Craig E Coleman, Plant and Wildlife Sciences
In this interim report I detail the results according to each of the six objectives proposed. A final report will be submitted when the experiments detailed in objective six are completed.
Objective 1. Assemble and annotate B. tectorum cDNA sequence data obtained from 454 pyrosequencing and compare to the transcriptomes of other grass species.
This objective has been completed. The project was carried out by undergraduate student David Elzinga. He has created an annotated set of unigenes for B. tectorum based on our 454 sequences and placed them in a BLAST-searchable database. The annotated cDNAs are critical preliminary data for a revised NSF proposal that I will be submitting with collaborators from BYU, the USDA Forest Service Shrub Laboratory and the University of Nevada, Reno in January 2011.
Objective 2. Identify SNPs from B. tectorum cDNA 454 sequence reads and use a validated set to genotype representative individuals from selected populations of cheatgrass.
This objective has been completed. Using the KASPar reagent system we have genotyped a representative sample of individuals from populations in the Intermountain Western United States as well as the native range of cheatgrass in Eurasia. Graduate student Keith Merrill working with undergraduate student Sudeep Ghimire validated over 100 single nucleotide polymorphisms from cheatgrass using the cDNA dataset. The work completed in this objective was also supported by an ORCA grant to Sudeep Ghimire. A poster will be presented by Sudeep Ghimire at the Plant and Animal Genome Conference in January 2011 and there are three manuscripts currently in preparation from this work:
Merrill KR, Coleman CE, Ghimire S, Meyer SE (2011) High throughput single nucleotide polymorphism (SNP) development and genotyping in Bromus tectorum. PAG XIX, San Diego, CA.
Meyer SE, Merrill KR, Ghimire S, Leger EA, Novak S, and Coleman CE. A rosetta stone for interpreting Bromus tectorum population genetics using multiple molecular marker systems. To be submitted to Heredity.
Meyer SE, Merrill KR, Ghimire S, Leger EA, Novak S, and Coleman CE. Cryptic invasion of the American Southwest by central Asian biotypes of Bromus tectorum. To be submitted to the Proceedings of the National Academy of Science USA.
Merrill KR, Ghimire S, Coleman CE, Meyer SE (2010) The development and use of single nucleotide polymorphism markers in assessing population genetic structure of Bromus tectorum. Manuscript in preparation.
Objective 3. Use fluorescent in situ hybridization to compare chromosome structure between cheatgrass lines.
This objective has been abandoned and will not be completed. Preliminary work on identifying hybridization probes was begun by undergraduate student Nada Oussible. The effort to obtain useful probes was costly in terms of time and resources and I felt that the overall success of the proposal would be better served if we shifted those resources to other objectives that had a better chance for completion.
Objective 4. Whole genome sequence assembly and annotation of P. semeniperda. This objective has been completed. The work was performed by graduate student Marcus Soliai and undergraduate students David Elzinga and Russell Hermansen. We have a draft sequence of one isolate of P. semeniperda that is fully annotated and available on-line through GBrowse at: http://soda2.cs.byu.edu/cgi-bin/gbrowse/bfod/. The genome is 40 Mb in size with approximately 10,000 gene models reported. We have made progress on assembling a draft sequence for a second isolate. While assembling the genome sequence it became apparent that the genome structure between the two isolates was significantly different, so the decision was made to assemble the two isolate genomes separately. Completion of the assembly of the second genome will require additional sequence data. A manuscript is currently in preparation to report the draft sequence and its comparison to P. tritici-repentis.
Soliai MM, Elzinga D, Hermansen R, Meyer SE, Coleman CE (2010) A whole-genome assembly of Pyrenophora semeniperda and its comparison to Pyrenophora tritici-repentis. Manuscript in preparation.
Objective 5. Identify SNPs from P. semeniperda whole genome sequence and use a set of them to genotype representative individuals collected throughout the intermountain west.
This objective has been completed. Graduate student Marcus Soliai worked with undergraduate student Jonathan Raney to validate 25 single nucleotide polymorphisms. These were used to genotype our collection of fungal isolates from the Intermountain Western United States and several isolates from Eurasia. This part of the project was in collaboration with scientists at Gonzaga University. A manuscript reporting this work is currently being prepared. Additionally, a manuscript reporting the technical details of identifying and validating fungal SNPs has been accepted for publication in the Methods in Molecular Biology series published by Humana Press.
Coleman CE, Merrill KR, Soliai MM (2010) Single nucleotide polymorphism discovery using 454 pyrosequencing of fungal DNA. In: Slotta T (ed) Methods in Molecular Biology, Human Press, in press.
Boose D, Harrison SL, Soliai MM, Raney JC, Coleman CE, Meyer SE, Stevens M. Population genetic structure and breeding system in a seed bank pathogen revealed using multiple molecular marker systems. To be submitted to American Journal of Botany
Objective 6. Obtain additional 454 sequence data from the whole genome of Ustilago bullata and assemble and annotate a draft genome sequence.
This objective has not been completed. Initial work to obtain genomic DNA and mRNA for producing additional 454 sequence data was done by undergraduate student Krystyna Winn as part of her effort to complete an Honors thesis. Because of health issues she was not able to complete the work. The project has now been taken over by undergraduate student Rachana Gyawali. She has obtained sufficient genomic DNA and cDNA for 454 sequencing and the sequencing will be completed soon followed by assembly and annotation. She has written an ORCA proposal for her work. A final report on this proposal will be submitted when this objective is completed.