Giovanni Giuliano, on behalf of the G2P-SOL consortium

Italian National Agency for New technologies, Energy and Sustainable Development

08/03/2019 , Italy-Israel Workshop, Casaccia, Rome

The G2P-SOL project: harnessing the genetic and phenotypic diversity of the four major

Solanaceae crops.

The G2P-SOL project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 677379

Coffea

Petunia

Nicotiana

Physalis

Capsicum

Cyphomandra

S. melongena

S. candidum

S. stramonifolium

S. tuberosum

S. lycopersicum

3rd food crop Model for tuber biology Production: 375 MT/y Autotetraploid, 0.9 Gb

2nd Solanaceous crop Model for climacteric ripening Production: 180 MT/y Diploid, 0.9 Gb

3rd Solanaceous crop Production: 51 MT/y Diploid, 1.2 Gb

4th Solanaceous crop Production: 38 MT/y Diploid, 3.3 Gb

Rubiaceae

The four G2P-SOL crops in the context of the Solanaceae family

The economic importance of Solanaceae

WorldwideandEuropeancropproductionsandvalues(source:Faostat2012).

Production(Mtons) Grossproductionvalue(Bn€)

Crop World Europe World EuropePotato 365 116 72 20

Tomato 162 21 70 14Eggplant 48 0.9 22 0.4

Pepper 31 2.8 22 2

G2P-SOLfour 606 156 186 39All horticulturalcrops

1,928 211 477 59

The centers of diversity of

the “G2P-SOL four”

Rain forest

Low temperatures

Deserts

Marginal soils High

altitudes

Arid environments

Aquatic environments

Tree tomato

The incredible adaptability of Solanaceae

The G2P-SOL consortium

Acronym Organisation name ENEA Agenzia Nazionale Per Le Nuove Tecnologie, L’Energia e lo

Sviluppo WUR Wageningen University JHI The James Hutton Institute HUJI The Hebrew University of Jerusalem IPK Leibniz Institut fuer Pflanzengenetik und

Kulturpflanzenzuchtung UPV Universidad Politécnica de Valencia

UniTO Università degli Studi di Torino INRA Institut National de la Recherche Agronomique CREA Consiglio per la ricerca e la sperimentazione in agricoltura ARO The Volcani Centre Eurice European Research and Project Office GmbH

IHAR Instytut Hodowli i Aklimatyzacji Roślin CIP Centro Internacional de la Papa

PHEN Phenome Networks Ltd. BATEM Bati Akdeniz Agricultural Research Institute

MVCRI Maritsa Vegetable Crops Research Institute

AVRDC AVRDC – The World Vegetable Center BLUMEN Blumen Group Spa

SATIVA Consorzio Sativa

Organisation name

Chinese Academy of Agricultural Sciences Seoul National University Boyce Thompson Institute Tomato Genetics Resource Center Michigan State University Limagrain Vegetable Seeds Association de créateurs de variétés nouvelles de pommes de terre Enza Zaden Hodowla Ziemniaka Zamarte Top Seeds International Esasem S.p.A. Semillas Fito Ramiro Arnedo Semillas Benson Hill Ltd

PARTNERS COLLABORATORS

Genebanks

Genetic accessions available in

G2P-SOL partner genebanks P

art

ne

r

Co

un

try

Tomato Of which

wild Potato

Of which wild

Pepper Of which

wild Eggplant

Of which wild

All four crops

Of which wild

AVRDC TW 8.260 812 0 0 8.235 464 3.713 1.499 20.208 2.775 IPK DE 3.840 26 6.020 2.845 1.530 63 110 0 11.500 2.934

HUJI IL 8.100 100 0 0 0 0 0 0 8.100 100

INRA FR 1.600 200 1.500 500 1.460 46 2.015 1.120 6.575 1.866 CIP PE 0 0 6.000 1.800 0 0 0 0 6.000 1.800

WUR NL 1.332 108 1.446 1.243 1.010 783 510 373 4.298 2.507 UPV ES 2.220 220 0 0 1.400 80 260 70 3.880 370

JHI UK 0 0 2.300 1.400 0 0 0 0 2.300 1.400

All others 390 50 390 300 850 56 280 40 1.910 446 Total 25.742 1.516 17.656 8.088 14.485 1.492 6.888 3.102 64.771 14.198

Genesys 46.407 38.247 30.485 7.235 122.374

The main G2P-SOL objectives

• Make an inventory of existing accessions and prebreeding materials for the four crops and of the available passport and phenotypic data

• Low density genotyping (>2,000 markers) of 30,000-40,000 accessions of the four crops and description of their population structures.

• Create and distribute core collections (300-500 genotypes) of the four crops. Streamline related regulatory and phytosanitary procedures.

• Pre-breed 2 existing traits/crop (1° wave) and 2 novel traits/crop (2° wave) into elite genetic materials

• High density genotyping (>20,000 markers) of the core collections and additional prebreding materials

• Phenotyping of the core collections in multiple locations over 2 years

• Genome wide association studies and QTL colinearity maps between the four crops

• Create an open access gateway with the project data

The four G2P-SOL genomes

Eggplant V3.0* Potato V1.0 Tomato SL2.50 Pepper V1.55

Genome Size ~1.2 Gb ~844 Mb ~900 Mb ∼3.3 Gb

Number of scaffolds 22.332 66.254 3.223 37.989

Ungapped length of

scaffolds 1,18 Gb (97%) 585.8 Mb (69%) 737.6 Mb (82%) 2.96 Gb (90%)

Ungapped length of

anchored scaffolds 825.5 Mb (69%) 585.8 Mb (69%) 719 Mb (80%) 2.67 Gb (81%)

N50 of scaffolds 2.6 Mb 1.3 Mb 16.5 Mb 2.4 Mb

Protein coding genes 35.922 35.004 34.725 34.899

Of which organellar 4.607 2.057 2.163 3.492

BUSCO genes present in

the annotation 892 882 893 750

Anchored genes 28.435 35.004 33.838 30.242

Total length of repeats 775 Mb (64.6%) 499 Mb (59.1%) 426 Mb (47.3%) 1,989 Mb (60.3%)

*Eggplant Genome Sequencing consortium, submitted

G2P-SOL inventory

Passport Phenotypic Images Tomato 23.931 12.859 10.324

Pepper 13.246 10.517 3.286

Eggplant 5.940 3.934 2.634

Potato 12.998 1.397 1.327 Total 56.115 28.707 17.571

Passport, phenotypic and image data collected

Examples of images

www.g2p-sol.eu/G2P-SOL-gateway.html

The next step: generation of an open access, interactive hub for Solanaceae Genetic Resources

www.g2p-sol.eu/G2P-SOL-gateway.html

G2P-SOL Gateway

Existing passport data

Existing phenotypes

New genotypes

New pre-breeding populations

New phenotypes

Genotyping workflow I

• 3 robust DNA preparation protocols selected and ring-tested (Qiagen, LGC, modified CTAB).

• each genebank choses preferite protocol, prepares DNA in 96-well format and ships to genotyping partners:

ENEA IPK JHI UniTO

• 1 control accession (reference genotype) included in well A1.

Genotyping protocols

GBS (Elshire et al, 2011) SPET (Nugen, unpublished)

Markers Throughput Cost (LD) Cost (HD) Freedom to operate

GBS Random Very high 17 € 30 € No (Keygene patent)

SPET Targeted Very high 17 € 30 € Yes

Characteristics of the two technologies

Low density genotyping

Species Enzymes Multiplexing

Target samples

Completed samples

High quality SNPs*

Potato PstI-MspI 96 5.700 6.000 pending

Pepper PstI-MspI 384 9.600 10.300 2.938

Species N. targets Of which CDS

Total samples

Completed samples

High quality SNPs*

Tomato 5.001 3.372 16.800 17.664 4.909

Eggplant 5.001 2.254 3.600 3.600 11.949

37.664 accessions and prebreeding lines genotyped as of Dec 31, 2018. 65% of active accessions from the partner genebanks.

Dendrogram of 13.824 tomato accessions

Identify duplicates

between and within collections (≤3%)

Identify mistakes in

taxonomy (≤4%) Identify monomorphic and

polymorphic alleles Identify introgressions from

wild species Choose accessions (400-

500) for the core collection

Hypothesized origin and domestication of tomato

Center of origin - Peru

Migration of cherry and cerasiforme genotypes

Secondary domestication in Mexico.

Introduction of Mexican landraces to Spain, Italy (1500s)

Introduction to N. America (1700s)

Transfer of traist from wild species (1900s)

11.03.2019

PCA analysis 13.824 accessions

Four major groups: green-fruited species, orange-fruited species, S. pimpinellifolium and S. lycopersicum

Incomplete separation between S. lycopersicum and its ancestor S. pimpinellifolium (admixture?)

S. lycopersicum var. cerasiforme falls in two different clusters, one between S. lycopersicum and S. pimpinellifolium, and one between S. lycopersicum and S. galapagense/cheesmaniae

Some taxonomic mis-classifications

Green-fruited species

S. pimpinellifolium

S. lycopersicum

S. galapagense + cheesmaniae S. galapagense

S. cheesmaniae

cerasiforme

Dendrograms of 9.569 pepper and 3.646 eggplant accessions

C. chinense

C. baccatum

C. pubescens

C. frutescens

67/3 (Ref)

(Ref)

S. aethiopicum

S. macrocarpon

Subgenus Solanum

Exchange of genetic materials and phenotyping in multiple locations

JHI

INRA

CREA UPV

Core collections of 450 accessions constructed and exchanged

4 EU «clearing centers», one per crop Responsible for the exchange of accessions

with non-EU partners and for phytosanitary clearance

The G2P-SOL phenotyping fields

The regulatory and biological hurdle

N bilateral SMTAs to be signed between partners for pepper, eggplant, tomato: 110 N of cumulative phytosanitary certificates for the same plants: 45.

The potato phenotyping fields

Problem: Potato germplasm exchange between America and Europe is extremely difficult due to phytosanitary regulations and photoperiodism.

Solution: 2 core collections, 1 South American and 1 European, with 50 genotypes in common for statistical homogenization

Phenotyping of core collections

T4.1 Yield and agronomic traits – Lead : CREA

T4.2.1 Fruit/tuber quality – Lead : HUJI

T4.2.2 Metabolic profiles – Lead : WUR-DLO

T4.3 Biotic stress Tolerance – Lead : INRA

T4.4 Abiotic stress Tolerance – Lead : ARO

Core collections

G2P-SOL gateway

Training activities

Both entry level and advanced training schools

www.g2p-sol.eu

Lessons learned Building a global information and exchange system for plant genetic resources is an

ambitious, but feasible goal. The technical tools are there, and the main obstacles are regulatory.

Two different regulatory regimes: International Treaty (SMTA, potato eggplant) and Nagoya protocol (Crop Wild Relatives, and potentially local varieties of tomato and pepper). Nagoya protocol would be a no go for exchange of genetic materials, especially crop wild relatives.

Some countries (eg China, Russia, Israel) are not signatory of the International Treaty. Most public and private entities find an SMTA to be preferable over no regulation. Most institutions, even in non signatory countries, are willing to sign an SMTA for all four crops, if this is for scientific research.

Regulatory and phytosanitary regulations are highly dishomogeneous, both internationally and within Europe (eg, direct exchange of pepper wild relatives between Turkey and Taiwan takes 2 years. Through Europe: 9 months).

Seeds are easy to smuggle, and you can buy seeds for thousands of species on Amazon, with no need for phitosanitary ceritificate or SMTA.

Simplify the International Treaty procedures and extend it to all crops and their interfertile wild relatives.

Build a global phytosanitary surveillance system, with genebanks as key partners.

Policy priorities

Acknowledgments

ENEA (Italy) P. Ferrante, G. Aprea, M. Sulli, O. Demurtas Wageningen University (The Netherlands) Y. Tikunov, A. Bovy, R. Finkers James Hutton Institute (UK) K. Mclean, S. Sharma, G. Bryan The Hebrew University of Jerusalem T. Pleban, D. Zamir IPK (Germany) A. Boerner, R. Brandt, M.T. Wallace, N. Stein Universitat Politècnica de Valencia (Spain) D. Alonso, P. Gramazio, S. Vilanova, M.J. Diez, J. Prohens University of Torino (Italy) A. Acquadro, L. Barchi, E. Portis, S. Lanteri INRA (France) J.E. Chauvin, F. Esnault, J. Salinier, V. Lefebvre CREA (Italy) L. Toppino, L. Bassolino, P. Tripodi, G.L. Rotino The Volcani Centre (Israel) I. Paran

EURICE GMbH (Germany) B. Dibari, V. Peuser Instytut Hodowli i Aklimatyzacji Roślin (Poland) E. Stefanczyk, J. Sliwka Centro International de la Papa (Peru) H. Lindqvist-Kreuze, , N. Anglin, D. Ellis Phenome Networks Ltd (Israel) Y. Semel BATEM (Turkey) F. Boyaci, A. Unlu Maritsa Vegetable Crops Institute (Bulgaria) G. Pasev, D. Ganeva, S. Grozeva, V. Tringovska The World Vegetable Center (Taiwan) R. Schafleitner

Blumen Group SpA (Italy) R. Tardani Consorzio Sativa (Italy) C. Milanesi