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