Location: Hard Winter Wheat Genetics Research2012 Annual Report
1a. 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.
1b. 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
3. Progress Report:
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.