondrion and these differences are important trait of fungal evolution, attracting broad interests from mycologists、geneticists and evolutionary biologists. Here, we analyzed the relationship between the mitochondrial genomic size and the number of introns in some representative species in Basidiomycetes, especially investigated related factors influencing the genome size of fungal mitochondrion.We also annotated the mitochondrial genome of Phlebopus portentosus strain PP33, the whole genome of which was sequenced deeply by our laboratory. Furthermore, population genetics of 64 sampled Phlebopus portentosus strains was conducted. The main results are as follows:(1 Mitochondrial genomes of 94 fungal species which had been published on the literature or NCBI database were collected to get the information of their size change, and the factors affecting the mitochondrial genome size change were find out. Relationship between mitochondrial genome-length and the number of introns within the 23 Basidiomycetes species has showed that the intron is the main factor affecting Basidiomycetes mitochondrial genome size. In addition, the 14 protein-coding genes of amino acid sequence of these 23 Basidiomycetes species mitochondrial genome from the NCBI database were downloaded, combined with the amino acid sequence of 14 mitochondrial genes, we studied their phylogenetic relationships, the results showed that different sub-systems were divide into different clades. Four basidiomycetes species of Pucciniomycotina gathered together into a small clade system at the top of the tree; three basidiomycetes species of Ustilaginomycotina followed accordingly to cluster into a small clade which placed in the middle of the systematic tree; the remaining 16 Basidiomycetes species belonging to Agaricales sub-systems gathered together into a large clade placed at the base of the systematic tree.(2)Based on the study of Basidiomycetes mitochondrial genome size variation and understanding of the fungal mitochondrial genome structure, we completed the analysis of Phlebopus portentosus strain PP33 mitochondrial genome, the length is 433O8bp, which contains 14 core genes involved in oxidative phosphorylation system on respiratory chain (COX1, COX2, COX3, cob, nadl, nad2, nad3, nad4, nad4L, nad5, nad6, ATP6, ATP8 and ATP6), two genes encode the ribosomal RNA of the large (rnL) and small (rns) subunits and a set of 23 different tRNAs. Three hypothetical ORF (orf1086, orf100, orf475) encoding the unknown protein were also found. There are only four introns exist on its Coxl gene, and open reading frame within the gene(s) encoding homing endonuclease on the four introns was found. Based on the above information, we annotated the whole mitochondrial genome of Phlebopus portentosus strain PP33, and draw the physical map.(3)Four intron primers at different locations of Coxl gene were designed based on mitochondrial genome sequences of PP33, and intron sequences of 84 Phlebopus portentosus species collected from Xishuangbanna and Puer region were amplified and sequenced. The amplification success rate of 76%, it is indicated that four intron primers could used to molecular studies of the Phlebopus portentosus. Population genetic analysis of Mantel tests showed no positive correlation between genetic distances and geographic distances in Phlebopus portentosus geographical populations. The AMOVA suggested that the major genetic variation (59%) was found within local populations, and 41% was found among local populations level, but the genetic differentiation among different regions contribute 0. Results of multilocus linkage disequilibrium analyses for four introns indicated recombination plays an important role in most Phlebopus portentosus’s natural populations.