Pérez de los Cobos et al. Horticulture Research (2021)8:11 https://doi.org/10.1038/s41438-020-00444-4 Horticulture Research www.nature.com/hortres ARTICLE Open Access Pedigree analysis of 220 almond genotypes reveals two world mainstream breeding lines based on only three different cultivars Felipe Pérez de los Cobos1,2, Pedro J. Martínez-García 3, Agustí Romero 1, Xavier Miarnau4, Iban Eduardo 2, Werner Howad2, Mourad Mnejja2, Federico Dicenta3, Rafel Socias i Company5, Maria J. Rubio-Cabetas5, Thomas M. Gradziel6, Michelle Wirthensohn 7, Henri Duval8, Doron Holland9, Pere Arús 2, Francisco J. Vargas1 and Ignasi Batlle1 Abstract Loss of genetic variability is an increasing challenge in tree breeding programs due to the repeated use of a reduced number of founder genotypes. However, in almond, little is known about the genetic variability in current breeding stocks, although several cases of inbreeding depression have been reported. To gain insights into the genetic structure in modern breeding programs worldwide, marker-verified pedigree data of 220 almond cultivars and breeding selections were analyzed. Inbreeding coefficients, pairwise relatedness, and genetic contribution were calculated for these genotypes. The results reveal two mainstream breeding lines based on three cultivars: “Tuono”, “Cristomorto”, and “Nonpareil”. Descendants from “Tuono” or “Cristomorto” number 76 (sharing 34 descendants), while “Nonpareil” 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; has 71 descendants. The mean inbreeding coefficient of the analyzed genotypes was 0.041, with 14 genotypes presenting a high inbreeding coefficient, over 0.250. Breeding programs from France, the USA, and Spain showed inbreeding coefficients of 0.075, 0.070, and 0.037, respectively. According to their genetic contribution, modern cultivars from Israel, France, the USA, Spain, and Australia trace back to a maximum of six main founding genotypes. Among the group of 65 genotypes carrying the Sf allele for self-compatibility, the mean relatedness coefficient was 0.125, with “Tuono” as the main founding genotype (24.7% of total genetic contribution). The results broaden our understanding about the tendencies followed in almond breeding over the last 50 years and will have a large impact into breeding decision-making process worldwide. Increasing current genetic variability is required in almond breeding programs to assure genetic gain and continuing breeding progress. Introduction cold climatic regions of both hemispheres. Almond world Almond [Prunus dulcis (Miller) D.A. Webb, syn. P. production (1,258,324 kernel tonnes) is led by the USA amygdalus (L) Batsch] is the most economically impor- (80%), Australia (6%), and Spain (5%)1. tant temperate tree nut crop worldwide. Due to increasing The origin of almond within the Amygdalus subgenus, demand, production areas are expanding into warm and including cultivated almond and its wild relatives such as P. fenzliana Fritsh, P. bucharica (Korsh.) Fedtsch, P. kuramica (Korsh.) Kitam., and P. triloba Lindl2,3 took Correspondence: Ignasi Batlle (ignasi.batlle@irta.cat) 1 Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Mas Bové, place ~5.88 million years ago4. Almond originated in the Ctra. Reus-El Morell Km 3,8, 43120 Constantí, Tarragona, Spain 2 arid mountainous regions of Central Asia, where it was Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Centre de Recerca en first cultivated around 5000 years ago5 and then moved to Agrigenòmica (CRAG), CSIC-IRTA-UAB-UB. Cerdanyola del Vallès (Bellaterra), 08193 Barcelona, Spain the Mediterranean region and later to California and the Full list of author information is available at the end of the article © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Pérez de los Cobos et al. Horticulture Research (2021)8:11 Page 2 of 11 southern hemisphere (South America, Australia, and the USA (with limited accessible genetic resources) and South Africa)6. Wide cultivation of almond, often under later in Russia and Mediterranean region (with more the more severe environments of Central Asia and the diverse germplasm available), rapid genetic advances were Mediterranean region, was possible because of the avail- achieved. In California, “Carmel” (introduced in 1966), as ability of a highly diverse gene pool, genetic recombina- “Nonpareil” pollinizer, was the first cultivar release with tion promoted by its self-incompatibility, and possibly, by extensive commercial impact. In Russia and the former interspecific hybridization and gene introgression invol- Soviet Union, several late-flowering and frost-hardy cul- ving other members of the Amygdalus subgenus. As a tivars were obtained in the 1950s with Primorskyi (date result, almond is an extremely variable species, with a high unknown) later used extensively for breeding in Europe. morphological and physiological diversity. This variability, In the Mediterranean region, late flowering, productive, measured with biochemical and molecular markers7–9, well-adapted, and resilient cultivars like Ferragnès (1973) has revealed that almond is the most genetically variable or Masbovera (1992) were released with great success. of the diploid Prunus cultivated species10,11. The French self-compatible cultivar Lauranne (1991) In the Mediterranean Region, 2000 years of almond showed a broad environmental adaptation, high produc- culture concentrated production to specific areas, where tion, and regular cropping. well-defined seedling ecotypes and local cultivars Although improved cultivars continued to be released, evolved2. By the turn of the 20th century, most of these the amount of progress per generation diminishes since almond-producing countries had identified locally desir- parents were continually drawn from the same gene able cultivars that were often seedling selections of pool13. This situation has resulted in a potential loss of unknown origin12. Thus, growers selected cultivars and genetic variability in new breeding stocks and cultivars. landraces, which represented a rich genetic diversity. Inbreeding depression in almond, expressed as low vigor, Most of these Mediterranean local cultivars have largely reduced flower number and fruit set, increased fruit disappeared from cultivation in the last 50 years13. abortion, lowered seed germination and seedling survival, Modern almond cultivation is based on a reduced number increased leaf and wood abnormalities, and loss of disease of cultivars (preferably self-compatible) grafted onto soil- resistance have been reported16–19. In addition, low self- adapted clonal rootstocks and cultivated under irrigated fruitfulness in self-compatible almond genotypes was conditions when possible. suspected to be due to inbreeding20. Modern almond breeding started in the 1920s with the Regarding breeding for self-compatibility, male parents making of controlled crosses and seedling selections to carrying the Sf allele and sharing the other S allele with the meet changing agronomic and market demands. Cur- female parent are commonly used. In addition, crossing rently, there are six active public breeding programs heterozygous self-compatible parents in breeding pro- worldwide: the USA (UCD-USDA), Spain (CITA, IRTA, grams has been suggested to obtain homozygous self- and CEBAS-CSIC), Australia (University of Adelaide), and compatible genotypes to be used in further breeding21. Israel (ARO). Some private breeding programs exist also Such breeding strategies can narrow the genetic variability in the USA. In addition, there were various breeding of crops when they lead to a reduced number of genotypes initiatives in Russia, France, Greece, Italy, and Argen- utilized as parents. tina13. Different breeding objectives were developed Summarizing, modern almond breeding and production according to regional agronomic, commercial, and market are dominated by a small number of widely distributed requirements. One of the main differences in the objec- and related cultivars. This situation can lead to a potential tives is nut shell hardness. Two types of almonds are bred: increase of inbreeding depression and genetic vulner- soft-shelled (in the USA and Australia mainly) and hard- ability, i.e., susceptibility of most of the grown cultivars to shelled (in most Mediterranean countries). Common aims biotic and abiotic stresses due to similarities in their of Mediterranean breeding programs are self- genotypes22,23. Therefore, it is needed to have up-to-date compatibility and late-blooming, as most traditional information of the relationships among genotypes used at almond cultivars are self-incompatible and early- breeding and production levels. blooming. Self-compatibility is controlled by a single Several almond populations have been analyzed with self-compatibility Sf dominant allele14. During the last 50 molecular markers in order to determine genetic varia- years, almond breeding for self-compatibility has mainly bility and relatedness9,24–26. However, these studies were used two sources of Sf, local landraces originated in Italy performed with material from limited geographic areas (“Tuono” and “Genco”) and related species such as P. and do not represent the current worldwide status of persica and P. webbii15. almond breeding stocks. Although genomic measures of Almond breeders have relied mainly on outcrossing inbreeding are more accurate than those obtained from and, occasionally, on introgression from other Prunus pedigree data27,28, pedigree-based analysis is a cost- species, for the development of new cultivars. Initially, in effective technique to estimate these parameters in Pérez de los Cobos et al. Horticulture Research (2021)8:11 Page 3 of 11 breeding populations and an alternative when genomic identical by descent, and it is calculated by the following measures are unviable. Several reports have evaluated formula34: inbreeding based on pedigree data in breeding popula- " # tions of fruit and nut tree crops29–32. In almond, a pedi- X 1n1 þn2 þ1 gree analysis of 123 different genotypes from the USA, Fx ¼ ð1 þ FA Þ ; 2 France, Spain, Israel, and Russia was reported33. However, their work was mainly focused on North American gen- where n1 = number of generations from one parent back otypes and did not include many cultivars that have to the common ancestor, n2 = number of generations subsequently been released worldwide. This study aimed from the other parent back to the common ancestor, and to determine the genetic structure of current breeding FA = inbreeding coefficient of the common ancestor. stocks and breeding tendencies over the last 50 years Pairwise relatedness (r) or coancestry coefficient, the using marker-verified pedigree data. degree of relationship by descent of two parents, equals the inbreeding coefficient of their prospective progeny. Materials and methods The genetic contribution (GC) of a founder to a cultivar Marker-verified pedigree data is calculated by the following formula35: Pedigree data of 220 almond genotypes (169 of known x  n X origin and 51 of unknown origin) were compiled from 1 available bibliography and breeding records. From the 220 GC ¼ ; 1 2 almond genotypes, 37 genotypes were no longer available (17% of the studied genotypes) as they were eliminated where n = number of generations in a pedigree pathway some time ago or were from discontinued breeding pro- between the founding clone and the cultivar and x = grams. To verify parental relationships of the rest of number of pathways between the founding clone and the genotypes (183), we used SSRs, SNPs, and self- cultivar. The three parameters were calculated using the incompatibility S-allele data from previous studies per- SAS INBRED procedure (SAS 9.4 SAS Institute, Cary, formed by the breeding programs taking part in this study NC, USA). (Supplementary Material 1). Marker data confirmed both In summary, the inbreeding coefficient measures the parents of 71 genotypes and one parent of four genotypes probability that two alleles in a locus are identical by (146 confirmed relationships) and found three erroneous descent and so copies of the same allele from a previous parentages. Two wrong parentages were found on the generation. The pairwise relatedness measures the prob- male parent of “Capella” and “Davey”, changing their ability that two alleles at any locus are identical by descent pedigree to open-pollinated and a third incorrect par- (copies of the same allele in a previous generation) entage on “Yosemite” female parent, eliminating this between two different individuals. F and r range from 0 to genotype from the analysis. 1, with values close to 0 indicating a low degree of After the corrections made, pedigrees of 169 genotypes inbreeding or relatedness and values close to 1 indicating of known origin (77 of them marker-verified, approxi- a high degree of inbreeding or relatedness. The genetic mately 54% of the available genotypes) were analyzed contribution estimates the proportion of genome that (Supplementary Material 1). The origin of the genotypes comes from the same individual. Thus, a child will have were 59 from Spain, 56 from the USA, 16 from Russia, 11 0.5 genome of either parent and a grandchild will have from Israel, 10 from France, 7 from Australia, 7 from 0.25 genomes of his grandparents. Greece, 2 from Argentina, and 2 from Italy. A pedigree data file was created. Each record in the file Analysis description contained one cultivar or selection name, the female To calculate F, r, and GC, parents of unknown origin parent and the male parent, in that order. Once entered, were assumed to be unrelated and noninbred. The seed these data were available for inbreeding analyses such as parent involved in all open pollinations was also assumed determining the number of times a cultivar appeared in a to be unrelated to the pollen parent. These assumptions, pedigree as a male or female genitor. Genotypes of known based on the fact that most almond cultivars are obligate origin were classified into two groups according to self- outcrossers because of their self-incompatibility, may lead compatibility: 104 self-incompatible and 65 self- to an underestimation of inbreeding. In the cases of compatible. genotypes of open-pollinated origin (OP), numbers OP1, OP2, and OP3 were given to the pollen parent in order to Inbreeding coefficient, pairwise relatedness, and genetic be distinguishable for genetic studies. Also, all mutants contribution were considered to have no genetic differences from the The inbreeding coefficient (F) is defined as the prob- original cultivar, thus GC = 1. Since the differences ability that a pair of alleles at any locus in an individual are between such mutants and the original cultivar are Pérez de los Cobos et al. Horticulture Research (2021)8:11 Page 4 of 11 expected to be caused by a few mutations in the DNA, this 29 and 21 descendants, respectively. “Mission” only simplification avoids the overestimation of inbreeding shared progeny with “Nonpareil” (Fig. 1). coefficients. Cultivars like Supernova and Guara were Analyzing the results by country, breeding programs considered as “Tuono” clones36,37. Regarding the different from the USA had two main founding clones, “Nonpareil” clones of the French paper-shell cultivar Princesse, used and “Mission”, with 46 and 24 descendants, respectively, in both the USA and Russian breeding programs, we out of 56. These two founders were followed by “Eureka” adopted the approach of Lansari et al.33 by analyzing both and “Harriott”, with 14 and 11 descendants each. Breeding clones as the same cultivar. Historical reports suggest that programs from Spain had three main founding clones, the Hatch series “Nonpareil”, “I.X.L.”, and “Ne Plus Ultra” “Tuono”, “Cristomorto”, and “Primorskyi”, with 32, 31, were seedling selections from an open-pollination pro- and 24 descendants, respectively. Cultivars from the dis- geny of the early-introduced cultivar Princesse. This cul- continued French program had three main founding tivar probably originated from the Languedoc region in clones from two geographical origins, “Cristomorto” and France6,38–40. Also, “Nikitskij” was selected in France in “Tuono” (from Italy) with nine and five descendants, 190241. Because their specific origins remain uncertain, respectively, and “Aï” (from France), with eight descen- we analyzed these genotypes as nonrelated, which, how- dants. The Australian program had only two main ever, could lead to an underestimation of inbreeding. founding clones, “Nonpareil” and “Lauranne”, with six Pedigree data were analyzed at four levels: worldwide, and five derived genotypes, respectively. The Israeli by country (Australia, France, Israel, Spain, and the USA), breeding program showed the most balanced pedigree by breeding program (when different programs exist with six main founding clones, “Marcona”, “Greek”, “Um within a country: CITA, IRTA, CEBAS-CSIC, and UCD- ElFahem”, “Tuono”, “Nonpareil”, and “Ferragnès”. USDA), and by genotypes carrying the Sf allele for self- The UCD breeding program had “Nonpareil” as the compatibility. main founding clone with 29 descendants. Cultivars Eureka, Mission, and Harriott had a slight influence on Results the pedigree with 14, 12, and 10 descendants, respectively. Founding clones Within Spain, CITA breeding program had Italian The entire almond pedigree traced back to 51 founding “Tuono” as the main founding clone with seven descen- clones (Supplementary Fig. 1). “Nonpareil”, “Cristo- dants. The IRTA breeding program showed three main morto”, “Mission”, and “Tuono” were the founders with founding clones, “Cristomorto”, “Primorskyi”, and the largest number of descendants in the pedigree: 140 of “Tuono” with 30, 19, and 16 descendants, respectively. the 169 genotypes of known parentage traced back to one The CEBAS-CSIC breeding program had three main or more of these founding clones (Fig. 1). No genotype founding clones, “Tuono”, “Ferragnès”, and “Primorskyi” was derived from all four cultivars, i.e., did not trace back with 15, nine, and eight descendants, respectively. The to the four founding clones. There were only five geno- French local cultivar Aï was also present in the three types that came from a three-way shared progeny, all of Spanish programs through the largely used French “Fer- them tracing back to “Tuono”–”Cristomorto“–”Nonpar- raduel” and “Ferragnès”. These two cultivars were the eil”. The largest two-way shared genotype sub in set were ancestors of 25 genotypes. “Tuono”–”Cristomorto” and “Nonpareil”–”Mission” with Analyzing the 65 genotypes carrying the Sf allele for self- compatibility, the founding clones that traced back to the origin of this allele are “Tuono”, “Genco”, and genotypes originated from introgression crosses with P. persica and Nonpareil Mission P. webbii. Tuono Cristomorto 38 5 Inbreeding coefficients 7 21 0 The mean inbreeding coefficient (F) of the 169 geno- 10 0 0 25 types of known parentage analyzed was 0.041 (Supple- mentary Material 2). Some 43 genotypes presented an 0 F > 0, with 14 over 0.250 (Table 1). 0 0 0 5 Considering the results within each country, programs 29 showing more inbreeding were France, the USA, and Spain with 0.075, 0.070, and 0.037 mean F, respectively (Supplementary Material 2). The programs from Australia Fig. 1 Descendants shared by “Tuono”, “Nonpareil”, “Mission” and and Israel had F = 0. The USA accessions ranged from “Cristomorto”. Venn diagram showing the number of descendants shared by “Tuono”, “Nonpareil”, “Mission” and “Cristomorto” F = 0 to 0.375 with 21 of the 56 genotypes having F > 0. The French cultivar Ferralise and selection FGFD2, Pérez de los Cobos et al. Horticulture Research (2021)8:11 Page 5 of 11 Table 1 Genotypes with the highest inbreeding coefficient Line nanme Female parent Male parent Origin Country Inbreeding A2-198 C1328 C1328 CEBAS-CSIC SPAIN 0.5 Solano 21–19 W 22–20 UCD USA 0.375 Sonora 21–19 W 22–20 UCD USA 0.375 Vesta Nonpareil Solano UCD USA 0.375 Ferralise Ferraduel Ferragnès INRA FRANCE 0.25 FGFD2 Ferragnès Ferraduel INRA FRANCE 0.25 21–19W Nonpareil A1-30 UCD USA 0.25 22–20 Nonpareil A1-30 UCD USA 0.25 6–27 Nonpareil Jordanolo UCD USA 0.25 Calif. 24–6 Eureka A5-25 UCD USA 0.25 Emerald Mission S2 PRIVATE USA 0.25 Profuse Nonpareil Jordanolo PRIVATE USA 0.25 Supareil Nonpareil Carmel PRIVATE USA 0.25 D01-462 A2-198 S5133 CEBAS-CSIC SPAIN 0.25 derived from the same reciprocal cross, had F = 0.250. Table 2 shows the GC of the mean founders by country. The Spanish selection A2-198 from CEBAS-CSIC, In the Australian breeding program, only two founders, showed the highest inbreeding coefficient (F = 0.500) as it “Nonpareil” and “Lauranne”, represented 71.4% of the is a selfing from selection C1328 and was raised to obtain total GC. The French breeding program was characterized homozygous SfSf individuals. by the extensive use of three founders “Cristomorto” (GC The UCD-USDA breeding program had a mean F of = 35.0%), “Aï” (GC = 30.0%), and “Tuono” (GC = 25.0%). 0.096. Within Spain, the CITA program had F = 0. The These cultivars together with “Ardèchoise” and “Tardy CEBAS-CSIC program had only three genotypes with F > Nonpareil” (both GC = 5.0%) accounted for 100% of the 0, but presented an average F of 0.048. The IRTA program total GC. The Israeli breeding program presented six holds 15 genotypes with F > 0 and a mean F of 0.043 main founders, “Greek” (GC = 20.5%), “Marcona” (GC = (Supplementary Material 2). Considering only the 65 self- 18.2%), “Um ElFahem” (GC = 13.6%), “Tuono” (GC = compatible genotypes, they had a mean F of 0.042, ran- 11.4%), “Nonpareil” (GC = 11.4%), and “Ferragnès” ging from 0 to 0.500 (Supplementary Material 2). (GC = 6.8%), which together accounted for 81.9% of the total GC. The USA breeding programs were largely Genetic contribution dependent on “Nonpareil” (GC = 43.7%) followed by “Nonpareil”, “Tuono”, “Cristomorto”, and “Mission” “Mission” (GC = 13.9%), “Eureka” (GC = 8.7%), and were the founding clones with the highest mean genetic “Harriott” (GC = 5.5%), which all accounted for 71.8% of contribution (GC, Fig. 2). These four cultivars accounted the total GC. The cultivars released by the three Spanish for 48.4% of the total GC worldwide. “Nonpareil” repre- breeding programs were based mainly on four founders: sented 20.5% of GC worldwide, “Tuono” and “Cristo- “Cristomorto” (GC = 23.7%), “Tuono” (GC = 22.6%), morto” were around 11%, and “Mission” slightly exceeded “Primorskyi” (GC = 15.6%), and “Aï” (GC = 7.5%), 5%. Nevertheless, the mean GC of these founding clones accounting for 69.4% of the total GC. within each country was variable. The breeding programs The UCD-USDA breeding program had the same most dependent on these founders were Australia and founders as the overall American programs, “Nonpareil” France, where “Nonpareil”, “Tuono”, and “Cristomorto” (GC = 43.2%), “Eureka” (GC = 14.8%), “Harriott” (GC = represented >60% of the total GC. Israel was the least 8.5%), and “Mission” (GC = 5.5%). Differences were dependent country as these founders represented ~25% of observed in the use of founding cultivars between Spanish the total GC. Cultivar Nonpareil was the founder with the breeding programs. The CITA program was mainly based highest mean GC in the USA and Australia, while in Spain on four cultivars “Tuono” (GC = 35.0%), “Belle d’Aurons”, and France were “Tuono” and “Cristomorto”. The cultivar “Bertina”, and “Genco” (GC = 10.0% each). These culti- Mission was used only in the American programs. vars were accounting for 65.0% of the total GC. The Pérez de los Cobos et al. Horticulture Research (2021)8:11 Page 6 of 11 A B 25 100 90 20 80 70 Founder 15 60 Other GC (%) GC (%) 50 Mission Cristomorto 10 40 Tuono Nonpareil 30 5 20 10 0 0 Nonpareil Tuono Cristomorto Mission USA SpainAustralia Israel France Founder Country Fig. 2 Mean genetic contribution (GC) of founders “Nonpareil”, “Tuono”, “Cristomorto”, and “Mission”. A Mean GC worldwide. B Mean GC by country CEBAS-CSIC program was based also on four founders, In the Australian breeding program, the selection “Tuono” (GC = 28.9%), “Ferragnès” (GC = 18.4%), A97001-1BT47 had the highest mean r with a value of “Genco” (GC = 12.5%), and “Primorskyi” (GC = 11.8%). 0.375. “Rhea” was not related with the rest of the geno- The IRTA program was based on four founding clones types, so its mean r was zero. The rest of the genotypes too: “Cristomorto” (GC = 39.9%), “Primorskyi” (GC = have a mean r between 0.188 and 0.333 showing a high 21.5%), “Tuono” (GC = 14.4%), and “Aï' (GC = 8.0%). The degree of relationship. self-compatible Italian cultivar Tuono was the Sf donor In the French breeding program, “Ferralise” had the most commonly used by the three Spanish programs. highest mean r (0.500). “Ferrastar” and “R1000” had the Within the 65 genotypes bred carrying the Sf allele, the lowest mean r, 0.167 and 0.111, respectively. The rest of 24.7% of the total GC came from “Tuono” (Supplemen- French genotypes had a mean r over 0.300, being the tary Material 3). breeding program with the most related genotypes. Genotypes from the Israeli program had a mean r under Pairwise relatedness 0.225. The highest r observed between the ten cultivars Pairwise relatedness (r) between all cultivars and released was 0.500 between two pairs: “Dagan”–”Gilad” breeding selections is shown in Supplementary Material 4. and “Fergil”–”Gilad”. Selection 54 showed r of 0.500 with Cultivars with the highest mean r worldwide are present “Kochba” and 0.250 with “Kogil-Pat”, “Samish”, and in Table 3. The genotype with the highest mean r was “Solo”. Figure 3 compares the breeding program with the “Nonpareil” followed by its mutants (“Tardy Nonpareil”, most related genotypes (France) with the breeding pro- “Jeffries”, and “Kern Royal”). “Vesta”, from the cross gram with the least related genotypes (Israel). “Nonpareil” × “Solano”, was next. Carina, Mira, and Within the Spanish breeding programs, the highest r Maxima (Australian genotypes originated from the cross among released cultivars was 0.500 (“Antoñeta”–”Marta” “Nonpareil” × “Lauranne”), followed. These three geno- and “Makako”–”Penta”). “Makako”–”Tardona” and “Pen- types were first generation of “Nonpareil”, second gen- ta”–”Tardona” had an r = 0.313. The CEBAS-CSIC’s eration of “Tuono”, and third generation of “Cristomorto”. selections A2-192 and C1328 had the highest r with a Table 4 shows the mean r among breeding programs by value of 1. In the CEBAS-CSIC program, “D01-462” had country. Programs from Australia and France had the the highest mean r (0.273). The genotypes with a higher highest mean r (0.256 and 0.357, respectively). In contrast, mean r in the CITA breeding program were “Guara” and Israel showed the lowest mean r. Comparing relatedness “Felisia” with values of 0.278 and 0.250, respectively. The results between countries, Spain and the USA breeding remaining CITA genotypes had a mean r under 0.200. programs were the least related. The most related Within the IRTA breeding program, the highest r among breeding programs were those of France and Spain and released cultivars was 0.563 (“Glorieta”–”Marinada”). also, Australia and France. Among IRTA’s selections, “29–47” and “35–164” showed Pérez de los Cobos et al. Horticulture Research (2021)8:11 Page 7 of 11 Table 2 Genetic contribution (GC) of mean founding Table 4 Mean of pairwise relatedness (r) among breeding clones by country programs from five different countries Founding clone Country of origin GC (%) GC total (%) Australia France Israel Spain USA Australia Australia 0.256 0.156 0.081 0.094 0.172 Nonpareil USA 39.3 71.4 Lauranne France 32.1 France – 0.357 0.070 0.195 0.022 France Israel – – 0.134 0.047 0.050 Cristomorto Italy 35.0 100.0 Aï France 30.0 Spain – – – 0.162 0.009 Tuono Italy 25.0 USA – – – – 0.232 Ardechoise France 5.0 Tardy Nonpareil USA 5.0 Israel Greek Israel 20.5 81.9 r values among selections and cultivars, four combinations Marcona Spain 18.2 had an r = 1 (“21–19 W”–“Solano”, “22–20”–“Solano”, Um ElFahem Israel 13.6 “21–19 W”–”Sonora”, and “22–30”–“Sonora”). In addition, Tuono Italy 11.4 two other pairs: “21–19 W”–“Vesta” and “22–20”–“Vesta” Nonpareil USA 11.4 had an r of 0.875 (Supplementary Material 4). Within the Ferragnès France 6.8 Spain UCD breeding program, “Vesta“, “Sonora”, and “Solano” Cristomorto Italy 23.7 69.4 had a mean r over 0.400. Tuono Italy 22.6 Among the group of 65 genotypes carrying the Sf allele, Primorksyi Russia 15.6 the mean r was 0.125. Grouping the genotypes by origin of Aï France 7.5 the Sf allele source (“Tuono”, “Genco”, and other Prunus USA spp), the mean r values were 0.210, 0.333, and 0.173, Nonpareil USA 43.7 71.8 respectively (Supplementary Material 4). Figure 4 shows Mission USA 13.9 the main self-compatibility sources used when breeding for Eureka USA 8.7 this character with “Tuono”, “Genco”, and other Prunus Harriott USA 5.5 species involved in 48, 4, and 13 genotypes, respectively. Discussion Table 3 Genotypes with the highest mean relatedness (r) Two mainstream breeding lines based on three different cultivars Genotype Mean r Our genetic study of almond breeding programs Nonpareil 0.153 worldwide demonstrated that the most widely used cul- Tardy Nonpareil 0.153 tivars were Nonpareil, Tuono, Cristomorto, and Mission. Jeffries 0.153 “Nonpareil” had a large influence in USA and Australian Kern Royal 0.153 programs, where soft-shelled nuts are bred. This reference Vesta 0.143 A97001-1bT4 0.137 cultivar was present in all the breeding programs studied Carina 0.136 (in some cases through its late-blooming mutant Tardy Mira 0.133 Nonpareil). The self-compatible “Tuono” and the late- Maxima 0.133 blooming “Cristomorto” were extensively used in the Mediterranean programs, where hard-shelled nuts are bred. “Mission” initially showed a considerable impor- the highest relationship with an r of 0.719. The selection tance worldwide, but deeper analysis demonstrated that it “29–47” had the highest mean r (0.350). The rest of was mainly influential in private American programs. IRTA’s genotypes had mean r over 0.130 (Supplementary Taking into account these results, we can establish two Material 4). In the USA breeding programs, “Nonpareil” main breeding lines based on the use of three different and its mutations (“Tardy Nonpareil”, “Jeffries”, and “Kern founders: the European programs based mainly on Royal”) and “Vesta” had a mean r over 0.400. “Indepen- “Tuono” and “Cristomorto” (hard shell), and the North dence” and “Bell” had a mean r equal to 0. The rest of American–Australian programs based on “Nonpareil” North American genotypes showed a high degree of (soft shell). The French and Spanish breeding programs relatedness between them. Two combinations, “Sola- were based directly on “Tuono” and “Cristomorto”. In the no”–“Vesta” and “Sonora”–”Vesta”, had r = 1, with French INRA program, the Italian cultivars Tuono and “Sonora”–”Vesta” r = 0.875. Analyzing the highest Cristomorto account for 60.0% of total GC and were Pérez de los Cobos et al. Horticulture Research (2021)8:11 Page 8 of 11 R1000 LEVAD SHEFA FERRAGNES MATAN FERRALISE 54 SAMISH FLTU18 FGFD2 FERGIL FERRASTAR KOGIL−PAT SOLO FERRADUEL MANDALINE STELIETTE GILAD KOCHBA LAURANNE DAGAN Fig. 3 Relationship matrix of genotypes from France and Israel. Genotypes from France are showed in the left side, genotypes from Israel are showed in the right side. Line thickness shows the degree of relationship, being the thicker lines the more related genotypes SB16, 2−44 SB2, 45−96 BELONA SB13, 25−75 SOLETA 4−26 BLANQUERNA SB6, 56−88 97, 1−232 MARDIA VIALFAS S4017 SB3 54−39E UCD8−160 A2−198 C1328 FELISIA S2332 PA3777 SOLSEL 5−15 F7 2−18 D01−462 AYLES Tuono R1000 D06−795 F5, 4−6 GUARA MANDALINE Genco D01−188 TARDONA MIRA A−192 CAPELLA FGTR13 FLTU18 Other Species A97001−1BT4 MARITA D00−078 MARTA ALMENARA CAMBRA CALIF. 24−6 CARINA TERESA FRANCOLI ANTONETA D00−360 LAURANNE STELIETTE S5133 PENTA MAKAKO VAIRO 13−565 MARINADA CONSTANTI VELA 21−321 24−53 MATAN INDEPENDENCE 35−164 29−148 23−173 30−297 Fig. 4 Relationship matrix of the 65 self-compatible genotypes carrying the Sf allele. Colors indicate origin of the Sf allele. Line thickness shows the degree of relationship, being the thicker lines the more related genotypes Pérez de los Cobos et al. Horticulture Research (2021)8:11 Page 9 of 11 present in the pedigree of all ten cultivars and selections of parents (“Nonpareil“, “Tuono”, and “Cristomorto”) and evaluated. Also, the local French late-flowering and their related genotypes, as we have shown for almond Monilinia-resistant cultivar Aï was a parent to both breeding. “Ferragnès” and “Ferraduel”. In the three Spanish breed- Among the group of the 65 genotypes carrying the Sf ing programs, the importance of “Tuono” and “Cristo- allele for self-compatibility, the mean r was 0.125. In morto” cultivars was very high, accounting to 46.2% of cherry self-compatible selections, coefficients of coan- total GC. These two cultivars were present in the pedigree cestry ranged from 0.102 to 0.25631 and thus were of of 53 out of 59 cultivars and breeding selections from similar magnitude. In Western Europe, the Italian cultivar Spain. These results can be explained by the large influ- Tuono was used extensively as a source of self-compat- ence of the French germplasm on the Spanish breeding ibility, late blooming, and spur-type cropping. More programs, causing a high relationship between the pro- recently, it has become important in Israel and Australia grams of both countries (mean r = 0.195). In the North (in Australia through “Lauranne” (“Ferragnès” × American breeding programs, “Nonpareil” accounts for “Tuono”)). This “Ferragnès” × “Tuono” cross also origi- 43.7% of the total GC and was present in the pedigree of nated the cultivar Steliette and was later successfully used 48 out of 56 cultivars and breeding selections from the in two of the Spanish breeding programs, resulting in USA. In Australia, ‘Nonpareil’ accounts for 39.3% of the three self-compatible cultivars: Cambra at CITA, and total GC and is present in the pedigree of 6 out of 7 Antoñeta and Marta at CEBAS-CSIC. Thus, these five cultivars and breeding selections. Also, “Lauranne” (32.1% cultivars are full siblings. In addition, in the USA, breeders of the total GC) reaches an importance similar to ‘Non- are using “Guara” (syn “Tuono”) as Sf donor. A similar pareil’, explaining the close relationship between the case occurred in sweet cherry with the cultivar Stella as it Australian and French programs (mean r = 0.156). Even was the most frequently utilized parent for self- in other countries with noncontinuous breeding initia- compatible selections in North America31. tives, such as Russia, Greece, or Argentina, the use of A lack of diverse germplasm may limit continued pro- “Nonpareil” as a founder was common. Israel was the only gress in almond breeding programs. This genetic limita- country where these cultivars had a relatively low influ- tion is of particular concern in the main producing ence. This may be due to the extreme Israeli climatic countries. Thus, Californian and Australian production conditions, forcing breeders to use locally adapted selec- rely mainly on ‘Nonpareil’ and closely related culti- tions as parents. In Spain, the use of locally adapted cul- vars46,47, while in Spain, some new Spanish cultivars like tivars such as Bertina at CITA as a donor for Polystigma Vairo and Penta, derived from second generation of ochraceum (Wahlenb.) Sacc. resistance was successful but “Tuono” and “Cristomorto”, as well as “Belona” and used only to a limited extent. Other examples of sec- “Soleta”, derived from second generation of “Genco”, are ondary founders include “Primorskyi”, used regularly as replacing traditional cultivars in new orchards. This trend late-blooming and Fusicoccum-resistance donor in two of is also favored by the almond industry needs. Only in the Spanish breeding programs (IRTA and CEBAS-CSIC) some regions of Central Asia, Middle East, and North and “Eureka” and “Harriott” in the North American Africa, local and well-adapted traditional selections still breeding programs. play an important role in commercial production26,48–50. Loss of genetic variability and increasing of inbreeding at Usefulness of pedigree data analyzing breeding breeding and production level tendencies Comparing our results on almond inbreeding with other Pedigree analysis is a cost-effective and well-established Prunus species, the mean inbreeding coefficient world- way to monitoring inbreeding and relatedness among wide of all genotypes (F = 0.036) was lower than that of controlled breeding populations. However, the veracity of Japanese plum42 and apple43 and several orders of mag- any analysis based on this kind of data relies on the nitude lower than those calculated for peach44,45 and accuracy of records collected across multiple institutions cherry31. Within almond, inbreeding and relatedness and by many breeders. In order to verify parental rela- coefficients obtained in this study were higher than those tionships of the genotypes under study, we used SSRs, reported by Lansari et al.33. While they documented only SNPs, and self-incompatibility S-allele data from previous ten genotypes with F > 0 (four of them with F ≥ 0.250), we analysis carried out by the breeding programs taking part found 43 genotypes meeting this condition (14 of them in this study. Our molecular marker analysis confirmed with F ≥ 0.250). Analyzing mean r by country, in the case 146 parentage relationships and found three errors (2% of France and the USA (with a number of cultivars error rate), which were corrected accordingly. Thus, the comparable in both studies), this coefficient increased. marker-based pedigree analysis performed showed only This loss of variability and an associated increase of small parental changes and corroborates the consistency inbreeding is due to the repeated use of a limited number of the results reached by this study. Pérez de los Cobos et al. Horticulture Research (2021)8:11 Page 10 of 11 However, several reports have demonstrated that large- data from the UCD breeding program. The authors thank two reviewers and scale genomic analysis may provide more accurate results the editor for their insightful and constructive comments. than pedigree analysis27,28. This kind of genome-based Author details pedigree analysis has already been performed in apple51. 1 Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Mas Bové, The recent publication of two almond reference gen- Ctra. Reus-El Morell Km 3,8, 43120 Constantí, Tarragona, Spain. 2Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Centre de Recerca en omes4,52 and the increasing availability of quality genomic Agrigenòmica (CRAG), CSIC-IRTA-UAB-UB. Cerdanyola del Vallès (Bellaterra), data opens opportunities to complement our study and 08193 Barcelona, Spain. 3Centro de Edafología y Biología Aplicada del Segura, obtain more complete and accurate pedigrees based on Consejo Superior de Investigaciones Científicas (CEBAS-CSIC), P.O. Box 164, 30100 Espinardo, Murcia, Spain. 4Institut de Recerca i Tecnologia genomic variability. This kind of studies can be useful Agroalimentàries (IRTA), Fruitcentre, PCiTAL, Gardeny Park, Fruitcentre Building, even when some genotypes were discarded due to 25003 Lleida, Spain. 5Centro de Investigación y Tecnología Agroalimentaria de breeding process, as is the case in our almond Aragón (CITA), Avda. Montañana 930, 50059, Zaragoza, Instituto Agroalimentario de Aragón IA2 (CITA-Universidad de Zaragoza), Zaragoza, pedigree work. Spain. 6University of California, 1 Shields Avenue, Davis, CA 95616, USA. Although almond showed a higher genetic variability 7 University of Adelaide, Waite Research, School of Agriculture, Food and Wine, than other Prunus species, the historical expansion of PMB 1, Glen Osmond, Adelaide, SA 5064, Australia. 8Institut National de la Recherche Agronomique (INRA), Domain St. Maurice CS 60094, 84143 almond from the Mediterranean region to California and Montfavet Cedex, France. 9Agricultural Research Organization, Newe-Ya’ar from California to Australia could have caused a bottle- Research Center, P.O. Box 1021, Ramat Yishad 30095, Israel neck effect in the breeding population under study. Dif- ferent studies have reported a high genetic relatedness Author contributions F.P.C. and I.B. conceived and designed the study. F.P.C. performed the analysis. between Australian and Californian cultivars9,53, possibly F.P.C., P.M., A.R., X.M., I.E., W.H., M.M., F.D., R.S., M.R., T.G., M.W., H.D., D.H., P.A., caused by the introduction of a limited number of cul- F.V., and I.B. wrote the paper. All authors approved the final version of tivars from Europe to these countries. In addition, the paper. breeding programs worldwide have used cultivars from Conflict of interest French origin as main founders as Aï, Princesse, Arde- The authors declare that they have no conflict of interest. choise, Nonpareil, IXL, Ne Plus Ultra, or Nikitskij. This situation could have led to an underestimation of relat- Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41438-020-00444-4). edness and inbreeding. 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