Plant Breeding. Opportunities of New Plant Breeding Techniques. Breeding projects at WUR-Plant Breeding

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1 Opportunities of New Plant Breeding Techniques Jan Schaart Plant Breeding Important for crop improvement Combining of genetic variation Based on the steps of crossing and selection Limitations polyploidy, heterozygosity self-incompatibility long generation time... Breeding projects at WUR-Plant Breeding Food: potato, tomato, cabbage, lettuce, onion, apple, quinoa, mushroom,... Bio-based: crambe, camelina, hemp, miscanthus... Ornamental: lily, tulip, rose... Improved breeding using New Plant Breeding Techniques New Plant Breeding Techniques (NPBT) Process & products New genetic techniques for crop improvement Similar breeding goals as conventional plant breeding but, faster, easier All NPBT have process that make use of a genetic modification (GM) step For most techniques: Ultimate products are free of genes that are not present in the species gene pool Plant Genetic modification (application of recombinant DNA) Screening for desired genomic modifications Removal of recombinant DNA Improved plant

2 Products with three levels of genomic modifications Range of techniques and concepts Cisgenesis. Addition of an extra copy of a existing gene to the plant genome. Modifications to an gene that is present in the plant genome 3. No direct modification to the genome (process used to facilitate breeding steps) Sequence-specific nuclease technology (ZFN, TALENs, CRISPR-Cas) Oligo-directed mutagenesis RNA-dependent DNA-methylation Grafting on a GM-rootstock Reverse breeding Induced early flowering CENH3-mediated haploid induction 7 8 Cisgenesis vs transgenesis Cisgenesis Transformation concept introducing gene sequences that originate from the species itself Cisgenesis: addition of an extra copy (allele) of a native gene Extra copy of a gene: superior allele Agrobacterium-mediated or direct gene transfer (cis)gene MdVf-promoter e.g. native apple Vf-gene MdVf-coding sequence (incl introns) MdVf-terminator Examples: Apple: Scab resistance Red flesh Potato: Phytophthora resistance transgene pcamv35 gus Tnos e.g. gus-marker gene E. coli Agrobacterium Cauliflower mosaic virus 0 Scab in apple Apple scab is major disease in apple Common culture: 0-30 sprays/season Classical introgression of Vf-gene in apple Different sources of scabresistances available Malus floribunda Vfgene Classical breeding is extremely time-consuming >50 years to breed Vfgene in commercial cultivar

3 Introduction of Vf-gene in apple through cisgenesis At WUR-Plant breeding: validation of Vf as suitable scab-resistance gene Introduction in Gala together following transgenic approach Greenhouse scab-resistance test Scab-resistance test Cisgenic Gala with Vf cisgenic Gala with Vf-gene (tree 67) Gala (tree ) Gala (tree 8) Gala + Vf, cisgenic (tree 67; SpVf-, NL) Next step: Durable resistance by gene stacking Vf+V5+Vr Gene stacking: Combining Vf with other scab resistance genes: Vr, cloned from M. pumila, tested in transgenic plants, ready for cisgenesis V5, cloned from selection and currently being tested Vf, V5 and Vr different type of genes, different mode of action Cisgenic red-fleshed Gala apple Red flesh: MdMyb0-gene from apple cultivar Red Field 7 3

4 Cisgenesis in potato for durable Phytophthora disease resistance Triple R-genes against Phytophthora infestans Stacking of three broad-host resistance genes from reated wild species: Rpi-sto (from S. stoloniferum) Rpi-blb3 (from S. bulbocastanum) Rpi-vnt. (from S. venturii) Field test before Tuber test after Field trial August 05 Durable potato cultivation Improved variety lines with various combinations of resistance genes Cultivation rotation scheme to support durable disease management Sequence-Specific Nucleases (SSNs) CRISPR-Cas9 Pre-designed restriction enzymes Cut at predefined DNA target sequence (so at your favourite sequence) Different types: Meganucleases Zinc Finger Nuclease (ZFN) TAL Effector Nuclease (TALEN) CRISPR-Cas9 4 4

5 What are SSNs doing? DNA repair: two outcomes SSNs induce double strand breaks (DSB) at a specific target site in genomic DNA DNA break SSN, e.g. CRISPR-Cas Homologous Recombination (HDR) SSN-induced DSB End Joining (NHEJ) repair template Repair of DSB by cell targeted mutation precise repair Applications: targeted mutagenesis Traditional mutagenesis Direct mutation of target genes no mutagenized populations required; no laborious and expensive screening methods (Tilling) Powerful tool to make mutations in multiple alleles: polyploids very difficult using traditional mutation methods Population of plants with genome-wide mutations (e.g.,000x) Select plants with mutations on target Breeding for removal of undesired mutations and homozygous state Directed mutagenesis Many crops have a polyploid genome Few plant with target mutations (e.g. 5x) Select plants with no or few off-target mutations Polyploids have multiple allelic versions of each gene eg: Potato, tetraploid (4x) Wheat, chrysanthemum, hexaploid (6x) Strawberry, octoploid (8x) If necessary: Breeding for removal of CRISPR-Cas9 DNA-construct and/or off-target mutations Knock-out mutation: all alleles have to be targeted Multi-allelic mutation with CRISPR-Cas! 5

6 Directed mutagenesis-projects at WUR-PBR ZFN-induced deletion in tetraploid potato CRISPR-Cas for mutagenesis in different crops: -potato (4x): S-genes, starch, carotenoids -tomato (x): S-genes, taste attributes -camelina, crambe (6x): oil composition, antinutritional factors -bread wheat (6x): gluten -chrysanthemum (6x): haploid induction -nicotiana (4x): tests ZFNs targeted towards sbeii All four alleles targeted in a single experiment Plant#5-: deletion in all four alleles! allele allele allele 3 allele 4 ZFN-target site 5-3 OsN3 susceptibility gene Deletion of OsN3 promoter S-gene for rice bacterial blight (caused by Xanthomonas oryzae pathovar oryzae (Xoo) Xoo produces effector-proteins that activate the expression of OsN3 Xoo effector protein OsN3 promoter expression ATG OsN3 coding sequence Xoo effector protein OsN3 promoter expression ATG OsN3 coding sequence Xoo effector protein no expression ATG OsN3 promoter OsN3 coding sequence TALENSinduced deletion RNAi: Silencing of OsN3-expression: Xoo resistance!...but OsN3 is essential gene in rice. Silencing results in stunted plants. 3 plant transcription factor expression ATG OsN3 promoter OsN3 coding sequence deletion TALENs-induced mutations in OsN3 promoter S-genes for Phytophthora New source of resistance?? 36 Li et al., (0) Nature Biotechnology,

7 Induced early flowering Application of early flowering E.g. in fruit trees: generation time 5-0 years Shortening juvenile phase: accelerated breeding Induction of early flowering: Over-expression of Flowering locus T gene (AtFT) Silencing of Terminal Flower gene (MdTFL) Flachowski et al. (0) in apple Overexpression of BpMADS4 gene (silver birch): breaking of juvenility: early flowering Speed breeding Scorza et al. (0) in plump Overexpression of FT (populus) FasTrack breeding Source: H. Flachowsky; JKI Stacking of resistance genes by speed breeding Speed-breeding scheme transgenic early flowering Flachowsky et al. 009 & 0 (adapted) (FB) Fire blight resistance Fire blight + apple scab resistance check for presence using DNA markers parent early-flowering transgene gametes parent Regia (FB-F7, Rvi, Rvi4) 008 X Golden Delicious X offspring X 009 BC -seeds early flowering selection of nontransgenic offspring check for absence of transgenes using DNA markers final product, no GM present Viral delivery of early flowering genes Conclusion Yamagishi et al (03) expression of early flowering genes using apple latent spherical virus (ALSV) Infection of seedlings: 90% produced fertile flowers in.5-3 months virus is not transmitted through seed New genetic techniques for crop improvement Similar breeding goals as conventional plant breeding faster easier new possibilities Products often indistinguishable from products of traditional breeding 7

8 Related Issues All NPBT have process that make use of a genetic modification (GM) step For most techniques: Ultimate products are free of genes that are not present in the species gene pool Products often indistinguishable from products of traditional breeding Thank you for your attention! jan.schaart@wur.nl Are products from these New Plant Breeding Techniques GMOs or not Consumer acceptance