Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): The specific objectives of this proposal are to: (1) Sequence and assemble the complete genome of P. triticina isolate 1-1, Race 1 (BBBD), which is estimated to be between 100 - 120 Mb, using a hybrid of 454 and ABI (Sanger) Fosmid-end sequence; (2) Annotate gene structure using computational methods, 200,000 454 reads of ESTs from each of four new cDNA libraries, and other available ESTs; (3) Evaluate P. triticina polymorphism and diversity by comparing the sequenced strain with three additional isolates using Illumina/Solexa sequence; (4) Prompt public release of all reads, assemblies, annotation, and discovered polymorphisms; and (5) Develop education, training and outreach programs. Approach (from AD-416): To achieve our objective we propose to: 1. Generate a whole genome shotgun assembly using an optimized combination of traditional and new sequencing technology data (Sanger and 454). 2. Annotate gene structure using computational methods, 200,000 454 reads of ESTs from each of four new cDNA libraries, and other available ESTs. 3. Evaluate the level of P. triticina DNA polymorphisms and diversity by sequencing three additional isolates of P. triticina using Illumina/ Solexa sequence. 4. Prompt public release of all reads, assemblies, annotation, discovered polymorphisms, and EST alignments. 5. Develop education, training and outreach programs Puccinia triticina is the name of the fungus that causes wheat leaf rust. Wheat leaf rust is one of the most important diseases of wheat worldwide. In order to understand the biology of this pathogen better, we have been working to obtain the entire genome sequence of this fungus. One race was chosen previously to prepare the original draft DNA genome sequence of the leaf rust pathogen. In the last year of the leaf rust genome project, we have expanded the DNA sequencing to include fifty- seven more North American races. These races were all collected in 2011 from around the US and represent members of North American lineages 2, 3 and 5. Races were selected based on virulence patterns, region of collection, and comparisons of the same race collected from different geographical regions. Also, 20 races from the five North American lineages were selected for RNA sequencing. These will be used to evaluate race-specific gene expression during stages of infection in the wheat plant. The race DNA sequencing is identifying race-specific DNA markers, changes in avirulence genes, and giving clues as to how new races evolve. A manuscript is being prepared that compares the genome of leaf rust with the other two wheat rusts. Another manuscript is being prepared describing the results of the race comparisons. This is the final year of this grant.
Impacts
(N/A)
Publications
|
Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): The specific objectives of this proposal are to: (1) Sequence and assemble the complete genome of P. triticina isolate 1-1, Race 1 (BBBD), which is estimated to be between 100 - 120 Mb, using a hybrid of 454 and ABI (Sanger) Fosmid-end sequence; (2) Annotate gene structure using computational methods, 200,000 454 reads of ESTs from each of four new cDNA libraries, and other available ESTs; (3) Evaluate P. triticina polymorphism and diversity by comparing the sequenced strain with three additional isolates using Illumina/Solexa sequence; (4) Prompt public release of all reads, assemblies, annotation, and discovered polymorphisms; and (5) Develop education, training and outreach programs. Approach (from AD-416): To achieve our objective we propose to: 1. Generate a whole genome shotgun assembly using an optimized combination of traditional and new sequencing technology data (Sanger and 454). 2. Annotate gene structure using computational methods, 200,000 454 reads of ESTs from each of four new cDNA libraries, and other available ESTs. 3. Evaluate the level of P. triticina DNA polymorphisms and diversity by sequencing three additional isolates of P. triticina using Illumina/Solexa sequence. 4. Prompt public release of all reads, assemblies, annotation, discovered polymorphisms, and EST alignments. 5. Develop education, training and outreach programs Wheat leaf rust is one of the most important diseases of wheat. Puccinia triticina is the name of the fungus that causes wheat leaf rust. In order to understand the biology of this pathogen better, we have been working to obtain the entire genome sequence of this fungus. The first version of the Puccinia triticina genome assembly was considered a rough draft due to the large number of unassembled sequencing reads. It was believed that more sequencing, with longer reads would improve the assembly. In 2011-12, more genomic DNA of P. triticina was sequenced using new advances in Roche 454 technology. A much improved second assembly of the Puccinia triticina genome was released with significant reductions in unassembled sequences. Because the cost of sequencing technology has dropped, we have been able to explore the differences among races of P. triticina. Using the genome assembly as a reference, 56 races of P. triticina were sequenced and aligned to identify single nucleotide polymorphisms. These races represent collections from different time periods, geographical regions, and sexual populations. Initial analysis has identified clusters of races that represent both sexual and asexual populations, as well as, diversity points that have led to major race shifts. Changes in the genome are also being associated with changes in effector proteins produced by the pathogen. Effector proteins control whether the interaction of host and pathogen results in susceptibility or resistance. In the next year, 50 more races will have their DNA sequenced and 15 races will have their RNA sequenced.
Impacts
(N/A)
Publications
|
Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) The specific objectives of this proposal are to: (1) Sequence and assemble the complete genome of P. triticina isolate 1-1, Race 1 (BBBD), which is estimated to be between 100 - 120 Mb, using a hybrid of 454 and ABI (Sanger) Fosmid-end sequence; (2) Annotate gene structure using computational methods, 200,000 454 reads of ESTs from each of four new cDNA libraries, and other available ESTs; (3) Evaluate P. triticina polymorphism and diversity by comparing the sequenced strain with three additional isolates using Illumina/Solexa sequence; (4) Prompt public release of all reads, assemblies, annotation, and discovered polymorphisms; and (5) Develop education, training and outreach programs. Approach (from AD-416) To achieve our objective we propose to: 1. Generate a whole genome shotgun assembly using an optimized combination of traditional and new sequencing technology data (Sanger and 454). 2. Annotate gene structure using computational methods, 200,000 454 reads of ESTs from each of four new cDNA libraries, and other available ESTs. 3. Evaluate the level of P. triticina DNA polymorphisms and diversity by sequencing three additional isolates of P. triticina using Illumina/Solexa sequence. 4. Prompt public release of all reads, assemblies, annotation, discovered polymorphisms, and EST alignments. 5. Develop education, training and outreach programs A draft genome sequence of the wheat leaf rust fungus, scientific name Puccinia triticina, has been released to the public. Recently, collaborators in the project have been working on refining the quality of the sequence by attempting to sequence large insert DNA libraries. Also, three different races of leaf rust have been sequenced and compared with the draft sequence. The comparison has revealed that there is significant genetic variation within the leaf rust population. To further understand the variation, 56 different races have been identified and the DNA has been isolated. In the next year of this agreement, these 56 races will be sequenced and the DNA compared to try to understand how the races develop and where do they originate. Also, the genome sequence is being used to understand how the fungus infects wheat and causes disease. Three effector proteins, which may trigger resistance reactions to the rust, have been identified and are currently being characterized. Progress on this agreement is monitored by regularly discussing program goals and approaches and by reviewing annual accomplishments reports.
Impacts
(N/A)
Publications
|
Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) The specific objectives of this proposal are to: (1) Sequence and assemble the complete genome of P. triticina isolate 1-1, Race 1 (BBBD), which is estimated to be between 100 - 120 Mb, using a hybrid of 454 and ABI (Sanger) Fosmid-end sequence; (2) Annotate gene structure using computational methods, 200,000 454 reads of ESTs from each of four new cDNA libraries, and other available ESTs; (3) Evaluate P. triticina polymorphism and diversity by comparing the sequenced strain with three additional isolates using Illumina/Solexa sequence; (4) Prompt public release of all reads, assemblies, annotation, and discovered polymorphisms; and (5) Develop education, training and outreach programs. Approach (from AD-416) To achieve our objective we propose to: 1. Generate a whole genome shotgun assembly using an optimized combination of traditional and new sequencing technology data (Sanger and 454). 2. Annotate gene structure using computational methods, 200,000 454 reads of ESTs from each of four new cDNA libraries, and other available ESTs. 3. Evaluate the level of P. triticina DNA polymorphisms and diversity by sequencing three additional isolates of P. triticina using Illumina/Solexa sequence. 4. Prompt public release of all reads, assemblies, annotation, discovered polymorphisms, and EST alignments. 5. Develop education, training and outreach programs The project is to sequence the genome of Race 1 (BBBD) of Puccinia triticina Eriks (Pt). BBBD was first collected in the 1950�s in Canada and is the most avirulent race of leaf rust that has been collected, meaning most leaf rust disease resistance genes in wheat protect against this race. The Genome Sequencing Center at the Broad Institute used next generation Roche 454 sequencing and released an early assembly of the genome in January of 2010. The genome is currently estimated at ~163 Mb with 11,638 recognizable genes. The current assembly is still very fragmented, and in July, the project was granted an extension for more sequencing. An additional library of 8 kb inserts from Race 1 was made and is currently in the que for sequencing. This will allow the computer programs to reduce the fragmentation in the assembly and increase the accuracy. In addition to Race 1, three other races with varying virulence on wheat were sequenced using Illumina sequencing. The sequences from these races will be used to compare races and determine where the genomes are different. Progress on this agreement is monitored by regularly discussing program goals and approaches and by reviewing annual accomplishments reports.
Impacts
(N/A)
Publications
|
|