The biology of naturally occurring insertions

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1 The biology of naturally occurring insertions Justin Vaughn, PhD Soybean Breeding and Molecular Genetics Laboratory University of Georgia, Athens 14 April 2014 Paris, France Titan Lane Valencia Morrell Woodruff

2 Plants do not like to be eaten. Seeds hate to be eaten.

3 Type I Type II Cucumber Sorghum Maize

4 A sample from the treasury of stable cultivars Bartlett Cabernet sauvignon Ogliarola Silver queen Russet Burbank Gros Michel

5 Of crop improvement strategies, genetic engineering should be the least likely to affect cultivar stability.

6 Strategies for crop improvement Cultivar x Cultivar Cultivar x Exotic Cultivar mutagenesis Genetically engineered cultivar (also clonal mutant or sport )

7 How are novel sequences created in natural and mutagenized populations? Cultivar x Cultivar Cultivar x Exotic Cultivar mutagenesis Genetically engineered cultivar

8 1,000,000 (1 MB) nucleotides compared; rice genome is 430 MB long

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10 Double-strand breaks can create large insertions but do not appear to activate transposable elements Enzyme-containing parent Site-containing parent

11 Induced double strand break repair creates complex insertions Soo-jin Kwon, unpublished

12 double-strand break ligation

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16 Progeny are phenotypically indistinguishable from parents Enzyme-containing parent Site-containing parent

17 Unanticipated function of the introduced sequence product Uncertainty about the direct impact on toxicity or nutrition In the absence of functional characterization Characterized function Feeding study / History of safe use Source: Another cultivar Exotic relative Transgenic or Synthetic Divergence from cultivar

18 Unanticipated function of the introduced sequence product Uncertainty about the direct impact on toxicity or nutrition In the absence of functional characterization Feeding study / History of safe use Source: Another cultivar Exotic relative Transgenic or Synthetic Divergence from cultivar

19 Unanticipated function of the introduced sequence product: background interaction? Interaction with genetic background? Another cultivar Exotic relative Transgenic or Synthetic Divergence from cultivar

20 How much novel sequence is expected to be introduced into the cultivar? Cultivar x Cultivar Cultivar x Exotic Cultivar mutagenesis Genetically engineered cultivar

21 How much novel sequence is expected to be introduced into the cultivar? Cultivar x Cultivar Cultivar x Exotic Cultivar mutagenesis Genetically engineered cultivar

22 >160 insertions (>1kb) per cultivar on average

23 Cultivar x cultivar cross would result in 50% of distinct insertions from the donor being introduced: 160 x 1,000 base-long insertions, or a minimum of 160,000 bases of novel sequence. The average length of a genetically engineered insertion is generally 6,000 bases long vs

24 How much novel sequence is expected to be introduced into the cultivar? Cultivar x Cultivar Cultivar x Exotic Cultivar mutagenesis Genetically engineered cultivar (also clonal mutant or sport )

25 8,554 insertions (>100bp) in G. soja relative to G. max ~5,000,000 bases total

26 An introgression of DNA from the wild species would result in: 4-15MB* of donor DNA and 20,000-75,000 bases of novel sequence The average length of a genetically engineered insertion is generally 6,000 bases long vs *Highly dependent on number of crosses and genomic region

27 How much novel DNA is expected to be introduced into the cultivar? Cultivar x Cultivar Cultivar x Exotic Cultivar mutagenesis Genetically engineered cultivar

28 Mutation Type Mutagenesis Transformation SNPs Deletions (< 5bp) Insertions (<5 bp) Table abbreviated for presentation No insertions >5 bp

29 Double-strand breaks can create large insertions but do not appear to activate transposable elements Minsoy Wm82 Breeding FN M Mutagenesis WPT Wm82 Transgenic Slide by Robert Stupar, University of Minnesota

30 Cultivar x Cultivar Cultivar x Exotic Cultivar mutagenesis Genetically engineered cultivar Knowledge about the function of introduced sequence(s) GE without tissue culture GE with tissue culture exotic x cultivar cultivar x cultivar sport ; clonal mutant chemical mutagenesis Number of bases added to the cultivar as a result of the improvement method

31 Are there non-transgenic organisms?

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33 Genetic engineering and cultivar preservation

34 Zenglu Li (UGA) Jeff Bennetzen (UGA) Soo-jin Kwon (KREI) Fang Lu (Dow AgroSciences) Holgar Puchta (KIT) Bob Stupar (UM) Scott Jackson (UGA) Wayne Parrot (UGA) Randy Nelson (USDA) Qijian Song (USDA) Perry Cregan (USDA)

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36 Unanticipated function of the introduced sequence product: background interaction? Interaction with genetic background? Source: Another cultivar Exotic relative Transgenic or Synthetic Divergence from cultivar

37 Unanticipated function of the introduced sequence product: background interaction? Flag leaf senescence Interaction with genetic background Wheat Lr34 resistance allele Risk et al Another cultivar Exotic relative Transgenic or Synthetic Divergence from cultivar

38 Unanticipated function of the introduced sequence product: background interaction? Flavanol enrichment Fruit coloration Interaction with genetic background Flavanoid-synthesis-related transcription factor from corn Source: Another cultivar Exotic relative Transgenic or Synthetic Bovy et al Divergence from cultivar

39 Even with single T-DNA insertions, structural rearrangement still exists

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42 1.4 inserts per transformant

43 Double-strand break repair mechanisms Nonhomologous endjoining (~65%) Single strand annealing (~30%) Synthesis-dependant strand annealing (~ %) T Helleday, et al. 2007

44 The myth of stability? 5 plants; 30 generations of self-pollination There is no cultivar stability without breeder selection.

45 Conventional breeding has never activated novel toxic pathways Of millions of conventional varieties, only 9 have been reported to have unintended effects Dermatitis and stomach aches One of the safest technologies All involved elevated levels of known toxins Part of OECD list When crops have known toxins, testing of new varieties has become customary

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