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We are studying fungal species in terms of their genetic isolation and adaptation. We now focus on one animal pathogenic genus, Coccidioides, and one plant-associated genus, Neurospora. <P>Additionally, we apply our knowledge to fungi of social importance as the need arises,e.g. in the past period, we also investigated the amphibian pathogen, Batrachochytrium and the animal pathogen, Paracoccidioides, and the plant-associated fungus, Aspergillus fumigatus.<P>First objective is phylogenetic species recognition and estimation of reproductive mode as clonal or recombining.The goal is to determine the limits of species based on genetic isolation in nature as measured by concordance of gene genealogies. Expected outcome is a phylogeny and results of tests for recombination or clonality.<P>Second objective is to compare fungal genomes within and among closely related species to identify genes important to adaptation.The goal is to discover gene family expansion/contraction, gene gain/loss, rapid gene evolution, and selection.The outcome will be a list of genes prioritized for gene disruption studies to determine if these genes are important to adaptation.<P>Third objective is to discover the genes important to species maintenance in sympatry by reinforced reproductive isolation.The goal is to identify genes that have evolved for reinforcement. We now are using transcription profiling in crosses that result in mature perithecia and aborted perithecia to identify genes in our known QTL that are important to reinforcement. The outcome will be genes whose disruption affects reproduction. <P>Fourth objective is to develop a community tool to rapidly associate phenotype and genotype in Neurospora. This project builds on our success with QTL analysis in objective3. Here, we are using both QTL and an approach developed for human genomics, Whole Genome Association (WGA). With QTL, the phenotypes are limited to those that vary in the parents or in the progeny, mapping population and the association is limited by the length of genetic blocks in linkage disequilibrium. WGA avoids these limitations because the population involves hundreds of wild isolates with more phenotypic trait variation than any pair isolates, and because the wild, outbred population has relatively shorter recombination blocs. The goal is to associate complex phenotypes with specific genes or other genome regions. The outcome is a collection of genes hypothesized to be important to the trait of interest. Yet again, disruption of these genes and assessment of the relevant phenotypes will tell if the approach has succeeded.<P>Fifth objective is to develop a work-flow to identify the fungi in environmental samples based on DNA. The goal is to identify fungi in environmental samples by extracting DNA from the sample, PCR amplifying a genome region that is diagnostic for fungal species (ITS of rDNA), sequencing the amplified regions, identifying the sequenced regions by phylogenetic comparison of the new, unknown sequences with known sequences. The outcome is a list of species and their abundance in an environmental sample. Output activities center on teaching and mentoring, conferences, workshops and symposia.
Non-Technical Summary: We developed molecular methods to recognize fungal species by genetic isolation in naure and continue to apply this approach to model and socially important fungi. We discovered mate choice in microbes and aim to find the genes that allow female fungi to reject suitors from other species. We have conducted the first comparative genomic study of a human pathogenic fungus and now aim to extend our efforts to account for population variation. To associate phenotypic and genetic variation, we have used quantitative trail locus mapping and now are moving to whole genome association mapping, following the lead of human geneticists. We have profiled RNA transcription in Coccidioides using microarrays of 1000 genes and now are moving to profiling all genes by sequencing the RNAs. We aim to automate identification of fungi to support ecological and agricultural research. <P> Approach: 1. To recognize phylogenetic species, we use concordance of multiple gene genealogies. This approach relies on sequence data of four to five genes that are polymorphic in a morphological species. Where gene genealogies move from congruence to conflict, species are identified. 2. For QTL, we mate parents with different character states for the phenotypic trait and with genetic variation. We then score 500 progeny for the trait and for genetic variation. Association of the trait and genetic markers places the QTL on the marker map. 3. For genome comparison, we assemble, annotate and align the genomes and then use tools appropriate for analysis at different levels of phylogenetic divergence. Gene family size change is studied with deep divergences and large numbers of genomes, gene gain and loss is studied at shallower divergences with just four taxa. Rates of evolution is studied at even shallower divergences with just three taxa, and selection is studied among very close relatives, i. e., sister species, with just two species. 4. Our WGA project focuses on 200 individuals from the Caribbean population of Neurospora crassa and we are assessing both transcription and characterizing single nucleotide polymorphisms through next generation sequencing of mRNAs. 5. To profile transcription in Coccidioides species we are using next-generation sequencing of mRNAs (RNA-seq). 6. To characterize fungi in environmental samples, we extract DNA, amplify the rDNA ITS region, and sequence it. Sequences are identified by phylogenetic analysis with known ITS sequences.