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Jiang Lab Role

Jiang Lab Role. Gene expression profiling of key potato biology (polyploidy, tuberization, late blight resistance) Provide services to the project for potato transformation and greenhouse late blight resistance evaluation. Gene Expression Profiling Progress. Polyploidy (20 Arrays)

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Jiang Lab Role

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  1. Jiang Lab Role • Gene expression profiling of key potato biology (polyploidy, tuberization, late blight resistance) • Provide services to the project for potato transformation and greenhouse late blight resistance evaluation

  2. Gene Expression Profiling Progress • Polyploidy (20 Arrays) • 12 arrays completed on leaflets • 8 arrays completed on root tips • Comparative profiling using cDNA-AFLP • Tuberization (22 Arrays) • 10 arrays completed on in vitro tuber development • 12 arrays completed on in vivo tuber development • Patatin gene family expression profiling • Late blight resistance (24 arrays) • 24 arrays completed on transgenic RB lines

  3. Transformation and Resistance Evaluation Potato Transformation Transgenic RB lines in Russet Burbank, Superior, Dark Red Norland Constructs from the Baker lab (VF36 with GSS-4 and GSS-5) Five constructs from the Jahn lab RNAi constructs for RPR and SGT, Two RB-tagging lines Late Blight Resistance Evaluation 635 clones, 2163 plants, in 29 inoculation experiments

  4. Publications from Jiang Lab Colton, L.M., Groza, H.I., Wielgus, S.M., and Jiang, J. (2006) Marker-assisted selection for the broad-spectrum potato late blight resistance conferred by gene RB derived from a wild potato species. Crop Sci.46: 589-594. Stupar, R.M., Beaubien, K.A., Jin, W., Song, J., Lee, M.-K., Wu, C., Zhang, H.-B., Han, B. and Jiang, J. (2006) Structural diversity and differential transcription of the patatin multicopy gene family during potato tuber development. Genetics (February issue) Stupar et al. (2006) Phenotypic and gene expression changes associated with autopolyploidization. To be submitted to Plant Cell within ~2 months.

  5. Ploidy and Gene Expression

  6. Potato Autopolyploidy series Wild diploid: AA(24 chromo.; Heterozygous) Anther culture: A(12 chromo.) Leaf disc: AA AAAA (24 chromo.)(48 chromo.) Homozygous Homozygous

  7. 1x 2xR3 2xR5 4x Series P77

  8. 1x 2xR3 2xR5 4x

  9. P77 Monomorphism M 1x 2xR3 2xR5 4x T B 1x 2xR3 2xR5 4x T B M

  10. 1x 2xR3 2xR5 4x

  11. Array experimental design Biological Rep 1 Biological Rep2 Biological Rep3 1x 2xR3 2xR5 4x 1x 2xR3 2xR5 4x 1x 2xR3 2xR5 4x

  12. 4x-1x; 2x-1x; 4x-2x 10 genes A B 2 genes 1x-4x; 1x-2x; 2x-4x 4x-1x 135 genes Ploidy- Upregulated Log(2) Expression Profiles Ploidy- Downregulated Log(2) Expression Profiles 155 genes 1x-4x 4x-1x; 2x-1x 248 genes 127 genes 1x-4x; 1x-2x 4x-1x; 4x-2x 22 genes 23 genes 1x-4x; 2x-4x Ploidy Ploidy

  13. Leaf ploidy array summary • ~10% of all genes showed significant expression changes over ploidy levels • ~50% of ribosomal protein genes showed significant expression changes over ploidy levels • ~75% of histone genes showed significant expression changes over ploidy levels

  14. Real-time PCR on selected genes 3 2 1 Fold Change Ploidy: 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 JM09 CZ25 CF69 IR76 CenH3 EW61 GF62 IO91 IZ46 DE81 CV58 IS17 Ribosomal protein genes Histone genes Cyclin Downregulated

  15. Conclusions • Ploidy affects plant growth rates and cell size • Gene families that change with ploidy • Ribosomal protein genes, histones, cyclins • Important for “cellular infrastructure” • “Ploidy genes” are context-dependent • Autopolyploid vs. Allopolyploid • We find much more subtle expression changes in autopolyploids than in allopolyploids

  16. RB-Associated Gene Expression 10 arrays were completed in 2005 to test the timing of transcriptome changes after late blight inoculation 24 arrays completed in the last three weeks for final profiling

  17. “Super-resistant” transgenic RB clone The late blight resistance phenotypes of two transgenic Katahdin lines under an intensive inoculation conditions. Left Panel: Left: A transgenic Katahdin clone containing a single copy of the RB gene; Middle: S. bulbocastanum clone PT29; Right: Katahdin control. The transgenic Katahdin plant, which is resistant to late blight under regular inoculation condition, shows a susceptible phenotype. Right Panel: Left: A transgenic Katahdin clone containing multiple copies of the RB gene; Middle: S. bulbocastanum clone PT29; Right: Katahdin control. In Southern hybridization, DNA from the transgenic plants was cut with HindIII (left lane) and EcoR1 (right lane) and hybridized with a vector probe

  18. Schematic of RNA pooling and microarray hybridization: Four identical plants from each of the three Katahdin plants (control Katahdin, Katahdin containing one RB gene, Katahdin containing multiple copies of RB gene) are sampled under each of the three time points (2, 5, and 10 hours after inoculation). Eight RNA samples will be isolated from these four plants. Challenged and unchallenged RNA samples from two plants will be pooled, amplified and labeled. The challenged aRNA from the first pair of plants is labeled with Cy5 and the unchallenged aRNA labeled with Cy3. Alternately, the challenged aRNA in the second pair of plants is labeled with Cy3 and the unchallenged with Cy5. Both labeled samples are mixed from each pair of plants and hybridized to the array. This procedure creates a dye-swap with two biological replicates.

  19. (A) Filtered data from Preliminary Array Experiment 1 is shown colored by the normalized intensity ratio (log transformed) along the vertical axis. The horizontal axis represents gene expression occurring 5, 15 and 25 hours post inoculation. (B) Filtered data from Preliminary Array Experiment 2 is shown colored by the normalized intensity ratio (log transformed) along the vertical axis. The horizontal axis represents gene expression occurring 2, 5 and 10 hours post inoculation

  20. Coming soon: Final results on gene expression profiling on RB-mediated late blight resistance pathway

  21. Acknowledgments Richard Veilleux Robin Buell Amy Hart Willem Rensink Brian Yandell National Science Foundation

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