Roger Wise USDA-ARS / Iowa State University Genetical genomics of Puccinia graminis TTKS response: A systems biology approach to rapid development of durable resistance for barley and wheat Genetical genomics of Puccinia graminis TTKS response: A systems biology approach to rapid development of durable resistance for barley and wheat

Gene Expression and QTLs The treatment of transcript accumulation as a Quantitative Trait is called eQTL mapping. Loci that regulate expression of genes or networks can be: -cis-acting QTL -trans-acting QTL

Requirements Experimental population that harbors genetic variation for resistance to the pathogenExperimental population that harbors genetic variation for resistance to the pathogen [QSM DH (Q21861 x SM89010)] [QSM DH (Q21861 x SM89010)] Global gene expression profiling platformGlobal gene expression profiling platform (Affymetrix Barley1 GeneChip) (Affymetrix Barley1 GeneChip) High-throughput genotyping platformHigh-throughput genotyping platform (QSM SFPs detected by Barley1 GeneChip) (QSM SFPs detected by Barley1 GeneChip) Database (BarleyBase/PLEXdb)Database (BarleyBase/PLEXdb)

Scoring of TTKS on QSM DH mapping population 0; ; Brian Steffenson

Barley1 GeneChip 22,840 Probe Sets (22,792 + controls) Close et al. 2004, Pl Physiology 134:

Single feature Polymorphisms (Steptoe and Morex) Raw Data: Steptoe Morex Robbie Waugh 22,000 Genes X 11 Oligos X 25nt = 6,325,000 bases [ >4,000 SFPs ]

Contig3660_at Expression Level Polymorphism Matt Moscou

Experimental Design Each flat contains up to 98 cones of (6) seedlings randomly arranged among reps and inoc & mock- inoc (Harvested 24 hours after inoculation). RNA from each cone of seedlings is hybridized to its own Barley1 GeneChip. Diversity Set (12 lines) Red Trays- Inoculated Blue Trays- Mock-inoculated Dew Chamber [79 DH lines + parents] x 2 inoc. treatments x 2 reps = 340 randomized samples QSM DH population (79 lines) 12 diversity lines x 2 inoc. treatments x 6 reps [= 144 randomized samples]

Data Analysis Identify subset of genes that show differential expression between TTKS-inoculated and mock- inoculated leaves. Identify subset of genes that show differential expression between TTKS-inoculated and mock- inoculated leaves. Map the eQTL that regulate the expression of these genes. Map the eQTL that regulate the expression of these genes. Identify regulators by hierarchical analyses. Identify regulators by hierarchical analyses. A locus that regulates one or more significant disease resistance networks is an obvious target for immediate use in breeding. A locus that regulates one or more significant disease resistance networks is an obvious target for immediate use in breeding.

Expectations ,000-8,000 SFP gene markers positioned on the barley population segregating for TTKS resistance; enables QTL mapping of TTKS resistance Marker-delineated positions of the eQTL that regulate networks of disease response genes; breeders can incorporate into their germplasm Elucidation of gene expression networks responsive to TTKS infection; provides biochemical targets for further analysis Genes that possess transcriptional activation elements that allow them to respond more aggressively to TTKS infection; provides key regulators for disease response.

What do we do with all that data? (65 Mb/chip = > 30 GigaBytes)

1000 Barley1 Hybs Uploaded

USDA-ARS/ISU Rico Caldo Matt Moscou Greg Fuerst Nick Lauter Jose Rodriguez Yan Meng Pingsha Hu Liu Xi Dennis Halterman Karin Werner Brent Kronmiller BarleyBase/PLEXdb Julie Dickerson Ethy Cannon Sudhansu Dash Lu Hong Lishuang Shen Dan Nettleton USDA-ARS/UMN Yue Jin Sam Gale Les Szabo Univ. of Minnesota Brian Steffenson Stephanie Dahl Knowledge For Tomorrow’s Solutions