Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSIllumina-based genetic linkage map for wheat leaf rust. David L. Joly 1,2 , Barbara Mulock 3 , Christina A. Cuomo 4 , Barry J. Saville 2 , Brent D. McCallum 3 , GuusBakkeren 2 . 1) Pacific Agri-Food Research Centre, Agriculutre and Agri-Food Canada, Summerland, British Columbia, Canada; 2) Forensic Science Programand Environmental & Life Sciences Graduate Program, Trent University, Peterborough, ON, Canada; 3) Cereal Research Centre, Agriculture and Agri-FoodCanada, Winnipeg, MB, Canada; 4) Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA 02142.Few genetic maps have been made for rust fungi; yet they are useful in identifying candidate loci for phenotypic traits or in unravelling chromosomalarrangements. This lack of maps is, in part, due to the obligate biotrophic nature of rusts and the difficulties in manipulating their life cycle in a way thatenables controlled crosses. Recently, the genome sequence of a wheat leaf rust (Puccinia triticina) isolate was determined and this prompted thesequencing of additional isolates using next-generation sequencing technologies. This has dramatically increased the amount of sequence informationavailable at a substantially decreased per base cost. Fifty-seven F2 progeny of a wheat leaf rust sexual cross between race 9 (SBDG) and race 161 (FBDJ)were sequenced using Illumina. In order to generate a high-resolution genetic linkage map, genome-wide single-nucleotide polymorphisms (SNPs) wereidentified. Employing the genome sequence information from the two parents and the F1 isolate, more than 25,000 SNPs were selected and used togenerate a genetic linkage map. Although they were obtained from different isolates, the genetic map and the reference genome were integrated,allowing the creation of pseudomolecules. Those represent a strong improvement over the currently fragmented status of the reference genome.Moreover, at least 9 seedling and 2 adult-plant avirulence genes were shown to segregate in this F2 population and candidate genes identified using thegenetic map are currently being investigated.Peering into the secret-ory life of Aspergillus nidulans with a little help from classical genetics. Miguel Penalva 1 , Areti Pantazopoulou 1 , Mario Pinar 1 ,Herbert N. Arst, Jr. 2 . 1) Cellular and Molecular Biology, Centro de Investigaciones Biologicas CSIC, Madrid, Spain; 2) Department of Microbiology, ImperialCollege, London, UK.Model fungi have survived the revolution of modern biology partly through their amenability to classical genetic analysis. Unquestionably, classicalgenetics lay at the root of the unmatched success of the yeast Saccharomyces cerevisiae, that exotic fungal visitor so pleasantly accepted into the parlourof true eukaryotic cells and in the conservatory of gene regulation that dominated the fungal community at the end of last millennium. Formerlyfashionable, classical genetics became nearly extinct with the advent of the ‘omics era’, their demise confirmed with each of the uncountabledevelopments of low-cost sequencing. However, we shall illustrate how extraordinarily powerful classical genetics can be, used in combination withsequencing techniques, to address general questions on the organization of the Golgi in eukaryotic cells. The Golgi is essential for secretion, and therefore,for hyphal growth. Thus, we begin with a sequenced, well-characterized heat-sensitive X ts mutation in an A. nidulans Golgi gene. An X ts strain ismutagenised to isolate suX suppressor mutations, reversing the absence of growth resulting from X ts at the restrictive temperature. Less interestingintragenic reversion/pseudo-reversion events are identified by the inability of any given suX X ts strain to produce single mutant X ts progeny when crossedto a wild-type. These mutations are next sequenced and archived. The remaining extragenic suppressors are allocated to one of the eight A. nidulanschromosomes by parasexual analysis, exploiting the rarity of mitotic recombination. Next, meiotic crosses between the suX X ts strain and a panel ofparental strains carrying markers in the suX chromosome are analysed to detect genetic linkage. Once linkage is detected, suX is further mapped to thesmallest feasible chromosomal interval. Candidate genes in the annotated genome interval, hopefully conspicuous at this stage to the educated eye, or, asa last resort, the whole interval between the genetic boundaries, are sequenced to identify the suppressor. The combination of gene mapping withsequencing eliminates the cumbersome identification of a single causative mutation (aka ‘a needle in a haystack’) hidden amongst the genetic variability ofthe mutant and parental strains, inherent to whole genome sequencing approaches.Domains of meiotic DNA recombination and gene conversion in Coprinopsis cinerea (Coprinus cinereus). Patricia J. Pukkila 1 , Wendy Schackwitz 2 . 1) DeptBiol, Univ North Carolina, Chapel Hill, NC, USA; 2) US DOE Joint Genome Institute, Walnut Creek, CA, USA.We have shown previously that rates of meiotic recombination are highly non-uniform along the assembled chromosomes of C. cinerea (Stajich et al.PNAS 107: 11889-11894, 2010). That study revealed an over-representation of paralogous multicopy genes in regions with elevated levels of meioticexchange. In addition, retrotransposon-related sequences were not found in large segments of the genome with low levels of meiotic exchange. However,the study was limited by the available markers, and only 31 Mb of the 36 Mb genome could be mapped. More recently, we have resequenced 45 meioticsegregants and 4 complete tetrads. We developed a simple script to detect crossover and gene conversion events involving over 75,000 SNPs spanning 35Mb. The data were analyzed using MSTmap (Wu et al. PLoS Genetics 4: e1000212, 2008). The new dataset revealed sub-telomeric recombination hotspotsat every chromosome end, and 36% of the crossovers were associated with uninterrupted tracts of gene conversion. The conversion tracts (2-8 SNPs) werequite short (8-219 nt), and the median distance between the flanking SNP markers was also small (500 nt). Since these subtelomeric hotspots correspondto sites of synaptic initiation in C. cinerea (Holm et al. Carlberg Res. Commun. 46: 305-346, 1981), these data may contribute to our understanding of howhomologous chromosome pairing and synapsis are coordinated with meiotic recombination. Supported by the U.S. Department of Energy Joint GenomeInstitute Community Sequencing Program. The work conducted by the U.S. DOE JGI is supported by the Office of Science of the U.S. Department of Energyunder Contract No. DE-AC02-05CH11231.A Hook protein is critical for dynein-mediated early endosome movement in Aspergillus nidulans. Jun Zhang 1 , Rongde Qiu 1 , Herbert Arst 2 , MiguelPeñalva 3 , Xin Xiang 1 . 1) Department of Biochemistry and Molecular Biology, Uniformed Services University, Bethesda, Maryland, USA; 2) Department ofMedicine, Imperial College London, London, UK; 3) Department of Molecular and Cellular Medicine, Centro de Investigaciones Biológicas CSIC, Ramiro deMaeztu 9, Madrid, Spain.It has been hypothesized that cytoplasmic linker proteins such as CLIP-170 facilitate motor-driven organelle transport by serving as an additional linkerbetween the organelle and the microtubule track. However, mammalian and fungal cells lacking CLIP-170 do not exhibit any apparent defects in vesicletransport. We recently found that in the filamentous fungus Aspergillus nidulans, the HooK protein ortholog, HookA, is critical for dynein-mediatedtransport of early endosomes. HookA mutants were obtained from a genetic screen for mutants defective in dynein-mediated early endosome movement,and the HookA gene was identified by a combination of classical genetic and whole-genome-sequencing approaches. The HookA protein is homologous tohuman Hook proteins containing a N-terminal microtubule-binding domain, a coiled-coil domain and a C-terminal cargo-binding domain, an organizationsimilar to that of CLIP-170. Both the N- and C-terminal domains of HookA are required for dynein-mediated early endosome transport, and HookAassociates with early endosomes via its C-terminal domain in a dynein-independent manner. Importantly, HookA physically interacts with dynein/dynactin,and this interaction is independent of the C-terminal early-endosome-binding domain but dependent upon the N-terminal microtubule-binding domain.Together, our results suggest that HookA may facilitate cargo-motor-track interactions during dynein-mediated transport of early endosomes.48