FREECLIMB Kick-off meeting

1. FREECLIMBKick-off meeting Meknes, April 1-2, 2019 Prof. Francesco Salamini Advisory Board

2. General comment The contents are those expected for such a project: long-based breeding methods, considerations of germplasm resources and multi-site collections, phenotyping and genotyping and related database, associations and genetic dissections, all Omics, MAS and GS models for resilience traits, protocols for abiotic stresses, pre-breeding materials, generation of novel genotypes. As part of the advisory Board iI am pleased that the project is essentially dominated by a genetic-genomic approach.

3. More general comments. It is recommended that all initiatives, sharings of activities, specific collaborations and all what reinforce the mutual interdependence of partners, should be given priority. This is particularly necessary for Exchange of students, Multi-site collections, Evaluation networks…….

4. Communications within the project A finding or a decision - relevant to the point of considering necessary its communication -implies that this should not happen too frequently. The case to be communicated has to be clearly written and short, not serving only to demonstrate that the sender is still a (surviving) part of the group. Exchange of pollen. In the project, a single unit should be organized which receive and distribute pollen. This pollen repository may be part of a new proposal following the current one.

5. Decision making Aspresented in the project, the process of decision making seemsrationale and preciselyorganized. It can be addedthat, within the rules designed for the project, a high degree of freedomhas to be left to operating scientists, not to limittheirpossibility of making unexpecteddiscoveries.

6. Specific comments. S genes A major issue in plant breeding is the current shift from R dominant gene, inducing resistance to micro-organisms, to S genes (S for susceptibility) and their derived recessive resistant mutants: this supports the present relevance, in pre-breeding and breeding, of recessive mutations.

7. Diseaseresistance: from R (resistance) gene to S (suscettibility) genes RGenes (NBS-LRR): theyencode the receptors of avirulence gene products of the microb: the resistance gene isdominant. SGenes: recessive mutations induce resistance or immunity Vite Geni Mlo/mlo Rhizomania in sugarbeet (Barzen et al. 1997. Mol. Bred. 3: 231) Melo (Hinze et al. 1991. PNAS, 88: 3691. Walter et al. 1993. MGG, 239:122. Buschges, et al. 1997. Cell 88:695).

8. Induction of diseaseresistance _______________________________________________________________________________________ Piant System Gene S targeted Pathogen/disease Result Ref. __________________________________________________________________________________________ Tomato TFSlMlo1 Mildew, M Resistance (1) TFDMR6 Pseudomonas, Xanth. Phytophthora Resistances (2) Arabidopsis TFeIF4E TuMV Resistance to TuMV(3) Cucumber TFeIF4E Virus CVYV, ZYMV and PRSMV Resistences(4) Grape TF MLO-7 M Indels in target Res NC(5) Melo TF DIPM-1, DIPM-2, DIPM-4 “Colpo di fuoco” Indels in target Res NC(6) FrumentoTFTaMLO-A1, TaMLO-B1, TaMLO-D1 M High tolerance (7) TTaMLO M Indels in target Res NC (8) T TaEDR1 (3) MResistenza(9) RisoTOsSWEET14 Bacterial blight Aumentodella Res. (10) TOsSWEET11, OsSWEET14 Bacterial blight Indels in promoter, Res NC(11) T OsMPK5 Fungi and bacteria Indels in target Res NC(12) AgrumiTCsLOB1 Citrus cancer From 83 to 98% reduction (13) __________________________________________________________________________________________ TF: transgene freeeT : transgenic NC: not yet controlled (1) Nekrasovet al., (2017). (2)deToledoThomazella et al., (2016). (3)Pyott et al., (2016). (4) Chandrasekaran et al., (2016). (5,6)Malnoy et al., (2016). (7) Wang et al., (2014). (8) Shan et al., (2013). (9) Zhang et al., (2017). (10) Li et al., (2012). (11) Jiang et al., (2013). (12)Xie e Yang, (2013). (13) Peng et al., (2017).

9. Specific comments. Genome editing • The current state of plantgeneticsadressesstongly the role of recessive mutants: • recessives in domestications • recessives in plantphysiology to asses gene functions • recessives in diseaseresistance. • Ifthisisrecognized, Genome editing becames a core technologyalso for this project. For instance, in the in the induction of diseaseresistanceor in the generation of improvedgenotypes from interspecific crosses.

10. Pavan, S., et al., Loss of susceptibility as a novel breeding strategy for durable and broad-spectrum resistance. Mol Breed, 2010. 25(1): p. 1-12. Zaidi, S.S., M.S. Mukhtar, and S. Mansoor, Genome Editing: Targeting Susceptibility Genes for Plant Disease Resistance. Trends Biotechnol, 2018. 36(9): p. 898-906.

11. Generation of improvedgenotypes from interspecific crosses. Where from noveltyoriginates? In interspecific crosses itisdifficult to selectrecombinants of appliedvalue. Why? The consideration of domesticationgenesmay help. They are usuallyidentifiedas recessive loss of function of specificdominantgenes. Ifthis can be consideredas a rule, in interspecific crosses inibitoryfunctionsactingacrossgenomes can be contributed by the two parental species. In interestingrecombinantgenotypes, the elimination, via editing, of relevantdomesticationgenesmayprovide new fenotypes of appliedinterest.

12. Conclusion on genome editing A unit or a WP should be createdspecificallydedicated to thistechnology. Like in the case of the Pollen repository, thisplatformmay be part of a new project following FRECLIMB.

13. Specific comments. Abiotic stresses The project has chosen to address a difficult theme. In fact, tolerance to stresses is manifested by traits difficult to be genetically understood and improved by selection. This will suggests to try always to link visual observations to measures of many specific traits. It may very well be that many recorded traits will not be found linked to the degree of expression of tolerance, but even the finding of only one association may reveal important. Recording traits in contrasting environments is very helpfull.

14. The identification of genes involved in drought resistance has provided evidence that the transcriptional factors known as CBF (DREB- repeat binding factors) can induce a kind of tolerance in tomato, wheat,colza, maize and rice. Difficult is how to detect tolerance.

15. GMO–induced resistance to abiotic stresses _________________________________________________________________ Resistance No. of mechanisms-approaches No of species transformed _________________________________________________________________ Stress due to drought 4 1 Tolerance to submersion 2 1 to salt 5 5 to cold 3 3 to heavy metals 3 2 _________________________________________________________________ Conclusion on abiotics: ithasbeennotedthat one necessity for severalcropsis to create a culture related to candidate genes for abiotic stresses. Thiswill help atalllevelsbutparticularly in trascriptomics, QTL cloning, generation of recessives via Editing. (Mathur, 2017. In Sustainable Agriculture Reviews, Lichtfouse Ed., Springer ,Switzerland).

16. Comments on Cropspecies. Citrus Five groups partecipate in defining key ideotypes, tolerance to Phytopthora and Mal secco, ironchlorosis, water stress, salinity and quality. The presentationhasmainlycoveredtolerance to diseases and breeding. Germplasmisproper and sufficient (root stocks included). Activity on Mal secco seems OK. Itmayneed a more cleardefinition of steps: suggestedis QTL finding, theircloning and use of Genome editing (butitwill take 10 years! Keep for next project!). The evaluation of germplasmhasfoundinterestinggenotypes for tolerance to Mal secco (with no PCR detection of the parasite, or positive to PCR).Thesegenotypes are starting points for crosses addressing the genetics of tolerance.

17. For Phytophtora in rootstoks the same steps can be consideredassuggestedabove (few candidate genes are alreadyavailable; seetable). Breeding activities should be helped by the knowledge, ifavailable, on domesticationgenes. In general: good presentationdescribing activities which can be made more clearlyconsidered in theirpriorities and logicalspeps.

18. Peach Concerningcollectionsesthablisment , the workingprofilepresentedseemscorrect. Major targets are tolerance to draught and calcareoussoils in scions, Monilia resistance, genotyping and laterassociation mapping and assistedselection. The experiment on draught stress iswellorganized (theymention the control of powderymildew by treatment, but…). Genotypingseemswellorganizedusing an array for the Milan referencepopulation. Pollenexchangeis to be organized, aswellas the exchange of personnel. A limit to operativity on thisspeciesis in vitro culture (seenasuseful for exchangingmaterials and for application of Genome editing; thisisrelevantbecause candidate genes for major traits are becomingavailable). The evaluation of this part of the project isfully positive.

19. Olive Four partners partecipating. Good collections are available and major targets are tolerance to abiotic stress , Verticilliumwilt, and fruitfly. Abiotic stresses considerheath, time of flowering, salinity. The experimentsconcerningfruitfly and Verticillumwill be carried out in all 3 years, in open fields and greenhouses+growthchambers. Ithasbeennotedthat one necesity for this, aswellas for othercrops, is to create a culture related to candidate genes for abiotic stresses. Thiswill help atalllevelsbutparticularly in trascriptomics, QTL cloning, generation recessives via Editing. Verticillium activities, trascriptomics and genotyping are welldescribed; a future deeperconsideration of how to realize QTL mapping and their use in MAS isnecessary, particularlybecause the choice of parents of crosses willheavilycondition the outcame.

20. Grape The germplasmdescribed and used in the project iffullyadapted to the scope and includessegregatingpopulations and a caucasianvarietalcollection. Tolerance to Downymildew (caused by Plasmophora viticola) isproperlyaddressedusing a populationderived by selfing a resistantV.viniferaaccession, and the caucasiancollection. Abiotic stresses include thoseinduced by drought and salinity and are studied via interspecific crosses (ex. in egiptianrootstocks). Phenotypingis OK asdescribed and genotyping, includingassociation mapping and linkage maps, is standard. The development of pre-breeding materialsbased on phenotypesis a matter of discussion: for the species, the time is ripe for genotypepyramidingbasedalso on Editing results: i.e. try to capitalize on knowledge of candidate genes for major traits.

21. Apricot The traits considered are tolerance to late frost, to Sharka and Monilia and to drought. The materialsavailable are OK in terms of presence of tolerantgeotypes; materials include resistantsaccessions, botanicalcollections, segregatingpopulations, and rootstocks.Thephenotyping for the several traits iscarefullyplanned, also in segregatingpopulations. Whathasbeendescribed for geneticdissection, Genome wide phenothiping, linkage and association mapping and GWAS is standard butneeds the definition of more precise protocols, the case also of the set of crosses dedicated to pre-breeding (whichwillalso use information from multiple testing locations).

22. Almond and interspecific crosses This part of the project hasbeenalsodiscussed under General comments. The programincludes the induction of resistances to viruses, late frost, Fusicoccum. Traits will be recorded in collections and segregatingpopulations. Genotypingisbased on SSR and Genome wide assistedselectionwill be introduced. The activities presented are essentiallyconsisting of pre-breeding operations. A questionhas to be seriouslydiscussed: isitonly the low geneticrecombination in interspecific crosses whichlimits the generation of usefullderivatives?