Breakthrough Technologies The Multisite PeachRefPop Collection: A True Cultural Heritage and International Scientific Tool for Fruit Trees12 Marco Cirilli,a,3 Sabrina Micali,b,3 Maria José Aranzana,c Pere Arús,c Annarosa Babini,d Teresa Barreneche,e Marco Bink,f Celia M. Cantin,c Angelo Ciacciulli,a José Enrique Cos-Terrer ,g Pavlina Drogoudi,h Iban Eduardo,c Stefano Foschi,i Daniela Giovannini,j Walter Guerra,k Alessandro Liverani,j Igor Pacheco,l Thierry Pascal,m Benedicte Quilot-Turion,m Ignazio Verde,b Laura Rossini,a,4,5 and Daniele Bassi a,4 a Department of Agricultural and Environmental Sciences, University of Milan, 20133 Milan, Italy b Consiglio Per La Ricerca In Agricoltura E L’analisi Dell’Economia Agraria, Research Centre for Olive, Fruit, and Citrus Crops, 00134 Rome, Italy c Institut de Recerca i Tecnologia Agroalimentàries, Centre de Recerca en Agrigenòmica Consejo Superior de Investigaciones Científicas, Institut de Recerca i Tecnologia Agroalimentàries, Universitat Autònoma de Barcelona, Universitat de Barcelona, Campus UAB, 08193 Barcelona, Spain d Phytosanitary Service, Regione Emilia-Romagna, 40128 Bologna, Italy e Université de Bordeaux, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement, Biologie du Fruit et Pathologie, F–33140 Villenave d’Ornon, France f Hendrix Genetics Research, Technology, and Services, 5830 AC Boxmeer, The Netherlands g Murcia Institute of Agri-Food Research and Development, La 30150 Alberca, Murcia, Spain h Hellenic Agricultural Organization ’Demeter’, Department of Deciduous Fruit Trees, Institute of Plant Breeding and Genetic Resources, 59200 Naoussa, Greece i Centro Ricerche Produzioni Vegetali, 47522 Cesena, Italy j Consiglio per la Ricerca in Agricoltura e L’Analisi Del’Economia Agraria, Research Centre for Olive, Fruit, and Citrus Crops, 47121 Forlì, Italy k Research Centre Laimburg, 39040 Vadena, Italy l Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, 7830490 Macul, Chile m Institut National de Recherche pour L’Agriculture, L’Alimentation et L’Environnement, Génétique et Amélioration des Fruits et Légumes, F–84143 Montfavet, France ORCID IDs: 0000-0002-2086-7467 (S.M.); 0000-0002-0240-8021 (M.J.A.); 0000-0002-7852-1418 (T.B.); 0000-0002-1278-2092 (M.B.); 0000-0001-9911-0194 (C.M.C.); 0000-0003-3041-6966 (J.E.C.-T.); 0000-0001-9804- 4543 (P.G.); 0000-0002-9963-2934 (I.E.); 0000-0002-7954-2202 (S.F.); 0000-0003-0977-390X (D.G.); 0000-0002-8662-4978 (W.G.); 0000-0003-2134-8527 (A.L.); 0000-0001-5057-0464 (I.P.); 0000-0003-4095-1399 (B.Q.-T.); 0000-0002-9139-955X (I.V.); 0000-0001-6509-9177 (L.R.); 0000-0002-8966-3534 (D.B.) Plants have evolved a range of adaptive mechanisms that adjust their development and physiology to variable external conditions, particularly in perennial species subjected to long-term interplay with the environment. Exploiting the allelic diversity within available germplasm and leveraging the knowledge of the mechanisms regulating genotype interaction with the environment are crucial to address climatic challenges and assist the breeding of novel cultivars with improved resilience. The development of multisite collections is of utmost importance for the conservation and utilization of genetic materials and will greatly facilitate the dissection of genotype-by-environment interaction. Such resources are still lacking for perennial trees, especially with the intrinsic difficulties of successful propagation, material exchange, and living collection maintenance. This work describes the concept, design, and realization of the first multisite peach (Prunus persica) reference collection (PeachRefPop) located across different European countries and sharing the same experimental design. Other than an invaluable tool for scientific studies in perennial species, PeachRefPop provides a milestone in an international collaborative project for the conservation and exploitation of European peach germplasm resources and, ultimately, as a true heritage for future generations. Since the Roman garden hortus, fruit tree orchards the selection of a multitude of local cultivars. These have represented distinctive features of the Mediterra- materials represent a cultural and genetic heritage of nean rural landscape, a synthesis of the interaction generations of farmers and a common good to preserve among genotype, environment, and human customs for present and future generations. (Biasi et al., 2012). The diversity of pedo-climatic con- Plants have evolved a range of adaptive mecha- ditions and production systems, along with the plas- nisms that adjust their development and physiology to ticity of the genotype and human traditions, has shaped variable external conditions, particularly in perennial 632 Plant PhysiologyÒ, October 2020, Vol. 184, pp. 632–646, www.plantphysiol.org Ó 2020 American Society of Plant Biologists. All Rights Reserved. Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Multisite PeachRefPop Collection species subjected to a long-term environmental expo- making their preservation and exploitation a suitable sure and interaction. Climate changes are impacting strategy for dealing with the changing climatic condi- cultivation environments, raising the need for more tions. (2) Systematic dissection of genotype-by-environ- resilient cultivars able to maintain performance across ment and/or genotype-by-management interactions as variable (and often unpredictable) weather conditions primary sources of variability for several important (Varshney et al., 2011; Luedeling, 2012; Ramírez and quantitative traits (Myles, 2013; Chagné et al., 2014). Kallarackal, 2015). Also, increasing the sustainability This is a critical point for genetic analyses of complex of fruit production (particularly in terms of resource traits, such as genome-wide association studies (GWAS), demands and disease management) requires leverag- where germplasm collections are characterized to iden- ing knowledge of the interactions between plants, soil, tify quantitative trait loci (QTLs) across different envi- and environmental factors and how they affect pro- ronments, or genome-wide selection (GS), used to predict ductivity and end-product quality (Coakley et al., 1999; genomic estimated breeding values. Singh et al., 2013; Parajuli et al., 2019). The comprehension of genetic, epigenetic, and physi- Peach (Prunus persica) originated in China (Li et al., ological mechanisms as well as the estimation of geno- 2019), later reaching Persia, the Mediterranean Basin, type-by-environment and/or genotype-by-management Europe, and the Americas; it is now the third most effects requires the development of multisite replicated cultivated fruit tree species in temperate regions. Beside collections and ad hoc experimental designs. The avail- its importance as a crop, peach is a recognized model ability of such types of resources is rapidly growing in for genetic and genomic studies in fruit trees, repre- annual species, while it has not yet been implemented in senting the ideal system for addressing two main perennial fruit trees. challenges in fruit tree breeding. (1) Understanding and During the past century, peach orchard systems have harnessing the allelic diversity within available gene- changed dramatically following innovations in orchard pools. Noteworthy for peach, the intercompatibility design, training systems, and agronomic management with related species of the Amygdalus subgenus (al- (Corelli-Grappadelli and Marini, 2008), other than cul- mond [Prunus dulcis], Prunus davidiana, Prunus kan- tivar evolution. Noteworthy, the first reported modern suensis, etc.) has long been considered a source of orchard was a peach plantation established in Massa natural variability, particularly for the introgression of Lombarda (Ravenna, Italy) at the end of the 19th cen- disease resistance (Gradziel, 2002; Foulongne et al., tury using the white-fleshed local cultivar population 2003). However, interspecific hybrids have had poor cv Buco Incavato (Bellucci, 1908). In the last de- applicability in current breeding programs (Cirilli cades, considerable breeding efforts have assisted the et al., 2017), although new genomics-based strategies intensification of cultivation techniques and the devel- could change this trend (Serra et al., 2016). Conversely, opment of horticultural quality concepts with the in- landraces and local ecotypes could be a source of troduction of novel, fit-for-purpose cultivars (Byrne resilience traits more straightforward to introgress, et al., 2012). In Europe, peach has a long cultivation history, tracing back to the Ancient and Middle Ages and characterized by the isolation and propagation of 1 This work was supported by PRIMA (grant no. FREECLIMB to chance seedlings operated by farmers and amateurs, D.B., I.E., P.D., and B.Q.T.), the European Commission (project Fruit- through which each country set its own pool of locally Breedomics; grant no. FP7–265582 to D.B., L.R., P.A., I.V., T.B., B.Q.T., adapted cultivars (Bassi and Monet, 2008). The para- and W.G.), MAS.PES, an Italian project aimed at apricot and peach digm shift to the modern controlled-crosses approach breeding (to D.B. and S.F.), the Spanish Ministry of Economy and in early United States breeding programs has been the Competitiveness (MINECO/FEDER grant nos. AGL2012–40228– foundation of the dramatic varietal improvement of the C02–01 and RTA2015–00050–00–00), and the CERCA Programme- last century, beginning with the introduction of seed- Generalitat of Catalonia. 2 ling materials from China in the mid-19th century (e.g. This work is dedicated to the memory of our colleague Chiara Ferrandi, who recently passed away. cv Chinese Cling, progenitor of most modern cultivars; 3 These authors contributed equally to the article. Faust and Timon, 1995; Byrne et al., 2012). The world- 4 Senior authors. wide spread of improved United States materials, fa- 5 Author for contact: laura.rossini@unimi.it. vored also by the limited activities in other countries, The author responsible for distribution of materials integral to the has resulted in a rapid replacement of landraces and findings presented in this article in accordance with the policy de- local accessions, particularly in Europe. From the sec- scribed in the Instructions for Authors (www.plantphysiol.org) is: ond half of the 20th century, however, novel programs Laura Rossini (laura.rossini@unimi.it). started in several European countries, although they Plant material: D.B., S.F., B.Q.-T., T.B., T.P., P.A., M.J.A., I.V., A.L., were mostly based on United States breeding stocks D.G.; host institutions: D.B., M.C., S.F., L.R., P.A., I.E., J.E.C.-T., P.D., with a marginal role for local cultivated germplasm. I.V.; phytosanitary controls: A.B.; site characterization: W.G., M.C.; experimental design: M.C., M.B.; phenotypic data analysis: A.L., This led to a consequent loss of many local cultivars, D.G., C.M.C.; genotypic data analysis: I.P., A.C., M.J.A., S.M.; in parallel with a progressive narrowing of the genetic core collection design and evaluation: S.M., I.V., M.C.; article writing: bases in modern cultivars (Aranzana et al., 2010; Verde M.C., S.M.; article revision: D.B., L.R., I.V., S.M., and all authors for et al., 2013). respective parts. Conception and coordination: L.R. and D.B. As awareness of genetic erosion in modern plant www.plantphysiol.org/cgi/doi/10.1104/pp.19.01412 breeding increased (Fu and Dong, 2015), the conservation Plant Physiol. Vol. 184, 2020 633 Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Cirilli et al. and exploitation of genetic resources has become a resulting in redundancies or risk of loss for unique fundamental aspect in crop breeding (Ford-Lloyd accessions. and Jackson, 1986). Considerable efforts have been This article describes the concept, design, and reali- made in the collection and characterization of many zation of the first multisite peach reference collection, plant germplasms (including fruit tree species), along PeachRefPop (PRP), across five locations in three Euro- with the development of approaches for their effec- pean countries (Italy, Spain, and Greece). Other than an tive management and utilization (Gepts, 2006). The invaluable tool for scientific studies, the PRP provides a concept of core collection, a subset of a germplasm milestone of an international collaborative project for collection of a species that captures most of the ge- the conservation and exploitation of European peach netic diversity while reducing redundancy, has rep- germplasm resources and, ultimately, as a true heritage resented an ideal solution for reducing costs and for future generations. increasing the efficiency of conservation programs (Frankel and Brown, 1984). Several allocation methods have been developed for selecting core collections, RESULTS attempting to maximize allelic richness or allele cover- age (MSTRAT, PowerCore, and GenoCore), minimize Criteria for the Construction of a Reference Panel of Peach or maximize genetic distance (GDOpt and SimEli), Accessions and Seedlings or simultaneously accommodating for multiple cri- teria (Core Hunter; Gouesnard et al., 2001; Kim et al., The PRP collection was built with the aim of 2007; Thachuk et al., 2009; Odong et al., 2011; selecting a reduced germplasm pool, reflecting the Krishnan et al., 2014). However, the effectiveness of original genetic and phenotypic diversity (Fig. 1) the sampling strategies varied depending on the and the cultural and socioeconomic value of peach objective of the core collection, the statistical ap- cultivation, for its exploitation in future breeding proach for its definition, and the measures for eval- programs. A four-step procedure was followed (ex- uating its quality (Odong et al., 2013). Furthermore, emplified in Fig. 2): (1) definition of the PRP domain; beyond statistical considerations, other aspects are (2) establishment of PRP size; (3) identification of the often considered by the institutions hosting the col- selection criteria; and (4) choice and allocation of the lection, such as historical and socioeconomic impor- entries. tance, relevance for breeding activities, popularity among growers and consumers, and distinctive Definition of the PRP Domain phenotypic characteristics. In peach, the absence of wild or feral populations To build a resource representing peach diversity makes ex situ collections the main valuable reservoirs and breeding history, the starting point was the ge- of allelic variability for many traits not yet exploited netic material characterized in the framework of the in current breeding programs. Remarkable progress FP7 European project FruitBreedomics in a coordi- has been achieved in the phenotypic and genotypic nated effort involving different universities and re- characterization of peach genetic resources (Badenes search institutions across Europe and China. A total et al., 2015; Cirilli et al., 2018; Yu et al., 2018), taking of 1,580 Prunus spp. accessions (comprising P. persica advantage of genome sequencing and the develop- and its hybrids with P. davidiana and almond) were ment of cutting-edge genotyping tools (Verde et al., phenotyped and genotyped with the 9K single-nucleotide 2012, 2017; Aranzana et al., 2019). In the framework of polymorphism (SNP) array, as previously described the European collaborative project FruitBreedomics (Micheletti et al., 2015). The inclusion of only peach (Laurens et al., 2018), a coordinated characterization (including P. ferganensis; Verde et al., 2012) among all of peach collections has been accomplished across the available Prunus spp. accessions was the leading relevant European repositories (Micheletti et al., 2015; concept behind the definition of the PRP reference Hernández Mora et al., 2017), promoting increased collection. Indeed, as a consequence of many factors utilization of resources and encouraging the shar- (genetic diversity, evolution history, mating system, ing of conservation responsibilities. For example, geographical distribution, etc.), sampling strategies the Prunus Working Group within the Fruit Net- for the inclusion of wild relatives (e.g. species of the work in the European Cooperative Program on Plant Amygdalus subgenus) may substantially differ from Genetic Resources is dealing with Prunus spp., includ- those for a cultivated species (e.g. peach; Brown and ing peach (Benediková and Giovannini, 2013). Never- Marshall, 1995). Moreover, to avoid limitations on the theless, long-term maintenance of collections remains exchange of plant material, the domain was restricted particularly challenging due to intrinsic vulnerabilities to European repositories. Based on these criteria, the (e.g. direct exposure to environmental variables and starting panel for building the PRP amounted to a pathogens) and costs for in vivo maintenance through total of 1,262 P. persica accessions (FB_1262). Besides vegetative propagation to preserve the original gen- accessions, seedlings from controlled crosses also otypes. Moreover, compliance to phytosanitary re- represent a valuable source of informative materials quirements hampers the sharing of resources among for both genetic analysis and breeding (or prebreeding) institutions, each having its own stock of materials, activities. For this reason, 1,467 individuals from 18 634 Plant Physiol. Vol. 184, 2020 Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Multisite PeachRefPop Collection Figure 1. Overview of the range of phenotypic diversity in the PRP. Images are as follows: co- lumnar and standard tree growth habit (top left and right images, respectively); heart-shaped, round, and flat fruit (top and third rows); range of fruit flesh, skin color, and overcolor (second and bottom rows); variation in flower morphology and color (third and fourth rows); and fruit size varia- tion (fourth row, first and last images). progeny and their parents (including an interspecific other through random sampling. The maximization cross with a P. davidiana accession), also analyzed procedure (M strategy by Schoen and Brown [1995]) during the FruitBreedomics project (Hernández Mora is based on the sampling of the total allelic diversity et al., 2017), were considered in the construction observed at marker loci in the least number of entries. process. By plotting the genetic diversity measured over the core size, a convex curve was obtained, indicating the Establishment of the PRP Size presence of redundancy across the European peach germplasm collection. The inflection point, corre- The definition of the size is one of the most criti- sponding to a plateau in the increase of diversity, was cal decisions for the establishment of a reference pop- observed at the level of core 26. At this core size, ulation. For fruit tree crops, the costs of in vivo 99.9% of the total genetic diversity was captured in maintenance are particularly onerous and, together the core obtained with the M method in comparison with long-term space availability in the field, the main with 93.5% with random sampling (Fig. 3). The out- limiting factor of running a germplasm collection. In the performance of the optimized versus the random perspective of analyzing the interactions between gen- selection was observed across all the core sizes, in- otype and environment and/or management practices, dicating that the maximization strategy was more or performing genetic studies such as GWAS and GS, efficient and was preferred for conservation purposes an adequate panel size and experimental design are key in our germplasm. factors for the power and reliability of statistical anal- According to some recent works in peach (for re- yses. On the other hand, for agrobiodiversity conser- view, see Aranzana et al., 2019), about 100 to 150 vation purposes, the least number of accessions to unrelated accessions usually provide an adequate include in a core set depends on the level of genetic resolution for identifying major loci or developing repetitiveness present in the original germplasm pool. prediction models. In light of all the above premises, The first step toward the establishment of the PRP size an ideal target number of 400 entries was deemed was the assessment of the allelic richness and redun- adequate for allocating a minimum of 150 accessions dancy observed at marker loci. Two series of core col- and a maximum of 250 seedlings from progeny (in- lections of incremental size were generated, one based cluding the parents) based on the outputs of the se- on the genetic diversity (maximization method) the lection criteria. Plant Physiol. Vol. 184, 2020 635 Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Cirilli et al. Figure 3. Assessment of allelic redundancy observed at marker loci in the starting panel (FB_1262). Core collections of incremental size were Figure 2. Graphical summary of the overall scheme followed for generated, based on the maximization (OPT) and random sampling selecting the PRP collection. From the starting panel of 1,262 acces- (RAN) methods in MSTRAT software, using a set of 445 SNPs. Data sions, 169 accessions were selected combining two sets: 69 acces- points represent averaged values over five independent repetitions for sions extracted from genetic and phenotypic diversity analyses and each size. taking into account the availability of whole-genome resequencing (WGRS) data; and 100 accessions selected by an empirical strategy from an experts panel considering breeding and traditional value genetic background, scientific relevance, and, above along with genetic structure. These were supplemented with 214 all, priority traits for breeding. seedlings from crossing populations of scientific importance and their respective 20 parents. The total number of entries in the PRP amounts to 403. Choice, Evaluation, and Description of the PRP Accessions Capturing the maximum amount of genetic diversity Identification of the Selection Criteria present in the entire collection while reducing redun- dancy was the primary driver for sampling the first PRP In spite of the genetic redundancy observed and ex- subset (the core set). For this purpose, the advanced M cluding the rare cases of synonymy, the vast majority method, implemented in the software PowerCore (Kim of the accessions are not overlapped across the vari- et al., 2007) through a modified heuristic algorithm, ous collections, being conserved for a multitude of was used to select a core from the initial panel of ac- reasons and purposes, including scientific research, cessions, based on a set of 3,894 filtered SNPs previously agrobiodiversity preservation, or support to breeding described by Micheletti et al. (2015). After superimposing activities. To reconcile these reasons with the aim of 17 accessions with available whole-genome resequenc- creating a feasible, usable, and multipurpose reference ing data, an ideal core of 69 accessions (PwC_69) collection to be shared among European institutions, a was extracted, representing a sampling size of 5.5% mixed approach was considered for selecting the ac- (Supplemental Table S1). Considering the many varia- cessions. A subset of entries was sampled using an bles that could affect the actual availability of materials analytical strategy, based on the criteria of maximizing for grafting, a flexible approach was further developed genetic (and phenotypic) diversity, also taking into ac- to rank each accession of the whole panel based on count the availability of whole-genome resequencing genotypic and phenotypic diversity. Four different sets data; the remaining entries were selected using an made up of 100 cores of 70 entries each were con- empirical strategy, leveraging the knowledge of an ex- structed with MSTRAT by setting different combina- perts panel (e.g. breeders, experienced scientists, and tions of genotypic data (nine subsets of SNPs extracted curators of each repository) and considering the tradi- approximately every 1.8 Mb to avoid linkage be- tional and historical value at national and/or regional tween them) and phenotypic data (seven qualitative levels, the relevance for breeders, growers, and con- and 10 quantitative traits, following transformation of sumers, taking into account agronomical or pomologi- the latter into categories; Supplemental Table S2). Ac- cal characteristics. Moreover, to maintain a balanced cessions were ranked in groups according to the av- representation of the genetic structure of the whole erage frequency of inclusion across the four sets collection, the empirical selection of accessions was (Supplemental Table S3). Combining the core popu- partially supported by information on population lation extracted by PowerCore with the MSTRAT structure (structure and principal component analy- ranking list resulted in a shortlist of 69 accessions (41 sis [PCA] data are available from Micheletti et al. and 28, respectively, indicated as Core_69), ensuring [2015]). Complementing the choice of accessions, the inclusion of the maximum possible level of ge- seedlings were selected based on the availability of netic diversity. For the completion of the final PRP_X detailed genotypic and/or phenotypic information, panel, the remaining 100 accessions (Priority_100) 636 Plant Physiol. Vol. 184, 2020 Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Multisite PeachRefPop Collection were empirically selected by experts, following the values, particularly for E-NE distance, indicating the above-specified criteria. presence of a certain redundancy within the panel. Estimates of genetic diversity were used to compare The population structure of peach germplasm was the starting panel FB_1262, the core collection obtained well represented in the PRP_X, in agreement with the by PowerCore (PwC_69), and the final set of PRP ac- presence of clusters of breeding-derived accessions cessions (PRP_X), composed by joining Priority_100 (further separated in peach- and nectarine-type groups), and Core_69 subsets. In addition, Core Hunter software Occidental traditional and admixed entries with preva- was used to create additional core sets, either of 69 or lent Oriental origins (Fig. 4A). Structure was also pre- 169 entries, based on the optimization of various crite- served in the other core sets, except for that selected ria, including allelic coverage (CV_169) and three through the E-E distance algorithm, tending to over- distance-based algorithms A-NE (AN_69 and AN_169), sample the admixed group (Supplemental Fig. S1). PCA E-NE (EN_69 and EN_169), and E-E (EE_69 and was also run to check the distribution of the PRP_X with EE_169). Concerning parameters accounting for allelic respect to the other sets, and the first two components diversity, all sets showed high and similar values for explained 15.9% and 8.4%, respectively, of the total the allelic coverage, while the number of effective al- variance detected. In the scatterplot, 95% confidence leles and expected heterozygosity were slightly lower ellipses show almost overlapping areas (except for for the Priority_100 subset (Table 1). The Shannon- EE_169), confirming that the PRP_X panel was well Weaver diversity index (SH index) was comparable distributed to represent the structure of the starting among the different subsets, ranging between 0.595 in germplasm (Fig. 4B). Finally, a neighbor-joining (NJ) tree, EE_169 and 0.534 in Priority_100. The SH index gen- based on the dissimilarity matrix between the whole erally displays higher values in the presence of a re- FB_1262 panel, was also built to assess the distribution duced redundancy (Peet, 1975). In contrast, values of of PRP accessions (Fig. 4C). observed heterozygosity tended to be more variable, A number of accessions of historical and regional ranging from a minimum of 0.202 in PwC_69 to a importance, mostly belonging to the Occidental tradi- maximum of 0.318 in AN_69. According to (Odong tional cluster, were included. For example, French cul- et al., 2013), distance-based criteria were used for fur- tivars dating from the late Middle Ages (cv Grosse ther evaluations, such as the minimization of A-NE Mignonne, cv Millecoton de Septembre, cv Reine des distance, particularly indicated for generalist collec- Verges, and cv Brugnon Violet; Okie et al., 2008), tra- tions (as the PRP), and maximization of either E-E or ditional nonmelting Spanish cultivars (cv Amarillo de E-NE, both suitable for core collection representing the Agosto 1, cv Calante, cv Campiel, cv Jesca, cv Groc extremes of the entire collection. A-NE distance gener- Abel, and cv Groc Alto; Badenes et al., 1998; Wünsch ally tends to decrease along with the increase of core et al., 2006), and the Italian cv Crasiommolo Rosso (a size, being minimized in the AN_169 and AN_69 core white-fleshed nectarine belonging to the ‘Sbergie’ type) sets (0.137 and 0.172, respectively), a priori optimized and cv Poppa di Venere, first reported at the end of the using this selection criterion. Despite the relative low 18th century (Majoli, 1790). The richness of the Italian performance of both Priority_100 and Core_69 (0.188 peach germplasm was also widely represented by ma- and 0.195, respectively), the PRP_X set showed low terials from several regions, including Sicily (cv Imera, values for this index (0.165), most probably as a con- cv Tardiva di Ficarazzi, cv Settembrina di Bivona, and sequence of the increased size. Regarding E-E and cv Gialla di Moavero; Marchese et al., 2005), Campania E-NE, PRP_X (as well as Priority_100) showed lower (cv Zingara Nera), Apulia (cv Percoco di Turi), Liguria Table 1. Genetic analysis and parameters for the different core subsets A-NE, Average distance between each genotype of the collection and the nearest entry; CV, percentage allelic coverage; E-E, average entry-to-entry distance; E-NE, average distance between each entry and the nearest entry; He, expected heterozygosity; Ho, observed heterozygosity; MR distance, average Modified Rogers genetic distance; Ne, number of effective alleles. MR Distance Set Name Ne SH Ho He CV EE A-NE E-NE FB_1262 1.621 0.547 0.292 0.367 0.995 0.285 0 0.131 PwC_69 1.675 0.574 0.202 0.39 0.987 0.318 0.203 0.237 EE_69 1.705 0.587 0.234 0.401 0.992 0.347 0.209 0.229 AN_69 1.645 0.560 0.318 0.378 0.977 0.286 0.172 0.210 EN_69 1.704 0.587 0.269 0.402 0.991 0.334 0.207 0.275 CV_169 1.638 0.556 0.285 0.375 0.995 0.302 0.163 0.212 EE_169 1.721 0.595 0.224 0.408 0.994 0.330 0.183 0.191 AN_169 1.643 0.559 0.300 0.377 0.987 0.290 0.137 0.203 EN_169 1.683 0.578 0.277 0.394 0.993 0.315 0.175 0.256 Core_69 1.713 0.593 0.247 0.406 0.988 0.303 0.195 0.212 Priority_100 1.597 0.534 0.283 0.356 0.979 0.277 0.188 0.179 PRP_X 1.647 0.563 0.270 0.379 0.988 0.290 0.165 0.180 Plant Physiol. Vol. 184, 2020 637 Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Cirilli et al. (cv Michelini), Emilia-Romagna (cv Buco Incavato, cv San Varano 2 and cv San Varano 3, and cv Rosa del West, this last used for the preparation of the famous cocktail Bellini), and Tuscany (cv Regina di Londa; Gallesio, 2003; Monte et al., 2006; Liverani and Giovannini, 2016). Early breeding materials, mainly from United States programs and founders of most of the currently cultivated materials, are also in- cluded, along with worldwide commercial cultivars (Supplemental Table S4). Finally, PRP accessions encompassed a wide range of phenotypic variability for traits related to fruit quality, resistance or tolerance against major diseases (brown rot, powdery mildew, leaf curl, aphids, and Sharka disease), tree growth habit, and phenology (Fig. 5; Supplemental Table S4). Choice and Description of the PRP Progeny Seedlings from 15 cross populations from the re- search and breeding activities of some European uni- versities and institutions were also added. Most of these materials were already described in depth (Hernández Mora et al., 2017). The leading criterion for the choice of seedlings was the effective segregation of priority traits in peach, mainly related to phenology (fruit develop- mental period and maturity date), fruit quality (fresh weight, soluble solid content, titratable acidity, texture, and aroma), and disease resistance (brown rot, pow- dery mildew, green peach aphids, and PPV; Table 2). A range of breeding materials was considered, such as F1, F2, and BC1 populations as well as hybrids with P. davidiana, particularly interesting as a source of PPV resistance (Decroocq et al., 2005). Experimental Design and Orchard Sites Description The PRP was established in five institutions from three countries (Greece, Italy, and Spain; Fig. 6A): (1) Institute of Agrifood Research and Technology (IRTA) in Gimenells, Catalonia region, Spain (ES); (2) Murcia Institute of Agri-Food Research and Development (IMIDA) in Mula, Murcia region, Spain (ES); (3) Centro di Ricerca per le Produzioni Vegetali (CRPV) in Imola, Emilia-Romagna region, Italy (IT); (4) Institute of Plant Breeding and Genetic Resources (IPB&GR) in Naoussa, Imathia region, Greece (GR); and (5) Research Centre for Olive, Fruit, and Citrus Crops (CREA) in Rome, Figure 4. Genetic structure and phylogenetic analysis of PRP acces- Italy (IT). sions. A, Population structure estimated in the whole panel (FB_1262) and PRP accessions (PRP_X), as estimated for K (number of a priori For each accession and seedling, a single mother cluster) equal to 4. OCB, Occidental breeding; OCT, occidental traditional; plant was propagated through grafting on a common ORT, oriental traditional, respectively. B, PCA analysis of the subsets cv GF677 rootstock by the same nursery. Plants were with a core size of 169 entries. Scores for each accession were obtained grafted in the same year (2015) to obtain trees of the from the work of Micheletti et al. (2015). The 95% confidence ellipses in same age. To ensure an adequate compromise between the scatterplot were estimated using PAST software. C, NJ phylogenetic the number of replicate trees and sustainable costs of tree. Blue squares indicate accessions with traditional and historical maintenance, an augmented design with replicated value, violet circles indicate the other PRP accessions, and colors reflect control checks was adopted in all sites except Rome, the population structure. hosting a partial copy of the PRP (accessions only, without randomization). 638 Plant Physiol. Vol. 184, 2020 Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Multisite PeachRefPop Collection Figure 5. Distribution of main pheno- typic traits in the PRP accessions. In the maturity date plot, UE, E, M, L, and VL indicate ultra-early, early, medium, late, and very late ripening accessions, respectively. SSC, Soluble solids con- tent. In the fruit texture plot, four major texture groups are shown: nonmelting (NM), melting (M), slow softening (SwS), and stony hard (SH). Accessions and seedlings were arranged in two between experimental sites, the three accessions cv Big blocks (M1 and M2) according to the following design Top, cv Springcrest, and cv Nectaross from the A group (Fig. 6B). The M1 block, composed of two subblocks were included with a higher replication and randomly (M1.1 and M1.2) each including the entire PRP collec- distributed over the M1.1 and M1.2 subblocks (at least tion of 169 accessions plus 20 cross parents (A group) five additional trees of each genotype for each sub- and 214 seedlings (S group), for a total of 403 genotypes block). The M2 block, composed of two subblocks in each subblock. Taking into account the physical (M2.1 and M2.2) each including half of the PRP collec- layout of the design for each location/field combination tion (85 accessions plus 10 cross-parents and 112 seed- (i.e. the number of rows and the number of positions lings). In each site, the M2.1 and M2.2 subblocks include per row), each accession and seedling was randomly the same set of entries (i.e. the same half of the collec- assigned to a position within a subblock (as illustrated tion), randomly assigned in each subblock, plus the for the Gimenells location in Supplemental Table S5). replicated control checks previously described (at least To assess and correct for spatial variation within and three additional trees of each entry for each subblock). Table 2. Description of the progeny used for establishing the PRP collection Trait abbreviations are as follows: BR, brown rot; FD, flowering date; FW, fruit weight; GPA, green peach aphid; MD, maturity date; PM, powdery mildew; PPV, Plum pox virus; SH, stony hard texture; SSC, soluble solid content; SwS, slow-softening texture; TA, titratable acidity. Type of Seedling Cross (Parents) Acronym Institution Trait(s) Progeny No. ‘Bolero’ 3 ‘Oro A’ B3O UMIL, Milan F1 9 MD, SSC, FW, skin overcolor, aroma ‘Contender’ 3 ‘Elegant Lady’ C 3 EL UMIL, Milan F1 14 BR, MD ‘Max 10’ 3 ‘Rebus 028’ M3R UMIL, Milan F1 9 MD, TA, SSC, FW, SwS ‘Sweetfire’ 3 ‘Garcica’ Sf 3 G UMIL, Milan F1 15 MD, TA, SSC, FW, SwS ‘Belbinette’ 3 ‘Nectalady’ Bb 3 Nl IRTA, Lleida F1 20 FD, MD, TA, SSC, FW ‘Big Top’ 3 ‘Nectaross’ Bt 3 Nr IRTA, Lleida F1 19 FD, MD, TA, SSC, FW ‘Big Top’ 3 ‘Armking’ Bt 3 Ak IRTA, Lleida F1 18 FD, MD, TA, SSC, FW ‘Subirana’ 3 ‘Feraude’ PN643 IRTA, Lleida F1 7 Fruit shape ‘Summergrand’ 3 P. davidiana P1908 SD INRA, Avignon F1 6 PM, PPV ‘Zephyr’ 3 [((‘Summergrand’ (S) 3 P. davidiana BC2 INRA, Avignon BC2 13 FD, PM, PPV, TA, SSC, FW P1908)) 3 S] ‘Pamirskij 5’ 3 ‘Rubira’ P3R INRA, Avignon F2 13 PM, GPA, foliage color FRF 1495 3 FRF 1148 (Ma 16-03-059) POP1376 CREA, Forli F1 17 PM, fruit pubescence IFF 983 3 Ma 25-01-042 POP1115 CREA, Forli F1 17 TA, SwS, aroma FRF 1695 3 FRF 1681 POP1095 CREA, Forli F1 19 SH FRF 813 3 FRF 691 POP1039 CREA, Forli F1 18 Skin overcolor Plant Physiol. Vol. 184, 2020 639 Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Cirilli et al. Figure 6. Experimental design and PRP orchards layout. A, Google Maps satellite images of the established PRP orchards across the different Eu- ropean sites. B, Experimental design of multisite PRP. A schematic example is provided for the Gimenells location. Accessions (A) and seedlings (S) in each block and subblock were completely randomized. The M1.1 and M1.2 subblocks each include a full copy of the collection (189 acces- sions, 214 seedlings) plus replicate checks (C) of the accessions cv Big Top, cv Nectaross, and cv Springcrest (five additional trees for each sub- block). The M2.1 and M2.2 subblocks include half of the PRP collection and each site has a different half, chosen according to a pairwise design scheme. To this end, accessions (excluding control checks) and seedlings were randomly assigned to eight disjoint subgroups (A1–A4 and S1–S4) of approximately equal size and four of them assigned so that each site shares at least one A or one S group with the other sites. In the ex- ample, each M2 subblock at Gimenells is com- posed of A1 and A2 (46 and 46 accessions, respectively, plus the three checks for a total of 95), S1 and S2 (54 and 54 seedlings, respectively, for a total of 108), and three other additional replicates for each check (nine trees). Gimenells shares the A1 and S1 groups with Mula, S2 with Imola, and A2 with Naoussa. The composition of the M2 block is not the same across months, January and August, respectively) to the sites (i.e. each site has a different half of the collection), warmer conditions of Mula (7.9°C 6 3.7°C and 27.1°C 6 chosen according to a pairwise design scheme (Fig. 6B). 2.6°C, respectively; Fig. 7A). The fulfillment of the First, excluding control checks, accessions and seed- chilling requirement (i.e. the period of cold temperatures lings of the PRP collection were randomly divided into needed for overcoming endodormancy) is a parameter eight disjoint subgroups, A1 to A4 (of 48, 47, 47, and 48 of utmost relevance for peach reproductive phenology. accessions, respectively) and S1 to S4 (of 54, 53, 54, and According to the chilling hours model (Weinberger, 53 seedlings, respectively), then four subgroups were 1950), assigning 1 h for each hourly temperature be- assigned so that each site shares at least one A or one S tween 0°C and 7.2°C, accumulation patterns widely with the other sites. For example, the Imola location ranged from 1,762 6 124 chilling hours at Naoussa to shares A4 and S3 with Naoussa, A3 with Mula, and S2 693 6 159 chilling hours at Mula. Also, precipitation with Gimenells. This partial replication design is such differently affected the selected sites, with Imola having that, within the full design, all subgroups were well the wettest conditions (964 6 218 mm per year) and both connected. Spanish locations having the driest (Gimenells 361 6 95 The geographic location of each site as well as the and Mula 336 6 75; Fig. 7B). basic climate and soil parameters are shown in Table 3. The sites covered a range of latitude from about 38°N in Mula (southeastern Spain) to 44°N in Imola (northern DISCUSSION Italy), while altitude spanned from near sea level in Imola and Rome (53 and 73 m, respectively) to 278 m at The concept of the PRP arises from the growing the Mula site. Although all sites are included in the awareness about current and common issues regarding Mediterranean zone, climates widely range from semi- ex situ peach conservation across European institu- arid in Mula (warm winter and hot summer) to sub- tions. Fluctuations in funding availability and intrinsic continental in Imola and Naoussa (with moderately cold constraints of living orchard collections threaten the winter). Average monthly temperatures (1999–2018 time long-term preservation of diversity resources, causing a series) varied from the colder regimes of Naoussa (1.7°C progressive loss of valuable materials. Reference or core 6 3.9°C and 23.5°C 6 2.9°C in the coldest and hottest collections have been designed for several fruit tree 640 Plant Physiol. Vol. 184, 2020 Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Multisite PeachRefPop Collection species, such as olive (Olea europaea; Khadari et al., 2003; Features include geographic coordinates, altitude, average annual minimum and maximum temperatures, and cumulative annual precipitation (data series 1999–2018). Chilling accumulation El Bakkali et al., 2013; Belaj et al., 2012), grape (Vitis S.O.M. 1.9 1.5 2.6 2.5 2.6 was calculated according to the Chilling Hours (CH) model as the sum of hourly temperatures between 0°C and 7.2°C during the dormant season. S.O.M., Soil organic matter content. % spp.; Laucou et al., 2011), cherry (Prunus avium; Campoy et al., 2016), apple (Malus domestica; Gross et al., 2013), and apricot (Prunus armeniaca; Krichen 7.7 7.2 7.8 6.8 7.7 pH et al., 2012). Nevertheless, they have mainly been cre- Soil ated for improving resource allocation in the context of a single institution or repository. The development of a Sandy, loam Sandy, loam Sandy, loam transnational and shared strategy provides the most Silty, loam Texture promising opportunity in the conservation approach. Actual establishment of the PRP has required huge Clay coordination efforts and faced the effective availability of materials, the difficulties of their exchange, and the success of clonal propagations (particularly for old, 224 195 159 124 133 Accumulation often unique, accessions). The sampling strategy for the Chilling 1,171 6 1,753 6 693 6 1,762 6 1,637 6 CH PRP has been defined to accommodate multiple pur- poses while maintaining the maximum possible diver- sity compared with the starting panel. The final panel of accessions was assembled by the combination of two different subsets: the first (Core_69), ensuring Cumulative Precipitation the preservation of the total allele number with the 731 6 155 964 6 218 818 6 160 minimum number of accessions, was extracted by 336 6 75 361 6 95 mm widely adopted maximization strategies, either using a class coverage criterion (in PowerCore) or SH index Climate (in MSTRAT), with the latter penalizing redundancy. The second subset, accommodating for other scopes (Priority_100), was chosen by experts with a robust knowledge of the genetic structure in peach, providing Average Annual Temperature a reliable criterion for assisting selection. (Minimum/Maximum) As a whole, genetic analysis supports that the PRP composition is highly representative of the diversity 11.2/18.9 12.0/24.5 9.5/17.9 8.1/17.2 9.1/18.9 of peach germplasms present in European collections, °C as it retains all the allelic variability present within the starting panel, specifically targets defined genetic clusters according to the genetic structure, and includes most relevant phenotypic traits. Indeed, differences among the various sampling strategies were negligible for allelic coverage, expected heterozygosity, and SH Altitude index, revealing a buffer effect toward optimization 79 53 278 119 259 m criteria. Such an effect could be expected, since peach Table 3. Basic pedo-climatic features of the five PRP locations has experienced a severe domestication bottleneck with a reduction of genetic diversity, followed by a strong Geographic Coordinates 41°479 N/12°339 E 44°209 N/11°459 E 40°379 N/22°069 E artificial selection during domestication and modern 41°399 N/0°239 E 38°3 N/1°259 O improvement (Verde et al., 2013; Yu et al., 2018; Li et al., 2019). This is also reflected in the narrow genetic bases of peach germplasm available across the main Euro- pean repositories. Thus, the high level of allelic redun- dancy allows selecting many different subpopulations able to retain the same amount of genetic variation. In spite of this, a preliminary validation using a distance- IPB&GR, Naoussa (Greece) based criterion not used in the selection stage showed a minimized A-NE index, the most indicative for evalu- IRTA, Gimenells (Spain) ating the quality of multipurpose collections (Odong IMIDA, Mula (Spain) CREA, Rome (Italy) CRPV, Imola (Italy) et al., 2013). Conversely, E-E and, particularly, E-NE Site indices were less optimized, due to a certain redun- dancy in the Priority_100 subset (i.e. a higher number of genotypes providing unique alleles). This was mainly due to the inclusion of accessions of traditional and breeding values, respectively belonging to the Occi- dental traditional and Occidental breeding clusters, Plant Physiol. Vol. 184, 2020 641 Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Cirilli et al. Figure 7. Climatic profiles of PRP sites. A, Trend of minimum and maximum daily air temperatures at the five PRP locations (averaged from the 1999–2018 time series). Thick lines show smoothed mean temperatures. B, Average monthly precipi- tation (in millimeters). characterized by a very narrow genetic background. difficulties in material propagation and exchange. The Clearly, the inclusion of these materials is crucial in the PRP aims to fill this gap, as the replicated design and overall perspective of balancing diversity and useful- the different pedo-climatic conditions across sites are ness, as they integrated various fundamental qualities, particularly indicated for the dissection of interactions such as popularity, prestige, tradition, and breeding. A between genotype and environment and/or manage- similar mixed strategy was also recently optimized for ment practice. The PRP locations cover major climatic creating a core collection for Swiss pear (Pyrus commu- zones of the Mediterranean area, from semiarid con- nis) germplasm (Urrestarazu et al., 2019). ditions of southern Spain to subcontinental condi- Climate challenges in peach-growing areas increase tions of northern Italy and Greece, determining a broad the need for resilient cultivars able to maintain pro- range of temperatures and precipitation patterns. In ductivity while showing an enhanced capacity for particular, sites are characterized by different rates and adaptation to suboptimal conditions. Nevertheless, re- amounts of chilling and heat accumulation, which will silience and adaptive traits often have a complex in- be particularly useful for the dissection of traits asso- heritance and a strong interaction with the environment ciated with reproductive phenology, such as blooming or cultivation practice (Kissoudis et al., 2016). The and fruit ripening time. The proximity of experimental partitioning of phenotypic variation into genotypic, sites to major production areas provides an added environmental, and their interaction components in- value for the translation of scientific outcomes. The in- volves ad hoc experimental designs and integration of clusion of both accessions and seedlings from various field data on a common set of genetic materials under a crosses allows the development and testing of statistical range of different environmental/management condi- approaches for genomics-assisted breeding, such as tions. Multienvironment trials have been extensively joint linkage-association analysis (Yu et al., 2008; Lu used to study genotype-by-management interactions, et al., 2010) and genome-wide selection (Resende carry out GWAS, and develop GS models for complex et al., 2012; van Nocker and Gardiner, 2014), or sys- traits in annual crops (Malosetti et al., 2013; Gutiérrez tematic QTL validation (Peace et al., 2014). Also, the et al., 2015; Zhu et al., 2018; Bustos-Korts et al., 2019) or integration of omics (including epigenomics) data may study genotype-by-environment interactions in forest improve our understanding of physiological changes in trees (Li et al., 2017). In contrast, such experimental response to environmental stimuli and constraints. designs are lagging in fruit trees, largely because of the The PRP multisite experimental design was estab- need for large and diverse germplasm sets for quanti- lished with a complete randomization of genotypes tative genetics analyses and the above-mentioned (accessions and seedlings) within each subblock and 642 Plant Physiol. Vol. 184, 2020 Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Multisite PeachRefPop Collection replicate checks to account for spatial variability. The materials, SNP genotyping, and phenotypic data for seven monogenic traits has been previously described (Micheletti et al., 2015). SNP geno- rationale behind the choice of such a design mainly typing data were obtained from the Genome Database for Rosaceae derived from the possibility of a direct comparison with (https://www.rosaceae.org/publication_datasets; accession no. tfGDR1013). standard reference varieties. A drawback of this ap- Phenotypic data reported in Supplemental Table S4 were obtained from proach is the relatively few degrees of freedom for Micheletti et al. (2015) and the FruitBreedomics database (http://bioinformatics. experimental errors, lowering the power to detect tecnoparco.org/fruitbreedomics/). differences among genotypes. The use of a designs (Patterson and Williams, 1976) and derived row- Construction of Core Subsets column designs (John and Eccleston, 1986) might be The advanced M strategy implemented in PowerCore v1.0 (Kim et al., 2007) statistically more powerful, especially to estimate con- using 3,894 SNP markers was carried out to extract a core subset able to capture trasts between genotypes and improve the estimation of all the alleles observed in the entire collection. The size of the final core col- spatial variation (e.g. due to different soil composition lection depends on the level of variability and redundancy present in the whole within the orchard). The identification of optimal de- panel and cannot be set a priori. Seventeen kernel accessions with available whole-genome resequencing data were superimposed through the preferential signs for a large number of genotypes is still challenging selection tool, which retains the accessions defined by the user without vali- (Cullis et al., 1998). The PRP will allow validation of the dation. The standard M strategy implemented in MSTRAT (Gouesnard et al., performance of this experimental design on fruit trees 2001) was also applied. The MSTRAT algorithm selects a subset of n accessions and provide a foundation for future planning of multi- from the N accessions of the entire collection by maximizing the number of site collections. alleles (and/or trait classes) at each locus. The sampling size estimated with PowerCore was set as the default parameter, and four sets of 100 core collec- The PRP has been grafted on a single ‘GF677’ root- tions were constructed by using different combinations of genotypic and phe- stock, a P. amygdalus 3 P. persica hybrid. ‘GF677’ is the notypic data. Due to the restraints in the number of variables MSTRAT is able to most widespread rootstock, mainly for its growth manage, different subsets of approximately 100 SNPs each were obtained vigor, excellent affinity, adaptation to limestone soils, through an ad hoc-developed Perl script program, by extracting one SNP every 1,800 kb, corresponding to the maximum boundary for linkage disequilibrium and tolerance to drought and replanting (Reighard and found in some subpopulations of the original plant material (Micheletti et al., Loreti, 2008). While the choice of a single rootstock is 2015). Seven qualitative and 10 quantitative traits (these last transformed into justified by the need of simplifying the experimental qualitative categories) were used as phenotypic data. For each run, the core size design, this precludes the assessment of scion-by-root- was set to 70, and 100 independent replicates with 100 iterations were gener- stock interaction. A number of Prunus spp. rootstocks ated. The SH index was used as a second criterion to classify core subsets. Redundancy was assayed through the Redundancy tool implemented in are currently available for peach, some of them har- MSTRAT, which samples two different sets of core collections of increasing size, boring interesting traits for resistance to soil pathogens as defined by the user, through the application of the maximization strategy or or abiotic stress conditions. Their integration into fea- random sampling. For this analysis, a subset of 445 SNP markers was pruned sible experimental designs will be the next challenge. from the whole set of 4,271 using Plink v1.07 with a window size of 50, a shift of 7, and a variance inflation factor of 2. Redundancy was assayed in the whole In perspective, the PRP should fulfill several pur- panel of accessions with a step of five in the first 100, five repetitions, and 50 poses, from research to education and traineeship of iterations. The Mixed Replica search algorithm implemented in the Core Hunter young breeders. A better understanding of diversity is II software (De Beukelaer et al., 2012) was used to generate a core collection of expected to encourage the use of broad-ranging germ- fixed size (either 69 and 169 entries) based on the optimization of the Modified plasm (maybe also in other existing ex situ collections) Rogers distance measure, with a weight of 1. For the evaluation of the quality of the different core subsets, genetic distance-based criteria were considered: the in breeding programs. In the last decades, the mission average genetic distance between all the entries of each core collection (E-E); the of many agriculture-oriented institutions has shifted average distance between each entry and the nearest neighboring entry for each from the traditional focus of establishing horticultural core collection (N-E); and the average distance between each genotype of the collections to a wider target of preserving germplasm entire collection and the nearest entry in each core collection (A-NE). The quality of each collection increased for lower values of A-NE (the maximum resources and agricultural heritage (Hammer et al., representation is obtained for AN 5 0, when each accession is represented by 2003; Havens et al., 2006). This objective is of utmost itself or by an identical duplicate) and higher values both for E-NE (maximizes importance for fruit tree species of ancient cultivation the average distance between each selected individual and the closest other history, such as peach. For these reasons, a number of selected item in the core) and E-E (maximizes the average distance between traditional and local cultivars (either old or relatively each pair of selected individuals in the core.). modern) have been included in the PRP as a safeguard of an integral part of the rural landscape and collective Genetic Diversity and Population Analyses memory. Since information and descriptions about lo- Genetic diversity measures were determined using GenAlex 6.41 (Peakall cal germplasms are scarce and often restricted to culti- and Smouse, 2006) and include number of effective alleles (the number of vation areas, their choice has been directly handled by equally frequent alleles required to give the observed level of heterozygosity), the curators of each repository, with the aid of experi- levels of observed and expected heterozygosity, and the SH index. Allelic enced breeders. coverage was calculated by the function allelic coverage implemented in Core Hunter II software. Population structure was inferred using the model-based clustering algorithm ADMIXTURE v1.22 (Alexander et al., 2009). From SNP data, the software identifies K a priori genetic clusters provided by the user, and MATERIALS AND METHODS for each individual, it estimates the probability of membership to each cluster. A preliminary analysis was performed by inputting successive values of K from 2 Data Sets to 6. The value of K that maximized the predictive accuracy was chosen based on a 10-fold cross-validation procedure with 10 different fixed initial seeds A set of 1,262 accessions was selected as representative of the peach (Prunus (Supplemental Fig. S2). PCA data were retrieved from a previous work persica) germplasm maintained in collections of four different European coun- (Micheletti et al., 2015). The 95% confidence ellipses in the scatterplot were tries (Supplemental Table S3). The complete list of institutions providing plant estimated using PAST software (Hammer et al., 2001). The phylogenetic tree Plant Physiol. Vol. 184, 2020 643 Downloaded from on October 6, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Cirilli et al. was built from a pairwise genetic distance matrix between individuals clustered Badenes ML, Martínez-Calvo J, Llácer G (1998) Analysis of peach germ- with the NJ method in TASSEL (Bradbury et al., 2007). Bootstrap replicates and plasm from Spain. Acta Hortic 465: 243–250 tree reconstruction were performed in MEGA7 software (Kumar et al., 2016). Bassi D, Monet R (2008) Botany and taxonomy. In D Layne, and D Bassi, eds, The Peach: Botany, Production and Uses. CABI, Wallingford, United Kingdom, pp 1–36 Experimental Design and Pedo-Climatic Analyses Belaj A, del Carmen Dominguez-García M, Atienza SG, Urdíroz NM, De In the experimental design, randomization was performed with the Genstat la Rosa R, Satovic Z, Martín A, Kilian A, Trujillo I, Valpuesta V, et al software (https://genstat.kb.vsni.co.uk/knowledge-base/hcitegen/). Meteo- (2012) Developing a core collection of olive (Olea europaea L.) based on rological time series from 1999 to 2018 were obtained from the European Centre molecular markers (DArTs, SSRs, SNPs) and agronomic traits. Tree for Medium-Range Weather Forecasts, except for Mula (Murcia, Spain), for Genet Genomes 8: 365–378 which data were available from a nearby weather station. Hourly temperature Bellucci A (1908) La Coltivazione del Pesco a Massalombarda. Tip Soc. series were obtained by linear interpolation of available trihourly data and Mazzini, Ravenna, Italy expressed in degrees Celsius. Cumulative precipitations were averaged and Benediková D, Giovannini D (2013) Review on genetic resources in the expressed in mm per month or year. Chill accumulation was calculated ECPGR Prunus Working Group. Acta Hortic 981: 43–51 according to the chilling hours model (Weinberger, 1950) as the sum of hourly Biasi R, Botti F, Cullotta S, Barbera G (2012) The role of Mediterranean temperatures between 0°C and 7.2°C during the dormant season (November 15 fruit tree orchards and vineyards in maintaining the traditional agri- to March 31). Soil texture was expressed according to U.S. Department of Ag- cultural landscapes. Acta Hortic 940: 79–88 riculture classification. Mineral composition, pH, and organic matter content Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, Buckler were determined according to standard procedures for soil analysis. ES (2007) TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics 23: 2633–2635 Brown AHD, Marshall DR (1995) A Basic Sampling Strategy: Theory And Supplemental Data Practice In Collecting Plant Genetic Diversity Technical Guideline. CABI Publishing, Wallingford, United Kingdom, pp 75–91 The following supplemental materials are available. Bustos-Korts D, Dawson IK, Russell J, Tondelli A, Guerra D, Ferrandi C, Supplemental Figure S1. Population structure estimated in the core sets Strozzi F, Nicolazzi EL, Molnar-Lang M, Ozkan H, et al (2019) Exome AN_169, EE_169, EN_169, and CV_169. sequences and multi-environment field trials elucidate the genetic basis of adaptation in barley. Plant J 99: 1172–1191 Supplemental Figure S2. Predictive accuracy (cross-validation error) of Byrne DH, Raseira MCB, Bassi D, Piagnani MC, Gasic K, Reighard GL, population stratification in both PRP_X and FB_1262 as determined by Moreno MA, Perez-Gonzalez S (2012) Peach. In ML Badenes, and DH ADMIXTURE software. Byrne, eds, Fruit Breeding. Springer, New York, pp 505–570 Supplemental Table S1. PowerCore output. Campoy JA, Lerigoleur-Balsemin E, Christmann H, Beauvieux R, Girollet N, Quero-García J, Dirlewanger E, Barreneche T (2016) Genetic di- Supplemental Table S2. MSTRAT outputs for the four settings. versity, linkage disequilibrium, population structure and construction of Supplemental Table S3. Accession ranking by MSTRAT frequencies. a core collection of Prunus avium L. landraces and bred cultivars. BMC Plant Biol 16: 49 Supplemental Table S4. PRP accession descriptions. Chagné D, Dayatilake D, Diack R, Oliver M, Ireland H, Watson A, Supplemental Table S5. Layout of subblock M1.1 as illustrated for the Gardiner SE, Johnston JW, Schaffer RJ, Tustin S (2014) Genetic and Gimenells location. environmental control of fruit maturation, dry matter and firmness in apple (Malus 3 domestica Borkh.). Hortic Res 1: 14046 Cirilli M, Flati T, Gioiosa S, Tagliaferri I, Ciacciulli A, Gao Z, Gattolin S, Geuna F, Maggi F, Bottoni P, et al (2018) PeachVar-DB: A curated ACKNOWLEDGMENTS collection of genetic variations for the interactive analysis of peach ge- We thank Claudio Buscaroli and Martina Lama (Centro Ricerche Produzioni nome data. Plant Cell Physiol 59: e2 Vegetali) for field assistance, Remo Chiozzotto (University of Milan) for lab Cirilli M, Rossini L, Geuna F, Palmisano F, Minafra A, Castrignanò T, assistance, and Michela Troggio (Fondazione Edmund Mach) for genotypic Gattolin S, Ciacciulli A, Babini AR, Liverani A, et al (2017) Genetic analyses. We are grateful to Fosco Vesely (University of Milan) for extracting dissection of Sharka disease tolerance in peach (P. persica L. Batsch). climate series. 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