Cisgenic late blight resistant potato is next step in conventional potato breeding

1. Cisgenic late blight resistant potato is next step in conventional potato breeding Evert Jacobsen Plant Breeding, Plant Sciences Group, Wageningen UR 21.06.2011 Brussel www.cisgenesis.com

2. Overview • Problems of late blight in potato • Problems of conventional potato resistance breeding • Cisgenesis as new conventional breeding method • R-gene cloning and R-gene stacking • Marker-free transformation and resistance • Comparison of safety issues

3. 12 - 15x sprays per season > 50% of all biocides in the Netherlands Environment: 1 424 ton active ingredients per year Costs Fungicides: 60 M€/yr Spraying: 60 M€/yr Losses : 30 M€/yr Total: 150 M€/yr (20 % of farm gate turn over) Sprays against late blight on potato in The Netherlands The problem

4. History: Late blight resistance breeding First half 20th century • Introgression of major R genes from wild species S. demissum(5 out of 11 R-genes have been used on agronomic scale) • quickly broken by the pathogen Second half 20th century • Breeding for field resistance (quantitative, polygenic) • Difficult and progress very slow • Chemical solution • development of resistance by the pathogen • regulatory restrictions and risk of unintended side effects visible in the long run • R genes revisited !!

5. Interspecific bridge crosses, in introgression breeding, takes 50 years Over 50 years ago – Bridge crosses for Phytophthora resistance S. acaule 4x  S. bulbocastanum2x (R genes)  AB hybrid 3x  colchicine doubling AB hybrid 6x  S. phureja 2x  ABP hybrid 4x  S. tuberosum 2x  ABPT material 4x R-gene + linkage-drag First resistant varieties came out, like cvs Toluca and Bionica, all with only 1 R-gene (Rpi-blb2) The Rpi-blb2–gene has already been broken. Stacking is next step

6. R r r r r r r R r r r r r r R R Cisgenesis Introgression breeding Receptor Donor Receptor The big difference between cisgenesis and conven- tional breeding R-gene from crossable plant byAgrobacterium tumefaciens + x Three examples of insertions No linkage drag Receptor F1 Insertion of cisgenic R-gene in 1 step without linkage drag x R r R BC1 R r BCn Linkage drag over 300 genes ... Introgression of R-gene with piece of donor DNA with hundreds of new genes/ alleles in multiple steps R r Quick and single step Slow and multiple steps

7. Unintended effects of introgression breeding • High likeliness of unintended effects • Linkage of R-gene with many other alleles from the wild species coding for altered morphology, agricultural traits, solanines, etc.. • Not all unintended effects can be removed easily by selection and backcrossing. Compensation breeding needed. • Self-monitoring of breeder protects for undesirable traits • Selection for low glycoalkaloid content is the most important health risk factor • Stacking of R-genes is increasing the problem of linkage drag with unintended effects massively

8. Cisgenesis: a new conventional breeding approach • It uses marker free genetic modification with only cisgenic resistance genes • It enables stacking of cisgenic R-genes • It introduces only R-genes (no glycoalkaloids or foreign genes) • Likeliness of unintended effects is very low • Prevention for unintended health effects by self-monitoring for low content glycoalkaloids

9. Screening of 1,000 Solanum accessions Sources of resistance Candidate R-genes

10. Groups of different resistance genes in potato • 24 Rpi-genes isolated • They belong to 8 linkage groups (in different species). Per group, they have the same specificity with Phytophthora isolates 1. Rpi-R1 2. Rpi-R2; -R2-like; -abpt ;–blb3; -mcd1-1; -edn1.1; -snk1.1 and 1.2; -hjt1.1; -hjt1.2; -hjt1.3 3. Rpi-R3a and –R3b 4. Rpi-blb1; -sto1and -pta1 5. Rpi-blb2 6. Rpi-vnt1; -nrs1 7. Rpi-mcq1; -phu1 8. Rpi-chc Dependent on late blight population all classes can contain useful genes for stacking

11. R-gene stacking in potato a triple R-gene vector with Rpi-sto1, -vnt1.1, –blb3 and kanamycin resistance selection marker

12. Relation between kanamycin resistance and resistance of triple R-genes 28 kanamycin resistant transformants were investigated: 23 had triple R-genes and 5 were missing all R-genes All 23 plants showed resistance reaction for all 3 R-genes Functional stacking of R-genes, using kanamycin resistance as selection marker for transformation, works in a very high frequency Does stacking of R-genes works?

13. Marker-free transformation A.tumefacienswithout transgenic selection genes coding for kanamycin or herbicide resistance

14. MF transformation: Relation between PCR selection and resistance of single/double R-genes 1. 37 Rpi-sto1: 20 resistant 2. 33 Rpi-vnt1.1: 26 resistant 3. 13 Rpi-sto1+Rpi-vnt1.1: all 13 resistant for both genes Stacked R-genes are frequently simultaneously biological active Does stacking of R-genes after MF transformation work? Over 50% of MF trans- formants were resistant

15. Cisgenes for resistance to P.infestans (Rpi-blb1)

16. Comparison between conventional introgression breeding and cisgenic insertion breeding Conventional: slow; R-gene stacking enlarges problem of linkage drag, more backcrosses needed; high likeliness of unintended effects; self-monitoring for low glycoalkaloids content; no existing varieties can be improved; value of new variety is determined in the years after release Cisgenic: backbone free plants (linkage drag free) can be selected; stacking without additional linkage drag problems; unintended effects in low frequency; no additional glycoalkaloids introduced; existing varieties with a “history of safe use” can be improved Cisgenic varieties are at least as safe as conventional ones

17. Relativelikelihood of unintended effects associated with various methods of plant breeding including gen. modification In absence of foreign DNA or genes from another species, an argument may be made that a GE plant is not transgenic and not unnatural, if only a native gene is transferred (page 58) At this moment EPA is in USA changing the regulation in order to exempt cisgenesis from GM-regulation National Academy of Sciences, USA, 2004 Approaches to assessing unintended health effects

18. Conclusion Cisgenic plant breeding is next step in conventional plant breeding Cisgenic late blight resistant varieties are at least as safe as conventionally bred potato varieties Unintended effects are minor, far within the base line of normal plant breeding