Roger WiseRoger Wise

USDA-ARS / Iowa State UniversityUSDA-ARS / Iowa State Universityhttp://wiselab.org/ http://plexdb.org/

http://barleygenome.org/

Genetical genomics of Genetical genomics of Puccinia graminisPuccinia graminis TTKS TTKS

response: response: A systems biology approach A systems biology approach

to rapid development of to rapid development of durable resistance for barley durable resistance for barley

and wheatand wheat

Gene Expression and QTLsGene Expression and QTLs

The treatment of transcript The treatment of transcript accumulation as a Quantitative Trait is accumulation as a Quantitative Trait is called eQTL mapping. Loci that regulate called eQTL mapping. Loci that regulate expression of genes or networks can be:expression of genes or networks can be:

-cis-cis-acting QTL-acting QTL-trans-trans-acting QTL-acting QTL

RequirementsRequirements

• Experimental population that harbors genetic Experimental population that harbors genetic

variation for resistance to the pathogen variation for resistance to the pathogen

[QSM DH (Q21861 x SM89010)][QSM DH (Q21861 x SM89010)]

• Global gene expression profiling platform Global gene expression profiling platform

(Affymetrix Barley1 GeneChip)(Affymetrix Barley1 GeneChip)

• High-throughput genotyping platform High-throughput genotyping platform

(QSM (QSM SFPs detected by Barley1 GeneChip)SFPs detected by Barley1 GeneChip)

• Database Database (BarleyBase/PLEXdb)(BarleyBase/PLEXdb)

Scoring of TTKS on QSM DH mapping Scoring of TTKS on QSM DH mapping populationpopulation

0;0; 1212 2121 33--210;210; 33++

Brian SteffensonBrian Steffenson

Barley1 GeneChipBarley1 GeneChip 22,840 Probe Sets 22,840 Probe Sets

((22,792 + controls22,792 + controls))

Perfect Match 25-merMismatch 25-mer600 bp5´ 3´AAAAAAAAAAAATGTAGGene SequenceClose Close et al.et al. 2004, Pl Physiology 134: 960-968 2004, Pl Physiology 134: 960-968

Single feature Polymorphisms (Steptoe and Morex)

Raw Data:Steptoe

Morex

Robbie WaughRobbie Waugh

22,000 Genes X11 Oligos X 25nt = 6,325,000 bases

[ >4,000 SFPs ]

Contig3660_atContig3660_at

Expression Level Polymorphism

Matt MoscouMatt 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 ofseedlings is hybridizedto its own Barley1GeneChip.

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 AnalysisData Analysis

• Identify subset of genes that show Identify subset of genes that show differential expression between TTKS-differential expression between TTKS-inoculated and mock-inoculated leaves. inoculated and mock-inoculated leaves.

• Map the eQTL that regulate the expression Map the eQTL that regulate the expression of these genes.of these genes.

• Identify regulators by hierarchical analyses.Identify regulators by hierarchical analyses.

• A locus that regulates one or more A locus that regulates one or more significant disease resistance networks is an significant disease resistance networks is an obvious target for immediate use in obvious target for immediate use in breeding. breeding.

ExpectationsExpectations

1. 4,000-8,000 SFP gene markers positioned on the barley population segregating for TTKS resistance; enables QTL mapping of TTKS resistance.

2. Marker-delineated positions of the eQTL that regulate networks of disease response genes; breeders can incorporate into their germplasm.

3. Elucidation of gene expression networks responsive to TTKS infection; provides biochemical targets for further analysis.

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

http://www.plexdb.org

1000 Barley1 Hybs Uploaded1000 Barley1 Hybs Uploaded

USDA-ARS/ISUUSDA-ARS/ISURico Caldo Rico Caldo

Matt MoscouMatt MoscouGreg FuerstGreg FuerstNick LauterNick Lauter

Jose RodriguezJose RodriguezYan MengYan Meng

Pingsha Hu Pingsha Hu Liu XiLiu Xi

Dennis Halterman Dennis Halterman Karin WernerKarin Werner

Brent KronmillerBrent Kronmiller

BarleyBase/PLEXdbBarleyBase/PLEXdbJulie DickersonJulie DickersonEthy CannonEthy Cannon

Sudhansu DashSudhansu DashLu HongLu Hong

Lishuang ShenLishuang ShenDan NettletonDan Nettleton

USDA-ARS/UMNUSDA-ARS/UMN Yue JinYue Jin

Sam GaleSam GaleLes Szabo Les Szabo

Univ. of MinnesotaUniv. of MinnesotaBrian SteffensonBrian SteffensonStephanie DahlStephanie Dahl

Knowledge For Tomorrow’s Solutions

http://barleygenome.org

http://barleycap.org