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.
A major component of the in-house project is to develop novel molecular tools to improve potato quality and disease resistance properties. Zebra Chip is an important emerging disease of potatoes in the United States caused by the vascular infection of potato plants with Candidatus Liberibacter solanacearum which is spread by a psyllid (Bactericera cockerelli Sulc ) vector. Potato plants either asymptomatic or infected with the Candidatus as well as the psyllid vector with and without the Candidatus were analyzed by high throughput sequencing, a relatively recent advance in the way genetic information is collected. This data was sufficient to verify the presence of pathogen sequences in the psyllid vector by comparison to a library of pathogens. Pathogen sequences were also detectable in the infected potatoes but only when mapping reads for the specific presence of the causal pathogen of the Zebra Chip disease.
Construction of antiviral transgenes with enhanced performance is one of the objectives of the in-house project. Transgenic lines containing inverted hairpin structures designed to produce small inhibitory RNA (siRNA) constructs directed towards the potato virus Y (PVY) exhibited resistance to PVY in both glass house studies in Idaho and field studies in New York. Using next generation sequencing, small RNAs (sRNA) of potato leaves were sequenced for two control varieties and four transgenic lines. The viral resistance phenotype was associated with strong siRNA production.