1. WHEAT DOUBLED- HAPLOIDS Melanie Caffe-Treml

2. Overview What are doubled-haploids? Applications Methods for wheat doubled-haploids production Speeding-up the development of new winter wheat cultivars for the Prairie Pothole region Summary

3. What are doubled-haploids? Heterozygous Completely homozygous Haploid Completely homozygous lines are developed from heterozygous parents in a single generation

4. × F1F1 F2F2 F3F3 F4F4 F5F5 F6F6 F7F7 F8F8 Frequency of homozygous 50 % 75 % 87.5 % 93.75 % 0 % 96.88 % 98.44 % 99.22 % × DH lines 100% homozygous F1F1 What are doubled-haploids?

5. Applications Development of new inbred cultivars: Selection on additive gene action Absence of dominance variation and segregation Speed-up the development of new lines New lines are highly homogeneous

6. Increased genetic gain Cross desirable parents Evaluate offspring Select highest performers Long term benefit: increased efficiency of “Parent building” From: Barkley, A. and F.G. Chumley. A doubled haploid laboratory for Kansas wheat breeding: An economic analysis of biotechnology option, March 15, 2011.

7. Applications Rapid development of inbred lines for hybrid production QTL mapping In combination with other techniques (e. g. genomic selection, marker assisted selection, mutagenesis, recurrent selection, backcross) www.passel.unl.edu www.extension.org

8. X Line 1Line 2 or Selfing (F 2 ) DH Induction Goal: Fixation of target alleles No. of genes F2F2 DH 10.250.5 20.06250.25 40.0040.0625 80.000020.004 160.000000000020.00002 Probability for Fixation of Target Genes Gene pyramiding Thomas Lübberstedt et al.

9. In: Foster et al. 2007. Trends in Plant Science 12:368-375 Backcross

10. Figure adapted from “Crop genomics: advances and applications,” by Peter L. Morrell, Edward S. Buckler & Jeffrey Ross-Ibarra, 2012, Nature Reviews Genetics 13, 86-96 coloradowheat.org Genomic selection

11. Adapted from Foster et al. 2007. Trends in Plant Science 12:368-375 Methods of haploid production

12. Methods of wheat DH production Anthers culture Dogramaci-Altuntepe et al. 2001. J. Hered. 92 (1): 56-64 Genotype dependent Albinos, chimera, chromosomal abnormalities,…

13. Methods of wheat DH production Isolated microspore culture Zheng M. Y. 2003. Plant Cell. Tissue and Organ Culture 73: 213-230.

14. Wheat × Maize chromosome elimination (Laurie and Bennett, 1986) × Fertilization Cell division and elimination of Maize chromosomes Haploid embryo Pollination 21 chromosomes from wheat + from 10 maize 21 chromosomes from wheat 2n = 20 2n = 42 Methods of wheat DH production

15. Mechanism of chromosome elimination Spatial separation of parental genomes Imperfect segregation of pearl millet chromosomes Formation of micronucleus Hetero- chromatinization, DNA fragmentation and disintegration of micronucleus Budding of pearl millet chromatin Release of pearl millet chromatin-containing micronucleus Germand et al. 2005. Plant Cell 17(9):2431-2438

16. Steps in producing winter wheat DH lines Vernalization Heterozygous “donor” plants Emasculation Pollination Hormone treatment

17. Haploid embryos

18. Embryo rescue

19. Steps in producing winter wheat DH lines Embryo rescue Haploid regeneration Vernalization Transplanting Colchicine treatment Vernalization Heterozygous “donor” plants Emasculation Pollination Hormone treatment

20. Colchicine treatment

21. Chromosome doubling Treatment with colchicine Alkaloid from Colchicum species Interfere with microtubule organization, inhibits spindle formation during mitosis in meristematic cells No separation of sister chromatids at anaphase  Doubled-haploid cell Some doubled-haploid tillers will develop on treated plants

22. Steps in producing winter wheat DH lines Embryo rescue Haploid regeneration Vernalization Transplanting Colchicine treatment Vernalization Heterozygous “donor” plants Emasculation Pollination Hormone treatment Transplanting Doubled- haploid seed harvest Seed increase

23. Speeding-up the development of new winter wheat cultivars for the Prairie Pothole region

24. Winter Cereals: Sustainability in action Ducks Unlimited / Bayer Crop Science “Improving agricultural productivity while maintaining habitat for wildlife” Winter wheat provides a better nesting habitat for waterfowl than spring sown crops www.wintercereals.us

25. WCSIA - SDSU Rapidly develop winter wheat variety with high yield, improved winter hardiness and disease resistance Use of doubled- haploids

26. Improvement to the methods SeasonClass Avg Nb embryos/ 100 florets % embryo with contamination Nb of haploids/ 100 embryos % of plants dead after transplanting % of plants successfully doubled 1HRW..14.0.43 2HRW15.721.866.663 37 (66 in GC – 28.8 in GH) 3HRS16.2710.266.139 ~ 37.3 (85.6 in GC – 23.5 in GH) 4HRW20.714.9607.358 5HRS16.27.756.5 1532 6HRW16.14.776.6 1234

27. Rootrainers Increased survival Space saving Saving on soil Saving on time

28. Barcode scanner Better record keeping Time saving

29. Data recording

30. DH production Season SpikesEmbryo Haploid plantlet Colchicine treated plant DH 1 45510731505122 2 66118331267441164 3 409226513581168678 4 769338825932173738 5 6551427 *** Total29499986536838331602 * In progress

31. Field evaluations 62 DH lines  yield plots in Aurora, SD + scab nursery 443 DH lines  rows in Aurora, SD 2014 season 2015 season 1097 DH lines  planting in Fall 2014

32. DH and overall breeding program StageActivityNumber of entries Crosses Two crossing cycles per year (September-December + February-June) 500 to 800 F1Increase in Arizona winter nursery or greenhouse500 to 800 F2Field plot evaluation (1rep, 1 location)500 to 800 F3Field plot evaluation (1 rep, 2 locations)300 to 600 F3:4Headrow field evaluation15,000 to 20,000 Early Yield Trial (EYT)Field evaluation (1rep, 1 location)500 to 1500 Preliminary Yield Trial (PYT) Field evaluation (2 reps, 5 locations) Screening in FHB inoculated nursery and DON testing Disease evaluation (mostly LR and SR) in Texas Quality testing SDSU lab 90 Advanced Yield Trial (AYT) Field evaluation (3 reps, 5 locations) Screening in FHB inoculated nursery and DON testing Disease evaluation (mostly LR and SR) in Texas Screening in WSMV nursery Quality testing SDSU lab Quality testing Kansas Quality Lab 35 Crop Performance Trial (CPT) Field evaluation (4 reps, 10 to 15 locations) Screening in FHB inoculated nursery and DON testing Disease evaluation (mostly LR and SR) in Texas Screening in WSMV nursery Quality testing SDSU lab Quality testing Kansas Quality Lab 8 to 10 DH Lines Parent for new crosses Increased genetic gain × n

33. Summary  Key is to get DH production efficiency to a high level to adequately sample initial genetic variation  Must have dedicated, full-time personnel and adequate facilities for DH production  As a breeding "tool", its biggest benefits are long- term, e.g. parent building, improving effectiveness of selection, hastening variety release over years

34. Acknowledgements Bill Berzonsky James Prus Robert Graf Steve Kalsbeck Jose Gonzalez Rodrigo Dos Santos Rick Geppert All the undergraduate students who have helped with the DH work Ducks Unlimited Bayer Crop Science

35. QUESTIONS ?