1. Need for a Public Doubled Haploid Facility ? Thomas Lübberstedt, Candy Gardner, Mike Blanco, Uschi Frei, Elisabeth Bovenmyer

2. Structure - DH Production in Maize: How ? - Why (Doubled) Haploids ? - Who uses DHs ? - Need for a Public DH Facility ?

3. 1)Haploid production - In vitro versus in vivo - Maternal versus Paternal 2) Haploid identification -Markers: Morphological or molecular - Cytological / flowcytometry 3) Genome doubling - Colchicine - Other Technologies

4. In vitro: Anther Culture

5. In vivo Haploid Induction F1F1 X,, F2F2,, X Maternal Inducer +,, + X Paternal Inducer (ig)

6. Workflow & Bottlenecks Maternal DH Induction StepChallenge Induction cross- Inducer availability - Induction rate Haploid (embryo) selection- Visual scoring - Background effects - Inducer DNA introgression ? Genome doubling- Toxic Colchicine - Alternative procedures - Background effects Selfing of double haploids- Chimera -> partial fertility

7. Identification of Maternal Haploids Endosperm Embryo H embryo F 1 embryo Lethal Outcrossed or self-pollinated DonorInducer Geiger 2009

8. Comparison DH Approaches in Maize ApproachStrengthWeakness In vitro- No need of inducer- Low induction rate - Genotype dependency - Need of tissue culture In vivo – paternal- Simple inheritance - cms conversion - Low induction rate (2%) In vivo – maternal-Limited genotype dependency - Induction rate (10%) - Background effects -Complex inheritance

9. Why (Doubled) Haploids ?

10. Speed in Line Development Founder line 1 xFounder line 2 F1F1 Selfing Inbred line DH Inbred line

11. Haploid Induction & Doubling of Allelic Diversity Materials Andrew Smelser et al., Plant Introduction Station, Ames

12. QTL MAPPING Type of Population StrengthWeakness F 2:3 - Speed of production - d and a estimates - Heterogeneous families RIL - Homogeneous families - Power of QTL detection - Slow production DH - Speed of producing homogeneous families - Power of QTL detection - Laborious production process - Lower recombination (>RIL) BC - Speed of production- Heterogeneous families - a and d confounded

13. Trait / Gene Stacking X Line 1Line 2 or Selfing (F 2 ) DH Induction Goal: Fixation of target alleles No. of genesF2F2 DH 10.250.5 20.06250.25 40.0040.0625 80.000020.004 160.000000000020.00002 Probability for Fixation of Target Genes

14. Usefulness in Line Development Seitz 2005

15. Rapid Line & Population Improvement Gordillo & Geiger 2008

16. Conclusions - Homozygous lines are obtained in short time - Maximal genetic variation among lines: increased selection gain - Reduces population size for gene pyramiding substantially - Increases power for QTL or QTP detection - Increases labor for line development – depending on technology

17. Who uses DHs ?

18. DH Application in Plant Breeding Maize: All major breeding companies (in vivo DH induction) Tuvesson et al. 2007, EU-COST Other species (Tuvesson et al.): Rye, Oat, Triticale, Potato, Cabbage

19. Need for Public DH Facility ?

20. Pro and Contra Public DH Facility (Maize) Contra: - Inducers are available in maize - Procedures are published -> Everyone can do it Pro: - Users might need DH materials only once or once in a while - Induction procedure requires know how (analogy to transformation) - Technology advance might make procedure more accessible - Possible platform for expansion into other species => Research opportunities in maize and other species And: Training of future plant breeders !! Technology & Application of DHs

21. Technological Challenges Colored Seed Smelser et al. 2009 Partial Fertile Questions relating to inducer: - Alternative/improved marker systems - Increased induction rate - Adaptation to US climate / photoperiod - Genetics / biology of induction mechanism Questions relating to genome doubling: - Genetic background effect ? - Optimized procedure (with colchicine) - Procedure without colchicine ? - Increased doubling / success rate

22. Conclusion Successful example of research oriented service facility at ISU: http://www.agron.iastate.edu/ptf/index.aspx New DH facility at ISU will soon be launched, if interested contact: Thomas Lübberstedt 515-294-5356 THOMASL@IASTATE.EDU Collaborators: Candy Gardner, Mike Blanco, Uschi Frei

23. Costs ? ServiceOn Campus FeeOff Campus Fee a)Haploid Seed Induction a)Selected Haploid Seed a)Doubled Haploid Seed Production (5-20 kernels) a)Doubled Haploid Line (200 kernels) - Summer / Winter pricing - GM seed ?