Location: Crop Improvement and Genetics Research2010 Annual Report
1a. Objectives (from AD-416)
The objective of this cooperative research project is to develop methodologies to explore large and repetitive plant genomes. The initial focus will be on comparisons of the diploid wild potato (Solanum bulbocastanum) with the tetraploid cultivated potato (S. tuberosum). The haploid genomes of each are estimated to be ~900 Mbp.
1b. Approach (from AD-416)
Genomic sequence information will be generated with Ilumina’s next generation sequencing technologies and analyzed in silico to identify potential subsequences to use as molecular markers and identify genes and sequences of interest for applied agronomic improvement. Assembled contigs will also be used to compare wild and domestic potato. This comparison may result in the development of single nucleotide polymorphisms (SNPs) that can be used as molecular markers by breeders. Identification of selected subsequences and novel genes will enable their isolation. Wet lab testing will be used to validate the utility of these subsequences and should enable the development of sequences that can be applied to the molecular genetic improvement of potato. Using standard Agrobacterium-mediated transformation, appropriate transgenes vectors will be constructed and the sequences mobilized into potatoes. Resulting transgenic lines will be examined for function, efficacy and to demonstrate proof of principle. We anticipate that this novel approach, if successful, will be applicable to the improvement of other plant species. Documents Non Funded Cooperative Agreement with University of Houston.
3. Progress Report
The agreement was established in support of Objective 3 of the in-house project, to develop tools to refine down-regulation technologies for metabolic manipulation as well as improve design characteristics of pathogen-resistance transgenes. The goal of this project is to use next generation sequencing to understand and elucidate large and complex potato genomes and use this information to analyze and develop tools to refine down-regulation technologies for metabolic manipulation as well as improve design characteristics of pathogen-resistance transgenes. The wild potato relative (Solanum bulbocastanum) is the ongoing subject of next generation sequencing with an Illumina Genome analyzer and to date 3.4 Gigabytes of sequence, or roughly 3x genome coverage, has been obtained for analysis. Preliminary assemblies have been mostly limited to high copy plastid and mitochondrial genomes, and sequence information will be analyzed further upon the release of the draft potato genomes (S. phureja and S. tuberosum) from the Plant Genome Sequencing Consortium. Using Next generation sequencing, small RNAs (sRNA) of potato leaves have been sequenced for two varieties of cultivated potatoes (controls) and for four transgenic lines containing inverted hairpin structures designed to produce small inhibitory RNA (siRNA) constructs directed towards the potato virus Y (PVY). Some of the transgenic lines showing positive phenotypes for disease resistance lines were successfully analyzed and demonstrated to have unique and strong siRNA production associated with viral resistance. Participants in this project have monitored the research through exchange of data, electronic communications, phone calls, and personal meetings.