Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Using the sequenced Fusarium oxysporum f.sp. lycopersici (Fol) as a reference genome, this proposed project will focus on four additional F. oxysporum strains to: 1. Generate deep Solexa sequencing coverage (~16X coverage) to survey overall sequence polymorphism and SNP distribution; 2. Create whole genome optical maps to study genome structural polymorphisms and to: a. identify strain specific genomic regions; b. characterize chromosomal variations such as inversion, translocation and large indels. 3. Sequence, assemble and annotate the small, polymorphic chromosomes for each selected strain using the isolated DNA from small polymorphic chromosomes. So far, we have accomplished all three objectives. For objective 1, we have: 1) deposited all the strains selected for the sequencing project at ARS Culture Collection (NRRL accession listed); 2) generated the Illumina data (>25X coverage) for 10 proposed strains 3) produced the Illumina sequence for the reference strain for the comparison 4) developed and tested a pipeline using MAQ to align the short Illumina reads to the reference genome assembly for SNP discovery and coverage analysis. For objective 2, we have: 1) constructed optical maps for all four strains; 2) conducted the pair-wise comparison using the map data, which confirmed our project plan to study genome structural polymorphisms and provide guidance for preparing samples for deep sequencing of the LS chromosomes. In accomplishing objective 3, we encountered difficulty in isolating sufficient DNA for the LS chromosomes to generate sequence. The revolutionary of new sequencing technologies makes the cost of generating sequence data so much lower. With that, we decided to generate whole genome sequence for these isolates instead. For objective 3, we have: 1) produced whole genome shotgun sequence for all 10 strains using the newest Illumina sequencing platform HiC. All the Illumina sequence data sets were deposited at NCBI trace repository for public access (http://www.ncbi.nlm.nih.gov/sites/entrez) under the "Study Accession" number SRP002087". 2) generated whole genome assemblies for all. The strategies to combine Illumina fragment reads (180 bp) and large insert libraries (4 kb and 8 kb) produced high quality assemblies for all the genomes with an average N50 scaffold over 2 Mb in size. All the assemblies are released through the Fusarium comparative genomics website (http://www.broadinstitute.org/annotation/genome/fusarium_group) in spring 2012. PARTICIPANTS: Andy Berg: PhD student at UMass Amherst PSIS graduate program Zhongying Huang: PhD student at UMass Amherst PB graduate program CHristina Stonoha: PhD student at UMass Amherst PB graduate program Jayashree Gandhi: UMass Commonwealth College Honor student dissertation. TARGET AUDIENCES: We have targeted our work toward undergraduate students by introducing the power of genomics in understanding interactions between organisms in the broad ecosystems. PI Ma presented the work at multiple instances including the Microbial and Plant Genomics Institute Fall Symposium at University of Minnesota and the Environmental Science Institute seminar at University of Massachusetts at Amherst. More importantly, the data generated through this project have been used to train 3 graduate students through the UMass Amherst Plant Biology Graduate Program and used in two high level classes in train both undergraduate and graduate students (PLSOILIN590C and PLSOILIN597Z). Analyze one of the genome assembly produced through this project, my student (Andy Berg) was invited to give an oral presentation of his research at the American Phytophatological Society annual meeting. PROJECT MODIFICATIONS: The revolutionary of new sequencing technologies makes the cost of generating sequence data so much lower. With that, we decided to generate whole genome sequence for all isolates proposed using the newest Illumina sequencing platform HiC. The performance of HiC platform has been tested at the Broad Institute, and is currently employed in production. To enhance the possibility to assemble these short next generation sequence reads, we will construct 2 sequencing libraries with 180-base and 5 kb insert sizes respectively. Overall the sequence data will provide more than 100X sequence coverage from the small insert lib and over 300X physical coverage from the other large insert libraries
Impacts
Members of the F. oxysporum species complex exhibit extraordinary genetic plasticity and cause some of the most destructive and intractable diseases across a diverse spectrum of hosts, including many economically important crops such as banana, cotton, canola, melons, and tomato. The primary solution to control such diseases is through the development of disease resistant plant cultivars. However, due to their persistence in the soil and the organisms ability to adapt, it is just a matter of time before the pathogens overcome the newly deployed resistance. The Fusarium comparative genomics project (2005-35600-16405) highlighted the existence of lineage-specific chromosomes that are enriched for transposable elements and encode genes that are pathogenicity related. The comparative analysis provides evidence for the horizontal transfer (HT) of four chromosomes accounting for 25% of the genome in an asexual, pathogenic fungal species, Fusarium oxysporum. The direct contribution of the chromosomes to pathogenicity is indicated by the fact that they encode known virulence factors such as effector proteins, necrosis-inducing peptides and a large array of enzymes targeting plant substrates, but lack genes involved in primary metabolism. The mobilization of these TE-rich and pathogenicity related chromosomes contributes to the rapid emergence of new pathogenic lineages of F. oxysporum in otherwise distinct and incompatible genetic backgrounds and provides the genetic basic for the remarkable genetic adaptability of the organism. The result implies that HT may offer previously unrecognized opportunities for genetic exchange and recombination in asexual lineages of lower eukaryotes. Our study deepens the understanding of evolutionary mechanism involving fungal pathogens development, which may have direct impact in developing novel management strategies in agricultural practices. The pair-wise comparison using the optical map data reveals the syntenic conservation for the 11 "conserved" chromosomes among all three strains. In addition to the 4 lineage specific (LS) chromosomes in the reference genome (Fol) identified through the Fusarium comparative project, additional LS chromosomes are identified in each of the mapping isolate, including one (~4 Mb) in the non-pathogenic isolate Fo47 (NRRL54002) and 4 (<2 Mb each) in the human isolate FOSC 3a (NRRL32931). This result provided the initial support to our hypothesis that genetic materials carried among the lineage-specific chromosomes play significant roles in organism adaptation among F. oxysporum species complex and laid out the guidance for preparing samples for deep sequencing of the LS chromosomes. The whole genome assemblies from different F. oxysporum pathogenic strains enable the identification of conserved "core" genome and highlight the novel sequences in each strain. This will enable us to study strain-specific virulent factors.
Publications
- Kumar, L., Breakspear, A., Kistler, C., Ma, L.-J. and Xie, X. 2010. Systematic discovery of regulatory motifs in Fusarium graminearum by comparing four Fusarium genomes. BMC Genomics 2010, 11:208. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853525/pdf/1471-2164-11- 208.pdf
- Ma L.-J. , and Fedorova, ND. A practical guide to fungal genome projects: strategy, technology, cost and completion. Mycology: An International Journal on Fungal Biology. 2010 Apr 01; 1(1): 9-24. 2010.
- Ma L.-J., J. W. Bennett, N. D. Fedorova. 2011. Harnessing the power of fungal genomics. Editorial: Fungal Biology in the Age of Genomics. Mycology: An International Journal on Fungal Biology. 2(3) 1.
- Grabherr M.G., Mauceli E., and L.-J. Ma*. 2011. Genome Sequencing and Assembly. Methods Mol. Biol., 722 J.-R. Xu and B.H. Bluhm, eds. Methods in Microbiology Vol. 722. 2011. Humana Press, pp1-9.
- Amyotte S. G., X. Tan, K. Pennerman, M. del M. Jimenez, S. J. Klosterman, L.-J. Ma, K. F. Dobinson, P. Veronese. 2012. Transposable elements in the phytopathogenic Verticillium spp.: insights into genome evolution and inter- and intra-specific diversification. BMC Genomics. 13: 314-334.
- Gao Q. et al. 2011. Genome Sequencing and Comparative Transcriptomics of the Model Entomopathogenic Fungi Metarhizium anisopliae and M. acridum. PLoS Genet 7(1): e1001264.
- Ma L.-J., C. van der Does, [60 coauthors], H. C. Kistler and M. Rep. 2010. Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium oxysporum. Nature 465:367-373.
- Klosterman S., K. [26 coauthors], K. F. Dobinson, L.-J. Ma. 2011. Verticillium comparative genomics yields insights into niche adaptation by plant vascular wilt pathogens. PLoS Pathogen 7(7): e1002137.
- Ma L.-J, M. Rep and H. C. Kistler. 2012. Evolution of plant pathogenicity in Fusarium species. In Sibley, L. D., B. J. Howlett, et al. 2012. Evolution of virulence in eukaryotic microbes. Hoboken, N.J., Wiley-Blackwell. 485-500.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Using the sequenced Fusarium oxysporum f.sp. lycopersici (Fol) as a reference genome, this proposed project will focus on four additional F. oxysporum strains to: 1. Generate deep Solexa sequencing coverage (~16X coverage) to survey overall sequence polymorphism and SNP distribution; 2. Create whole genome optical maps to study genome structural polymorphisms and to: a. identify strain specific genomic regions; b. characterize chromosomal variations such as inversion, translocation and large indels. 3. Sequence, assemble and annotate the small, polymorphic chromosomes for each selected strain using the isolated DNA from small polymorphic chromosomes. So far, we have accomplished the objective 1 and 2. For objective 1, we have: 1) deposited all the strains selected for the sequencing project at ARS Culture Collection (NRRL accession listed); 2) generated the Illumina data (>25X coverage) for 10 proposed strains 3) produced the Illumina sequence for the reference strain for the comparison 4) developed and tested a pipeline using MAQ to align the short Illumina reads to the reference genome assembly for SNP discovery and coverage analysis. For objective 2, we have: 1) constructed optical maps for all four strains; 2) conducted the pair-wise comparison using the map data, which confirmed our project plan to study genome structural polymorphisms and provide guidance for preparing samples for deep sequencing of the LS chromosomes. In accomplishing objective 3, we encountered difficulty in isolating sufficient DNA for the LS chromosomes to generate sequence. The revolutionary of new sequencing technologies makes the cost of generating sequence data so much lower. With that, we decided to generate whole genome sequence for these isolates instead. Current, we are planning to generate Illumina reads for 12 strains using the newest Illumina sequencing platform HiC. All the Illumina sequence data sets were deposited at NCBI trace repository for public access (http://www.ncbi.nlm.nih.gov/sites/entrez) under the "Study Accession" number SRP002087". Public release of the Fusarium Comparative web site: http://wwwdev.broad.mit.edu/annotation/genome/fusarium_group/ PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: We have targeted our work toward undergraduate students by introducing the power of genomics in understanding interactions between organisms in the broad ecosystems. PI Ma presented the work at multiple instances including the Microbial and Plant Genomics Institute Fall Symposium at University of Minnesota and the Environmental Science Institute seminar at University of Massachusetts at Amherst. PROJECT MODIFICATIONS: The revolutionary of new sequencing technologies makes the cost of generating sequence data so much lower. With that, we decided to generate whole genome sequence for all isolates proposed using the newest Illumina sequencing platform HiC. The performance of HiC platform has been tested at the Broad Institute, and is currently employed in production. To enhance the possibility to assemble these short next generation sequence reads, we will construct 2 sequencing libraries with 180-base and 5 kb insert sizes respectively. Overall the sequence data will provide more than 100X sequence coverage from the small insert lib and over 300X physical coverage from the other large insert libraries.
Impacts
Members of the F. oxysporum species complex exhibit extraordinary genetic plasticity and cause some of the most destructive and intractable diseases across a diverse spectrum of hosts, including many economically important crops such as banana, cotton, canola, melons, and tomato. The primary solution to control such diseases is through the development of disease resistant plant cultivars. However, due to their persistence in the soil and the organisms' ability to adapt, it is just a matter of time before the pathogens overcome the newly deployed resistance. The Fusarium comparative genomics project (2005-35600-16405) highlighted the existence of lineage-specific chromosomes that are enriched for transposable elements and encode genes that are pathogenicity related. The comparative analysis provides evidence for the horizontal transfer (HT) of four chromosomes accounting for 25% of the genome in an asexual, pathogenic fungal species, Fusarium oxysporum. The direct contribution of the chromosomes to pathogenicity is indicated by the fact that they encode known virulence factors such as effector proteins, necrosis-inducing peptides and a large array of enzymes targeting plant substrates, but lack genes involved in primary metabolism. The mobilization of these TE-rich and pathogenicity related chromosomes contributes to the rapid emergence of new pathogenic lineages of F. oxysporum in otherwise distinct and incompatible genetic backgrounds and provides the genetic basic for the remarkable genetic adaptability of the organism. The result implies that HT may offer previously unrecognized opportunities for genetic exchange and recombination in asexual lineages of lower eukaryotes. Our study deepens the understanding of evolutionary mechanism involving fungal pathogens development, which may have direct impact in developing novel management strategies in agricultural practices. The whole genome SNPs discovered using the Illumina sequences enable a comprehensive, un-biased study of phylogenetic relationship among the selected isolates. The data provides a solid foundation to study the evolutionary processes governing the pathogenicity development of this group of pathogens. The pair-wise comparison using the optical map data reveals the syntenic conservation for the 11 "conserved" chromosomes among all three strains. In addition to the 4 lineage specific (LS) chromosomes in the reference genome (Fol) identified through the Fusarium comparative project, additional LS chromosomes are identified in each of the mapping isolate, including one (~4 Mb) in the non-pathogenic isolate Fo47 (NRRL54002) and 4 (<2 Mb each) in the human isolate FOSC 3a (NRRL32931). This result provided the initial support to our hypothesis that genetic materials carried among the lineage-specific chromosomes play significant roles in organism adaptation among F. oxysporum species complex and laid out the guidance for preparing samples for deep sequencing of the LS chromosomes.
Publications
- L.J. Ma, C. van der Does, et al. 2010, Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium oxysporum. Nature 465:367-373.
- Rep, M. and Kistler H.C. 2010. The genomic organization of plant pathogenicity in Fusarium species. Current Opinion in Plant Biology. DOI 10.1016/j.pbi.2010.04.004.
- Kumar, L., Breakspear, A., Kistler, C., Ma, L.-J. and Xie, X. 2010. Systematic discovery of regulatory motifs in Fusarium graminearum by comparing four Fusarium genomes. BMC Genomics 2010, 11:208.
- Ma L.-J. , and Fedorova, ND. A practical guide to fungal genome projects: strategy, technology, cost and completion. Mycology: An International Journal on Fungal Biology. 2010 Apr 01; 1(1): 9-24. 2010.
- Invited lectures. Li-Jun Ma. March 31, 2010. Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium oxysporum, 10th European Conference on Fungal Genetics, Noordwijkerhout, The Netherlands.
- Li-Jun Ma. March 28, 2010 Exploring lineage-specific (LS) chromosomes in F. oxysporum species complex (FOSC) Fusarium Satellite Meeting, Amsterdam, the Netherlands.
- Li-Jun Ma. Feburary 24 2010 Understanding fungal biology through comparative genomics. JCVI, Maryland, USA
- Li-Jun Ma. Understanding fungal genome evolution and niche adaptation using comparative genomics. September 2010. University of Minnesota Microbial and Plant Genomics Institute Fall Symposium. St. Paul MN
- Li-Jun Ma. Verticillium Comparative Genomics -- pathogenicity of wilt pathogens. January 2010. USDA Microbial Sequencing Program Awardee Workshop. The XXI International Plant and Animal Genome Conference. San Diego, CA
- Corby Kistler July 22, 2010. Wilt v. Blight: Common Course and Divergent Paths to Plant Disease. USDA NIFA AFRI Awardee Workshop on Microbial Functional Genomics, Washington, DC.
- Corby Kistler September 15. 2010. Common Course and Divergent Paths to Plant Disease: Host specialization of Fusarium species revealed by comparative genomics. Crop Science Department, University of Illinois, Champagne/Urbana.
- Corby Kistler November 3, 2009. What has been learned from six years of genomic research on the Fusarium head blight pathogen. 6th Canadian Workshop on Fusarium Head Blight, Ottawa, Canada.
- Corby Kistler November 30, 2009. Discovery of regulatory motifs in Fusarium graminearum by comparative genomics. Aarhus University, Denmark.
- Corby Kistler December 4, 2009. Comparative genomics of Fusarium species Copenhagen University, Denmark.
- Corby Kistler March 28, 2010 Fusarium comparative genomics reveals lineage-specific chromosomes related to pathogenicity. Fusarium Satellite Meeting, Amsterdam, the Netherlands.
- Corby Kistler March 31, 2010 Fusarium graminearum as a Model for Human Niemann-Pick Type C Disease, 10th European Conference on Fungal Genetics, Noordwijker-hout, The Netherlands.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: The objective 1 has been accomplished. We have generated deep Solexa sequencing coverage (> 25X coverage) for all 10 proposed strains to survey overall sequence polymorphism and SNP distribution. In addition, we have sequenced the reference strain using the same sequence strategy as control data. Along the process, we have deposited all the strains selected for the sequencing project at FGSC; and developed and tested a pipeline using MAQ to align the short Illumina reads to the reference genome assembly for SNP discovery and coverage analysis. At the same time, a steady progress has also been made towards the accomplishing of the objective 2: to study genome structural polymorphisms of four selected strains by identifying strain specific genomic regions; and characterizing chromosomal variations such as inversion, translocation and large indels among different pathogenic strains using optical mapping approach. So far, high quality protoplast samples for the four selected isolates were prepared. The optical maps for two strains are completed. The construction of the other two are started. The pair-wise comparisons of the map data revealed multiple lineage-specific (LS) chromosomes in each of the mapped isolate as we anticipated. The result confirms the feasibility of our project plan and provides guidance to achieve our next objective. In the coming year, we will specifically isolate the LS specific chromosomes to be sequenced, assembled and annotated to study the contribution of these chromosomes in host-specificity and pathogenicity development. PARTICIPANTS: Li-Jun Ma: Broad Institute of MIT and Harvard; H. Corby Kistler: University of Minnesota; Shiguo Zhou, University of Wisconsin-Madison. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Members of the F. oxysporum species complex exhibit extraordinary genetic plasticity and cause some of the most destructive and intractable diseases across a diverse spectrum of hosts, including many economically important crops such as banana, cotton, canola, melons, and tomato. The primary solution to control such diseases is through the development of disease resistant plant cultivars. However, due to their persistence in the soil and the organisms' ability to adapt, it is just a matter of time before the pathogens overcome the newly deployed resistance. The Fusarium comparative genomics project (2005-35600-16405) highlighted the existence of lineage-specific chromosomes that are enriched for transposable elements and encode genes that are pathogenicity related. The comparative analysis provides evidence for the horizontal transfer (HT) of four chromosomes accounting for 25% of the genome in an asexual, pathogenic fungal species, Fusarium oxysporum. The direct contribution of the chromosomes to pathogenicity is indicated by the fact that they encode known virulence factors such as effector proteins, necrosis-inducing peptides and a large array of enzymes targeting plant substrates, but lack genes involved in primary metabolism. The mobilization of these TE-rich and pathogenicity related chromosomes contributes to the rapid emergence of new pathogenic lineages of F. oxysporum in otherwise distinct and incompatible genetic backgrounds and provides the genetic basic for the remarkable genetic adaptability of the organism. Our study will provide a broad survey of the existence of the LS chromosomes and reveal specific virulence factors that contribute to host specificity and adaptation of each pathogenic isolate. The knowledge gained through this project will deepen the understanding of evolutionary mechanism involving fungal pathogens development and has the potential in contributing to the development of novel management strategies in agricultural practices.
Publications
- Ma, L.-J. 2009. Lineage-specific chromosomes related to pathogenicity revealed by Fusarium comparative genomics. March 2009. Department seminar. UMASS Amherst. Massachusetts.
- Ma, L.-J. 2009 What can you learn from Comparative Genomics. Fusarium workshop. March 2009. The 25th Fungal Genetics Conference, Asilomar, California.
- Ma, L.-J. 2009 Genome Innovation revealed by Fusarium comparative genomics. January 2009. Plant and Animal Genome XVII Conference. San Diego CA.
- Kistler, H.C. 2009. Comparative genomics of plant-pathogenic Fusarium species. Department of Plant Pathology, University of Minnesota, St. Paul, MN, January 26, 2009.
- Kistler, H.C. 2009. Comparative genomics of plant-pathogenic Fusarium species. Department of Plant Biology and Pathology and the New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, NJ, April 3, 2009.
- L.-J. Ma, C. van der Does, K. Borkovich, J. Coleman, M.-J.Daboussi, A. Di Pietro, M. Dufresne, M. Freitag, M. Grabherr, B.Henrissat, P. M. Houterman, S. Kang, W.-B. Shim, C. Woloshuk, X. Xie, J.-R.Xu, J. Antoniw, S. E. Baker, B. H. Bluhm, A. Breakspear, D. Brown, R.Butchko, S. Chapman, R. Coulson, P. M. Coutinho, E. Danchin, A. Diener, L. Gale, D. Gardiner, S. Goff , K. Hammond-Kosack, K. Hilburn, A. Hua-Van, W. Jonkers, K. Kazan, C. Kodira, M. Koehrsen, L. Kumar, Y.-H. Lee, L. Li, J.Manners, D. Miranda-Saavedra, M. Mukherjee, G. Park, J. Park, S.-Y. Park, R. Proctor, A.Regev, C. Ruiz-Roldan, D.Sain, S. Sakthikumar, S. Sykes, D. Schwartz, G. Turgeon, I. Wapinski, O. Yoder, S. Young, Q. Zeng, S. Zhou, J. Galagan, C. Cuomo, C. Kistler , and M. Rep. 2009 Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium oxysporum. Nature (under revision)
- Ma, L.-J. 2009. Promises of fungal comparative genomics. September 2009. China Fungal Genome Initiative Symposium. China Mycological Society, Shanghai, China.
- Ma, L.-J. 2009. Genetic plasticity and pathogenicity development revealed through Fusarium comparative genomics. August 2009. APS. Portland, Oregon.
- Ma, L.-J. 2009. Lineage-specific chromosomes related to pathogenicity revealed by Fusarium comparative genomics. May 2009. Lockwood Lecture. Agricultural Experiment Station. Connecticut.
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