1. Breeding and Genetics Tools Dr. Brent Hulke Research Geneticist

2. Tools available/under development 1.Germplasm Resources 2.SNPs/genomic resources 3.Doubled Haploid 4.Evolution of the term “conventional breeding”

3. Germplasm Resources Wild species –Perennial species – “introgressions” by C.C.Jan –Annual species that are not H. annuus – “introgressions” by Seiler and Qi –Helianthus annuus itself and post- introgression taming – crosses by Hulke

4. Germplasm Resources Diverse breeding programs –Multiple independent programs in Europe, South America, USA –Landraces of confection, in particular –Open Pollinated Varieties available in Plant Introduction Station collection in Ames

5. Germplasm Resources The hybrid breeding system itself –Advantages: faster improvement because you are improving “female” and “male” lines simultaneously and with respect to each other Take advantage of hybrid vigor that doesn’t exist in pure line varieties (soybean, wheat, barley, beans) –Disadvantages: “perfect” flowers make it difficult to make test hybrids and requires development of male sterile lines – extensive backcrossing – really expensive

6. Genomic Resources / SNPs SNP = single nucleotide polymorphism –About 7500 quality SNPs available today Arabidopsis thaliana EIN2 (Ethylene Insensitive 2) homologus gene sequences in cultivated Helianthus annuus lines SNP

7. Genomic Resources / SNPs Traditional, 2 parent mapping – cross two distantly related lines different for a trait of interest, genotype and phenotype population, run analysis – takes ~ 2 years Association mapping – No crosses made, use phenotypes and genotypes from hundreds of lines from diverse backgrounds – takes months to years Genomic selection – No special crosses made, just use existing breeding lines and phenotypes – genotype with many markers and make model

8. Genomic Resources Single gene –Disease resistance Downy mildew Sunflower rust Verticillium wilt –Herbicide resistance –High oleic –Insect resistance??? Many genes –Disease resistance Phomopsis Sclerotinia stalk rot Sclerotinia head rot –Insect resistance??? –Yield –Oil content –Confection v. Oilseed Single gene traits and Many gene (quantitative) traits require different applications of SNP technology

9. Doubled Haploid 4 techniques –Anther culture –Microspore culture from anthers –Foreign species pollen as inducer –Mutant stock pollen/ovule as inducer None have been fully successful / proven to date

10. Doubled Haploid Anther Culture –Proper balance of chemical inducers, generation of callus from haploid pollen tissue, and regeneration of embryoids from callus is under investigation –We are able to get callus development at low- moderate levels (best in interspecific hybrids) –Root and shoot formation has not resulted from this callus, which will require additional experiments to overcome

11. Doubled Haploid Microspore culture –Callus derived from individual pollen grains instead of whole anthers –Culture conditions currently still being optimized

12. Doubled Haploid Foreign pollen –Seeds of potential non-Helianthus annuus species identified Criteria: high pollen production, ease of cultivation to harvest the pollen –A X B line crosses were made of confection and oilseed backgrounds to make a sterile F1 hybrid –Hybrids will be pollinated with foreign pollen in two months, seeds collected in three months, and seedlings germinated four months from now and assessed for haploidy.

13. Doubled Haploid Development of H. annuus genetic stock with haploid inducing ability –Novel mutations will be induced in sunflower this summer in our nursery –Winter nurseries will be used to derive mutant lines for testing –Lines will be evaluated for spontaneous development of haploid progenies –Could produce both maternal and paternal haploids (could be used for “instant CMS” and DH population development) – similar to Stock 6 in corn

14. Doubled Haploid Goals: –Reduce line development time from 5-7 years to under 2 years –Improve genetic purity of lines and hybrids –Provide instantly “true breeding” material to select optimum progreny using SNPs –Make development of “female” inbreds a single cross operation

15. “Conventional” Breeding With new technology, definitions will change, as well as abilities Genomic prediction and marker based selection based on markers will define what we actually advance and put in yield trials Doubled haploid will allow us to test new hybrids without years of inbreeding/backcrossing to CMS Additional germplasm resources from other species may provide new genes to exploit without transformation – not common in other crops