1. Light response QTL in Arabidopsis thaliana: LIGHT1 cloning Justin Borevitz Ecology & Evolution University of Chicago naturalvariation.org

2. Talk Outline Natural Variation in Light Response QTL Mapping/ fine mapping Gene Expression candidates KO candidates Aquilegia: new model organism Natural Variation in Light Response QTL Mapping/ fine mapping Gene Expression candidates KO candidates Aquilegia: new model organism

4. Light Affects the Entire Plant Life Cycle Light response variation can be seen under constant conditions in the lab Natural Variation under selection? Test in field

7. Quantitative Trait Loci

9. SNP377 SM184 SM50 SM35 SM106 G2395 SNP65 SM40 SEQ8298 TH1 MSAT7964 MAT7787 CER45 5.50 5.87 6.34 7.01 7.30 7.44 7.60 7.79 7.96 8.13 8.29 8.65 9.32 MbMarker Near-Isogenic Lines for LIGHT1 Ler / Cvi mm 81N-J17A-A/J114124189Ler 624333Plants Line RVE7 GI 194 3 5.0 5.8 5.15.95.7 5.8 Phenotype

10. 7.440 7.480 7.500 7.540 7.460 7.520 7.560 7.580 7.600 7.620 7.640 7.660 7.680 7.700 7.720 7.740 7.760 7.780 NIL189 NIL194NIL114 7.540 Mb 7.675 Mb Candidate Interval: 135 Kb 34 predicted genes Original Marker Mb LIGHT1 Fine-mapping (Ler / Cvi)

11. LIGHT1 (Ler / Cvi) in different light environments Ler189 194114Ler189 194114Ler189 194114

12. 50 genes down regulated in NIL189 compared to Ler 5 genes up regulated in NIL189 compared to Ler Gene Expression polymorphism between Ler and NIL189

13. At1g21600 only gene in the LIGHT1 interval that is misexpressed

14. At1g21600 is circadian-regulated

15. No quantitative hypocotyl differences in 22 lines TDNA lines in the LIGHT1 region

16. LIGHT1: At1g21600

17. Tilling: splice junction TDNA: Salk_024431 Ler: F29L Tilling: D103N Tilling Intron; C to T Tilling: A30= At1g21600 an allelic series of artificial mutations

18. At1g21600 is a single copy gene in multiple plant species

19. LF 5’ end of At1g21600 is not well conserved At1g21600 The candidate QTN has mutated several times independantly

20. Natural Varition in At1g21600 3 nt and 1 AA change between Ler and Cvi 10 accessions sequenced for 5’ end of At1g21600 Mt-0 (North Africa) shares AA change with Ler (Poland) Sequenced 96 accessions for At1g21600, LIGHT1 candidate gene Magnus Nordborg

21. At1g21600 AA change is associated with Hypocotyl length and Flowering time Hypocotyl data: P-value Red light + PhyB 20.01828 Red light + PhyB 70.01627 White light + PhyB 70.04189 Blue light + PhyB 10.03489 Flowering time data: LD True Leaf Number + FRI0.01482 LD Days to Flowering + FRI0.004912

22. LIGHT1 (Ler / Cvi) : future projects Further fine-mapping: Screen 4000 NIL189 Het plants Phenotype KO mutants (25 of the 34 candidate genes) Sequence some candidate SNPs across accessions Transgenics (complementation / functional analysis)

23. Seasons in the Growth Chamber Changing Day length Cycle Light Intensity Cycle Light Colors Cycle Temperature

24. Aquilegia (Columbines) Recent adaptive radiation, 350Mb genome

25. > 20k dbEST 11/14/2003 Animal lineage: good coverage Plant lineage: crop plant coverage

26. NSF Genome Complexity 35,000 ESTs 5’ and 3’ 350 arrays, RNA and genotyping –High density SFP Genetic Map Physical Map (BAC tiling path) –Physical assignment of ESTs QTL for pollinator preference –~400 RILs, map abiotic stress –QTL fine mapping/ LD mapping Develop transformation techniques Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus Nordborg (USC) Justin Borevitz (U Chicago) Jeff Tompkins (Clemson)

27. NaturalVariation.org Salk Todd Michael Olivier Loudet Joanne Chory Detlef Weigel Joseph Ecker Scripps Sam Hazen University of Chicago Xu Zhang Evadne Smith UC Davis Julin Maloof University of Chicago Xu Zhang Evadne Smith UC Davis Julin Maloof Salk Todd Michael Olivier Loudet Joanne Chory Detlef Weigel Joseph Ecker Scripps Sam Hazen