Progress 10/01/08 to 09/30/11
Outputs
OUTPUTS: The overall goal of this project was to develop genetic and genomic tools for the grape powdery mildew fungus, Erysiphe necator. The specific goals are: 1) to sequence a large number of expressed genes (the transcriptome) from two or more different mildew strains, 2) to develop polymorphic genetic markers using these sequences, and 3) to apply these polymorphic markers to genetic linkage mapping. Objectives 1 and 2 were fully accomplished, whereas objective has remained unsolvable because it is not possible to make controlled crosses with E. necator. We used Roche 454 GS-FLX technology (known as 454 sequencing) to sequence the transcriptome of one isolate of E. necator. These efforts have resulted in very high quality sequences and excellent coverage of the transcriptome. Comparative analyses showed that the transcriptome of E. necator is much like other powdery mildew fungi. We obtained sequences from cDNA from 55 additional isolates using Illumina sequencing, which gives millions of short reads. These sequences have been aligned with the 454 transcriptome reference sequence. We have also conducted bioinformatic analyses to assemble and compare the transcriptome sequences more comprehensively. During the course of this project, we developed 12 polymorphic microsatellite or simple-sequence repeat (SSR) markers for a genotyping system. Using the transcriptome sequences from 55 isolates, plus the reference isolate, we have identified thousands of single-nucleotide polymorphisms (SNPs). We used microsatellite markers to genotype large samples of E. necator from the eastern US and Europe; similar analyses are underway using SNP data. In the process of studying genetic variation using molecular markers, we also discovered variation in the virulence and pathogenicity of E. necator. This pathogen is host-specific on muscadine grapes (Vitis rotundifolia). Equally important, our diverse collection of E. necator from cultivated and wild grapevines allowed us to demonstrate race-specific resistance in grapevines and race variation in E. necator for the first time. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Obtaining high quality transcriptome sequence has been a major breakthrough in the genetics/genomics of E. necator. Before this project began, there were approximately 4-5 gene sequences from E. necator represented in publicly available databases (e.g., GenBank). Our transcriptome sequence has added thousands of gene sequences. Transcriptome sequencing has allowed us to develop SSR markers for population genetic analyses. These polymorphic markers are being used to address epidemiological questions concerning migration between different areas or between different host species, e.g., from wild species to cultivated vineyards, and within vineyards. For example, we found no genetic differentiation among populations on different host species of wild grapevines in the US, except for those on muscadine grapes in the southeastern US. This is an important finding because muscadine is a source of powdery mildew resistance that is being bred into other grapevine species. We also found that genotypes of this fungus are spatially aggregated within vineyards, most likely because of short-distance dispersal. Approximately 99% of the transcripts in the 454 sequence were also found in the sequences from these additional isolates. This close match underscores the utility of the 454 reference sequences for resequencing with the cheaper Illumina technology. Using transcriptome data, and comparative genomics with Blumeria graminis, the cereal powdery mildew fungus, we were able to identify the mating-type locus and to develop a PCR marker for mating type. The data from these additional transcriptomes are being analyzed for single-nucleotide polymorphisms (SNPs). The extent of polymorphism and distribution with respect to geography and phenotype are the objectives of subsequent analysis. We conducted population genetic analyses on E. necator from the eastern US and Europe using microsatellite markers. This study confirmed the results from multilocus sequencing, but provided more resolution because of higher levels of polymorphism than found previously. We found that a subpopulation of E. necator in the southeastern US that is very similar to one of the two genetic groups or lineages, group A, found in introduced populations in Europe and Australia. This A-like group is genetically distinct from the rest of the US population and is the likely progenitor of the introductions into Europe in the 1800s. Our emphasis on population genetics and diversity aided in breeding programs because it allowed us to identify races in E. necator and race-specific resistance in breeding lines of grapes.
Publications
- Brewer, M.T., Cadle-Davidson, L., Cortesi, P., Spanu,P.D., Milgroom, M.G., 2011. Identification and structure of the mating-type locus and development of PCR-based markers for mating type in powdery mildew fungi. Fungal Genetics and Biology 48 704-713.
- Frenkel O, Portillo I, Brewer MT, Peros J-P, Cadle-Davidson L, Milgroom MG, 2011. Development of microsatellite markers from the transcriptome of Erysiphe necator for analyzing population structure in North America and Europe. Plant Pathol, DOI: 10.1111 j.1365-3059.2011.02502.x.
- Wakefield, L., Gadoury, D. M., Seem, R. C., Milgroom, M. G., Sun, Q. and Cadle-Davidson, L. 2011. Differential gene expression during conidiation in the grape powdery mildew fungus, Erysiphe necator. Phytopathology 101: 839-846.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The overall goal of this project is to develop genetic and genomic tools for the grape powdery mildew fungus, Erysiphe necator. The specific goals are: 1) to sequence a large number of expressed genes (the transcriptome) from two or more different mildew strains, 2) to develop polymorphic genetic markers using these sequences, and 3) to apply these polymorphic markers to genetic linkage mapping. We used Roche 454 GS-FLX technology (known as 454 sequencing) to sequence the transcriptome of one isolate of E. necator. Our efforts have resulted in very high quality sequences and excellent coverage of the transcriptome. We increased our sequencing efforts in 2010 and now have approximately 168 Mbp of sequence, assembled into 19,959 contigs, with an average length of 1051 bp and 18.2 sequencing reads per contig. To determine the quality of the coverage in these sequences, we searched for 458 core conserved eukaryotic genes and found contigs with all but five of them. This suggests that our transcriptome sequencing provides excellent coverage of the genes expressed in E. necator. However, a preliminary analysis of genes identified in the transcriptome of E. necator has shown that approximately 100 genes that are broadly conserved in yeasts and other ascomycetes are missing in E. necator. These 100 genes match almost perfectly with the genes absent from draft genomes of other powdery mildew fungi. This reduction in gene number likely reflects the deletion of unnecessary genes for functions provided by the host in an obligate biotrophic interaction. Sequencing of cDNA from another isolate was done using Illumina sequencing, which gives millions of short reads. These sequences were aligned with the 454 transcriptome sequence. Approximately 99% of the transcripts in the 454 sequence were also found in the sequences from this second isolate. This close match underscores the utility of the 454 reference sequences for resequencing with the cheaper Illumina technology. We have also obtained additional transcriptome sequence for 5-6 additional isolates using Illumina but these data are still being analyzed. We used the transcriptome sequences to search for microsatellite or simple-sequence repeat (SSR) markers. To date, we have developed 11 polymorphic markers; we have the potential to develop many more markers by further mining the transcriptome. Although we have continued to make crosses between isolates of E. necator in the laboratory for linkage mapping, so far, we have not succeeded in obtaining enough progeny isolates from any one cross to be useful for linkage mapping. After many unfruitful attempts, this part of the project has been suspended. Instead of applying genetic markers to linkage mapping, we are applying these markers to the analysis of the population genetics of E. necator. So far, we have genotyped approximately 100 isolates from the US and 60 from Europe. We found much greater genetic diversity in the US than in Europe, a result consistent with the hypothesis that E. necator is native to the US and was introduced to Europe. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Obtaining high quality transcriptome sequence has been a major breakthrough in the genetics/genomics of E. necator. Before this project began, there were approximately 4-5 gene sequences from E. necator represented in publicly available databases (e.g., GenBank). Our transcriptome sequence has thousands of partial gene sequences. Transcriptome sequencing has allowed us to develop SSR markers for population genetic analyses. These polymorphic markers are being used to address epidemiological questions concerning migration between different areas or between different host species, e.g., from wild species to cultivated vineyards. For example, we found no genetic differentiation among populations on different host species of wild grapevines in the US, except for those on muscadine grapes in the southeastern US. This is an important finding because muscadine is a source of powdery mildew resistance that is being bred into other grapevine species. Although not an original goal of this project, we have used the transcriptome sequence to find genes in E. necator homologous to genes known to function in fungicide resistance in other fungi. We have identified four genes in E. necator in the family of ATP-binding cassette (ABC) transporter genes. We obtained nucleotide sequences of one gene (Cyp51) known to have a major impact on resistance to demethylation inhibitor fungicides (DMIs). We also studied the expression of ABC transporter genes and Cyp51 using quantitative PCR to see how expression correlates with resistance to DMIs. We found that expression of Cyp51 and at least one ABC transporter correlates well with the resistance phenotype. Therefore, genotype and expression data may be useful for predicting the resistance of individual isolates.
Publications
- Brewer M.T. and Milgroom M.G. 2010. Phylogeography and population structure of the grape powdery mildew fungus, Erysiphe necator, from diverse Vitis species. BMC Evolutionary Biology, 10:268.
- Frenkel O., Brewer M.T. and Milgroom M.G. 2010. Variation in pathogenicity and aggressiveness of Erysiphe necator from different Vitis species and geographic origins in the eastern United States. Phythopathology 100:1185-1193.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: The overall goal of this project is to establish genetic and genomic tools for studying the biology of the grape powdery mildew fungus, Erysiphe necator (formerly known as Uncinula necator). This fungus is an obligate parasite of grapevines and cannot be cultured on artificial medium, i.e., it can only grow on living grape leaves, making it very difficult to work with experimentally. The specific goals are: 1) to sequence a large number of expressed genes (the transcriptome) from two or more different mildew strains, 2) to develop polymorphic genetic markers using these sequences, and 3) to apply these polymorphic markers to genetic linkage mapping. Advances in DNA sequencing technology has made it possible to obtain large amounts of sequence data cheaply. We used Roche 454 GS-FLX technology (known as 454 sequencing) to sequence the transcriptome of one isolate of E. necator. Our initial efforts have resulted in very high quality sequences and excellent coverage of the transcriptome. We obtained approximately 82 Mbp of sequence from normalized cDNA. These sequences were assembled into 32,405 contigs, with an average length of 591 bp and 9.1 sequencing reads per contig. To determine the quality of the coverage in these sequences, we searched for 458 core conserved eukaryotic genes and found contigs with all but 5 of them. This suggests that our transcriptome sequencing provides excellent coverage of the genes expressed in E. necator. Sequencing of cDNA from another isolate was done using Illumina (formerly Solexa) sequencing, which gives millions of short reads. These sequences were aligned with the 454 transcriptome sequence. Approximately 99% of the transcripts in the 454 sequence were also found in the Illumina sequences from this second isolate. This close match underscores the utility of the 454 reference sequences for resequencing with the cheaper Illumina technology. Using the transcriptome sequence as a reference, we searched for microsatellite or simple-sequence repeat (SSR) motifs and designed primers flanking these regions. SSRs are often polymorphic and therefore useful markers for population genetics and linkage mapping. We screened for polymorphisms in 35 SSRs and obtained 16 that were polymorphic. We have continued to make crosses between New York isolates in the laboratory for linkage mapping. So far, however, we have succeeded in only obtaining a maximum of nine progeny from any one cross. We will need to obtain much larger numbers (a minimum of approximately 100) for successful linkage mapping. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Obtaining high quality transcriptome sequence has been a major breakthrough in the genetics/genomics of E. necator. Before this project began, there were approximately 4-5 gene sequences from E. necator represented in GenBank. Our transcriptome sequence has thousands of partial gene sequences. Additional transcriptome sequencing from other isolates (now underway) is likely to increase the length contigs and the number of genes represented. Specific to this project, transcriptome sequencing has allowed us to develop SSR markers for linkage and population genetic analyses. We have obtained enough polymorphic SSR markers to date for population genetic analyses; more will be needed for linkage analyses. These polymorphic markers can also be applied to address epidemiological questions concerning dispersal between different areas or between different host species, e.g., from wild species to cultivated vineyards. Although not specifically a goal of this project, we have used the transcriptome sequence to find homologs of genes known to function in fungicide resistance. We have identified four genes in E. necator in the family of ATP-binding cassette (ABC) transporter genes. We will study the expression of these genes using quantitative PCR in the next year to see how they correlate to resistance to demethylation inhibitor fungicides (DMIs). Many other types of questions concerning gene expression can now be undertaken in this important pathogen. Linkage mapping will depend on the successful crossing of mildew strains in the laboratory. The most significant constraint is getting the sexual spores (ascospores) to germinate and infect grape leaves. Overwintering of spores is required, which is being done several ways to optimize crossing techniques. These efforts are continuing.
Publications
- Brewer MT, Milgroom MG (2008) Phylogeography and sequence diversity of genetic lineages of the grapevine powdery mildew fungus, Erysiphe (Uncinula) necator, in North America, Europe, and Australia. Phytopathology 98, S25-S26 (abstract).
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The overall goal of this project is to establish genetic and genomic tools for studying the biology of the grape powdery mildew fungus, Erysiphe necator (synonym Uncinula necator). This fungus is an obligate parasite of grapevines and cannot be cultured artificial medium, i.e., it can only grow on living grape leaves, making it very difficult to work with experimentally. The specific goals are: 1) to sequence a large number of expressed genes (the transcriptome) from two different mildew strains, 2) develop polymorphic genetic markers using these sequences, and 3) apply these polymorphic markers to genetic linkage mapping. Collectively, these would represent a major step forward in developing genetic resources for studying this fungus. This project began in October 2008, so we few tangible outputs to report for the first three months. We have cultured two strains of E. necator on grape leaves in the laboratory and purified total RNA from harvested fungal tissues. The RNA has been submitted to the Cornell Biotechnology Resource Center for cDNA synthesis and genome sequencing using 454 technologies. We expect to receive transcriptome sequences in early 2009. We have begun making crosses between New York isolates in the laboratory for linkage mapping when markers are available. Isolates were grown on leaves in the lab so that they could mate and produce offspring for genetic analyses. Spores must mature for several months in a controlled environment that simulates winter conditions before further analysis can be done. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Although we are still developing methods, we expect to acquire transcriptome sequence data with which to develop genetic markers in early 2009. Linkage mapping will depend on the successful crossing of mildew strains in the laboratory. The most significant constraint is getting the sexual spores (ascospores) to germinate and infect grape leaves. Overwintering of spores is required, which is being done several ways to optimize crossing techniques.
Publications
- No publications reported this period
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