Design your genome! How to exchange chromosomes and organelles between Arabidopsis ecotypes. (and why we d like to do so) Erik Wijnker International Conference on New Plant Breeding Molecular Technologies 9 &10 October, Jaipur, India
Content: Generating cybrids Exchanging cytoplasm Reverse breeding Exchanging chromosomes Applications
The GFP-tailswap haploid inducer GFP CENH3
The GFP-tailswap haploid inducer GFP CENH3
Generating cybrids with GFP-tailswap X Heterozygote Haploid
Generating cybrids with GFP-tailswap X Heterozygote Haploid Doubled Haploid
Cybrids show new phenotypes 0.8 Nuclear DNA Col-0 Ler-0 Ws-4 Bur-0 Ely Shah C24 0.7 0.6 Col-0 Ler-0 Ws-4 Bur-0 Ely Shah C24 Photosynthesis 0.5 0.4 Mark Aarts 0.3 Jeremy Harbinson 0.2 Joost Keurentjes 0.1 0 7 9 11 13 15 17 19 21 23 Accessions 300 chosen for high or low rates of photosynthesis 250 +78% Plant size Padraic Flood 400 350 200 150 100 50 Ely WT Plant age Ely with Col cytoplasm 0 7 9 11 13 15 17 19 21 23 Plant age
Generating inducer lines with different cytoplasms X Heterozygote An inducer in a new cytoplasm!
A complete cybrid testpanel Col Ler WS-4 Bur Ely Sha C24 Col Ler WS-4 Bur Ely Sha C24
Generating cybrids: Cytoplasm in Arabidopsis inherits maternally Cybrids arise through maternal genome elimination The maternal nuclear genome eliminates The maternal organellar genome remains
Content: Generating cybrids Exchanging cytoplasm Reverse breeding Exchanging chromosomes Applications
About selection in breeding: X Be a breeder in the... selection quiz! This is a F1 (a hybrid) Can What you is the identify nicest its corncob? parents?
Two crucial observations: X 1) Offspring selection: easy! 2) Parent selection: very difficult!
X Breeding with complex heterozygotes is very difficult
and thus plant breeders generate as much random variation as possible? no!
Classical vs reverse breeding X Classical breeding Selection Reverse Breeding
Crossover suppression RNAi:DMC1
Segregation of non-recombinant chromosomes
Doubled haploid production
Parent selection
Hybrid recreation x x x Erik Wijnker 2011
Reverse breeding = Exchanging chromosomes 1) Crossover suppression Non-recombinant chromosomes segregate 2) Regenerate balanced spores as doubled haploids These are chromosome substitution lines These are parental pairs Erik Wijnker 2011
Reverse breeding Kees van Dun Cilia Lelivelt Bastiaan de Snoo Rob Dirks Joost Keurentjes Shima Naharudin Hans de Jong Maruthachalam Ravi Simon Chan Dirks et al. (2009). Plant Biotechnology Journal 7(9):837-845 (review) Wijnker et al. (2012): Nature Genetics 44(4):467-470 Wijnker et al. (2014): Nature protocols 9: 761 772 Erik Wijnker 2011
Content: Generating cybrids Exchanging cytoplasm Reverse breeding Exchanging chromosomes Applications Erik Wijnker 2011
The big questions in heterosis What are the contributions of: research: Cytoplasm effects Maternal and Paternal effects DNA Methylation, small RNA s and Gene Expression
A Chromosome Substitution Library
Isogenic hybrids x x x x x x x x x x x x x x x x
A CSL in Columbia cytoplasm
A CSL in Landsberg cytoplasm
A CSL in any cytoplasm
Erik Wijnker Group of Arp Schnittger, University of Hamburg, Germany Erik.wijnker@wur.nl Papers on Reverse Breeding: Dirks et al. (2009). Plant Biotechnology Journal 7(9):837-845 (review) Wijnker et al. (2012): Nature Genetics 44(4):467-470 Wijnker et al. (2014): Nature protocols 9: 761 772
On residual crossovers: Meiotic aberrations (laggards, polyads) increase strongly when crossovers are completely absent. For efficient crossover suppression few crossovers may be allowed. Dirks et al., 2009: Plant biotechnology journal 7(9):837-845
Reverse breeding generates transgene free offspring Transformants Possible DH s X Recruit parents without RNAi-construct
Columbia wt On what chromosome is the locus for early flowering?
Columbia wt Epistatic interactions:
Columbia wt Epistatic interactions:
Epistatic interactions: