Spanish Journal of Agricultural Research 14(3), e0706, 16 pages (2016) eISSN: 2171-9292 http://dx.doi.org/10.5424/sjar/2016143-8638 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) RESEARCH ARTICLE OPEN ACCESS Comparative analysis of traditional and modern apricot breeding programs: A case of study with Spanish and Tunisian apricot breeding germplasm Mohamed A. Batnini1, Lamia Krichen1, Hedia Bourguiba1, Neila Trifi-Farah1, David Ruiz2, Pedro Martínez-Gómez2, and Manuel Rubio2 1 Université Tunis El Manar, Faculté des Sciences de Tunis, Laboratoire de Génétique Moléculaire, Immunologie et Biotechnologie, Tunis, Tunisia. 2 CEBAS-CSIC, Departamento de Mejora Vegetal, Murcia, Spain Abstract Traditional plant breeding is based on the observation of variation and the selection of the best phenotypes, whereas modern breeding is characterised by the use of controlled mating and the selection of descendants using molecular markers. In this work, a comparative analysis of genetic diversity in a traditional (Tunisian) and a modern (Spanish) apricot breeding programme was per- formed at the phenotypic and molecular level using simple sequence repeat (SSR) markers. Seven phenotypic traits were evaluated in 42 Tunisian apricot accessions and 30 genotypes from the Spanish apricot programme. In addition, 20 SSR markers previously described as linked to specific phenotypic traits were assayed. Results showed that modern breeding using controlled crosses in- creases the size of the fruit. The fruit weight average observed in the Tunisian cultivars was of 20.15 g. In the case of traditional Spanish cultivars the average weight was 47.12 g, whereas the average weight of the other progenitors from France, USA and South Africa was 72.85 g. Finally, in the new releases from the CEBAS-CSIC breeding programme, the average weight was 72.82 g. In addition, modern bred cultivars incorporate desirable traits such as self-compatibility and firmness. Cluster and structural analysis based on SSR data clearly differentiates the genotypes according to their geographic origin and pedigree. Finally, results showed an association between some alleles of PaCITA7 and UDP96003 SSR markers with apricot fruit weight, one allele of UDAp407 marker with fruit firmness and one allele of UDP98406 marker with fruit ripening. Additional key words: traditional breeding; modern breeding; molecular markers; SSRs; phenotype; association genetic. Abbreviations used: CEBAS-CSIC (Centro de Edafología y Biología Aplicada del Segura-Consejo Superior de Investigaciones Científicas); FCA (factorial correspondence analysis); Fis (inbreeding coefficient); Fst (fixation index); GD (genetic distances); He (expected heterozygosity); Ho (observed heterozygosity); LG (linkage group); MAF (major allele frequency); MAS (marker as- sisted selection); NJ (neighbour joining); Nm (mean value of gene flow); nt (nucleotide); PIC (polymorphism information content); PPV (Plum pox virus); SSR (simple sequence repeat). Authors’ contributions: Performed the agronomical characterization of the Tunisian germplasm: MAB, LK, HB and NTF Performed the agronomical characterization of the Spanish germplasm: DR, MR. Analyzed the data: MAB and PMG. Citation: Batnini, M. A.; Krichen, L.; Bourguiba, H.; Trifi-Farah, N.; Ruiz, D.; Martínez-Gómez, P.; Rubio, M. (2016). Comparative analysis of traditional and modern apricot breeding programs: A case of study with Spanish and Tunisian apricot breeding germplasm. Spanish Journal of Agricultural Research, Volume 14, Issue 3, e0706. http://dx.doi.org/10.5424/sjar/2016143-8638. Received: 14 Sep 2015. Accepted: 12 Jul 2016. Copyright © 2016 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial (by-nc) Spain 3.0 Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Funding: Spanish Ministry of Economy and Competitiveness (AGL2013-41452-R); Seneca Foundation of the Region of Murcia (19879/GERM/15); Tunisian Ministry of Higher Education, Scientific Research and Technology. Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Pedro Martínez-Gómez: [email protected]. Introduction from different private and public institutions. Tradi- tional breeding, based on the observation of pheno- Plant breeding programmes involve the application typic variation and the selection of the best phenotypes, of different genetic techniques in order to obtain new was the first approach used around the world. Modern varieties with improved productivity, fruit quality and breeding, on the other hand, is characterised by the use resistance to existing diseases. Plant breeding is prac- of controlled mating and by the subsequent monitoring ticed by farmers and by professional plant breeders of the recombination obtained using molecular markers 2 Mohamed A. Batnini, Lamia Krichen, Hedia Bourguiba, Neila Trifi-Farah, David Ruiz et al. (Breseghello & Coelho, 2013), although in many spe- neous accessions of ‘Bargougs’ (the most common cies breeding the marker assisted selection (MAS) apricot cultivar in the middle of Tunisia) propagated protocols are still nor on development. by seeds and issued from the Gafsa Oasis, Degache, In the case of stone fruit (Prunus) species, breeding Tozeur, Nefta and Midess (Fig. 1 & Table 1). In ad- must address specific challenges arising from the nature dition, 30 genotypes from the apricot breeding pro- of the tree, such as the extended juvenile period and a gramme of CEBAS-CSIC in Murcia (Spain) were complex physiology. Traditional Prunus breeding based included in this study, including new releases, ad- on selection and propagation of the best individuals vanced selections and progenitors from different geo- open-pollinated have been practiced for thousands of graphic areas (Table 2). years. Nowadays, these traditional methods continue to be the basis for breeding and production in different countries and for many species. In the 1920s, the Uni- Phenotypic evaluation versity of California (USA) had the first modern breed- ing programme based on controlled pollinations in The following seven agronomic traits of breeding almond. Breeding programmes began using molecular interest were studied: flowering time evaluated every markers in the early 1990s in characterisation, recom- 1-2 days until 50% of the flowers were completely bination monitoring and the selection of desirable traits opened (F ) from very early (before February 16) to 50 (Martínez-Gómez et al., 2003). very late (after March 12); ripening time considered Apricot (P. armeniaca L.) breeding in Tunisia in- when the fruit had suitable firmness and colour at the volves the application by farmers of traditional methods commercial maturity stage from early (before May 10) based on the observation of variation and the selection to very late (after June 20); flower compatibility of the best phenotypes followed by propagation by evaluated by bagging as self-compatible when fruit set seeds or grafting (Carraut & Crosse-Raynaud, 1950; was presented or self-incompatible with null set (Bur- Bourguiba et al., 2010). Spanish apricot production, gos et al., 1993); fruit weight measured in g, very light on the other hand, has traditionally been based on local (< 20 g), medium (20-40 g), heavy (40-60 g), and very cultivars. Nevertheless, a process of varietal renewal heavy (>80 g); flesh firmness from soft (less than 15 is now underway in Spain with the introduction of new N) to firm (more than 30 N; and skin and flesh colour varieties from modern breeding programmes such as as determined by a Minolta Chroma Meter (CR-300, the CEBAS-CSIC programme in Murcia. This breeding Minolta, Ramsey, USA). Pairwise standard genetic programme is based on the use of controlled crosses to distances (GDs) were calculated between groups of obtain new populations with desirable traits. Further- cultivars based on the mean character difference of the more, the selection for certain traits like self-compat- assayed phenotypic traits constructing an unrooted ibility (Badenes et al., 2000) or sharka (Plum pox virus, Neighbour Joining (NJ) tree using DARwin software PPV) resistance (Rubio et al., 2014) is done on the v.5.0 (Perrier & Jacquemoud-Collet, 2006). In addition, basis of molecular markers, enabling researchers to Factorial Correspondence Analysis (FCA) was carried select genotypes having those traits of interest in their out to provide a synthetic representation of the genetic progenies. variability of the studied accessions. The objective of this work was to perform a com- parative analysis of genetic diversity in Spanish and Tunisian apricot germplasm, from a traditional and a Molecular characterisation and association modern apricot breeding program respectively, at the genetic studies phenotypic and molecular level using simple sequence repeat (SSR) markers. Total genomic DNA was isolated using an optimized variation of the procedure described by Doyle & Doyle (1987). Extracted apricot genomic DNA was PCR- Material and methods amplified using 20 primer pairs flanking SSR sequenc- es distributed among the whole genome and previ- Plant material ously described as linked to specific phenotypic traits (Table 3). Amplified PCR products were separated by The plant material assayed consisted of 42 Tunisian electrophoresis using 3% Metaphor® agarose (Biowit- apricot accessions, including 23 cultivars issued from taker, Maine, USA) gel (1 X TBE buffer) stained with traditional selections and propagated by grafting from GelRedTM Nucleic Acid Gel Stain® (Biotium, Hatwad, the north (Ras Jbel and Testour), the centre (Kair- CA, USA). A 1 Kb Plus DNA Ladder was used as mo- ouan), and the south (Gabes, Mareth), and 19 sponta- lecular size standard. Polymorphic alleles were scored Spanish Journal of Agricultural Research September 2016 • Volume 14 • Issue 3 • e0706 Comparative analysis of traditional and modern apricot breeding programs 3 Bouk H'med (V13A) Bouthani (V20B) Addadi Ahmar (V15) Chechi Bazza (V28D) Oud Aouicha (V71) North OOuudd EGln Haaa j (TVa2h7aBr) (V70) Oud Hmida (V21D) Amor El Euch (V5A) Oud Rhayem (V18A) Bayoudhi (V11A) Bedri (V1A) Khad Hlima (V2A) er Zbidi (V7A) nt e C Fourati (V38C) Bargoug (B40F, H, K, L, M) Bargoug (B46C, D, E) Bargoug (B44B, C, D, E, F, H) Bedri Louzi (V57B) Bargoug (B42A, C, G) Thaleth (V58A) Bargoug (B43A, B) Amor El Euch (V51A, C) Baccour (V41C) Bedri (V48A, H) h ut o S Figure 1. Location of apricot cultivars selected from the Tunisian breeding programme. as present or absent (1/0). Band scoring was analysed Structure analysis using SYNGENE® GeneTools gel analysis software (Cambridge, UK). The SSR allelic data obtained were To infer the genetic structure of the apricot material, used to estimate diversity parameters including the total a model-based Bayesian clustering method imple- number of genotypes and alleles, major allele fre- mented in the STRUCTURE 2.2 program (Pritchard et quency and observed heterozygosity (Ho) as well as al., 2000) based on SSR data was used. STRUCTURE expected heterozygosity/gene diversity (He). For this was run using a model with admixture and correlated purpose, the software GENETIX® 4.05 (Belkhir et al., allele frequencies, with the assumed number of ge- 2004) was used. Moreover, GENEPOP 4.0 software netic clusters (K) ranging from 1 to 10, with 10 inde- (Raymond & Rousset, 1995) was used to calculate pendent replicate runs for each K value. Statistical Wright’s F-statistics. Polymorphism information con- parameters based on the rate of change in the log prob- tent (PIC), defined as the parameter for calculating the ability of data between successive K values was calcu- discriminating and informative power of SSR markers, lated to confirm the exact estimation of the most was calculated for each SSR locus using PowerMark- likely number of K clusters using the Structure Har- er 3.25 software (Liu & Muse, 2005). Genetic relation- vester application (Earl & von Holdt, 2011). ships among the defined apricot groups were assessed, genetic distances were calculated and a dendogram was constructed using unrooted NJ analysis with 1000 Results bootstraps over 20 SSR loci as implemented in DAR- win software. Factorial Correspondence Analysis Phenotypic diversity and phenetic (FCA) was carried out to provide a synthetic represen- relationships tation of the genetic relationship based on molecular markers. SSR allele mining was also conducted on The study revealed the great phenotypic variability specific alleles associated with specific phenotypic trait of the studied agronomic traits (flowering-time, ripen- expression. ing time, self-compatibility, fruit weight, flesh firmness, Spanish Journal of Agricultural Research September 2016 • Volume 14 • Issue 3 • e0706 4 Mohamed A. Batnini, Lamia Krichen, Hedia Bourguiba, Neila Trifi-Farah, David Ruiz et al. Table 1. Apricot cultivars selected from the Tunisian breeding programme with their propagation mode and origin. The follow- ing seven agronomic traits were studied: self-compatibility, flowering and ripening time, fruit weight and firmness, and skin and flesh colour. Cultivar Propagation Origin Compatibility[1] Flowering Ripening Weight Firmness Skin colour Flesh colour Addadi Ahmar V15 Grafting Ras Jbel S-C Medium Early Medium Medium Soft Yellow Green Yellow Amor El Euch V5A Grafting Kairouan S-I Medium Early Medium Firm Light Orange Orange Amor El EuchV51A Grafting Mareth S-I Medium Early Medium Medium Soft Yellow Green Yellow Amor El Euch V51C Grafting Mareth S-I Medium Early Very Light Medium Soft Yellow Green Yellow Baccour V41C Grafting Mareth S-I Medium Early Very Light Medium Soft Yellow White Bargoug B40F Seedling Gafsa S-I Medium Medium Very Light Medium Soft Yellow Green Yellow Bargoug B40H Seedling Gafsa S-I Medium Medium Very Light Soft Yellow Green Yellow Bargoug B40K Seedling Gafsa S-I Medium Medium Very Light Soft Yellow Green Yellow Bargoug B40L Seedling Gafsa S-I Medium Medium Very Light Soft Yellow Green Yellow Bargoug B40M Seedling Gafsa S-I Medium Medium Very Light Soft Yellow Green Yellow Bargoug B43A Seedling Nefta S-I Medium Early Very Light Soft Yellow White Bargoug B43B Seedling Nefta S-I Medium Early Very Light Soft Yellow White Bargoug B42A Seedling Tozeur S-I Early Early Very Light Medium Soft Yellow Yellow Bargoug B42C Seedling Tozeur S-I Early Early Very Light Medium Soft Yellow White Bargoug B42G Seedling Tozeur S-I Early Early Light Medium Soft Light Orange Light Orange Bargoug B44B Seedling Degache S-I Early Early Very Light Medium Soft Yellow Green Light Orange Bargoug B44C Seedling Degache S-I Early Early Very Light Medium Soft Yellow Green Yellow Bargoug B44D Seedling Degache S-I Early Early Very Light Medium Soft Yellow Green Light Orange Bargoug B44E Seedling Degache S-I Early Early Very Light Soft Light Orange Light Orange Bargoug B44F Seedling Degache S-I Early Early Very Light Soft Yellow Yellow Bargoug B44H Seedling Degache S-I Early Early Very light Soft Yellow Green White Bargoug B46C Seedling Midess S-I Early Early Very Light Soft Yellow Green Yellow Bargoug B46D Seedling Midess S-I Early Early Very Light Soft Yellow Green Yellow Bargoug B46E Seedling Midess S-I Early Early Very Light Medium Soft Yellow Green Light Orange Bayoudhi V11A Grafting Kairouan S-C Very Late Medium Very Light Medium Soft White White Bedri V1A Grafting Kairouan S-I Early Early Very Light Firm Yellow White Bedri V48A Grafting Mareth S-I Early Early Very Light Soft Yellow White Bedri Thani V48G Grafting Gafsa S-I Early Early Very Light Soft Yellow White Bedri Louzi V57B Grafting Gabès S-I Medium Early Light Soft Yellow Green Yellow Bouk H’med V13A Grafting Ras Jbel S-I Medium Medium Light Medium Soft Yellow White Bouthani V20B Grafting Testour S-C Late Early Light Firm Yellow White Chéchi Bazza V28D Grafting Testour S-I Very Late Late Light Soft White White Fourati V38C Grafting Sfax S-I Medium Early Light Soft Yellow White Khad Hlima V2A Grafting Kairouan S-I Medium Early Light Medium Soft Yellow White Khad Hlima V2C Grafting Kairouan S-I Medium Early Light Medium Soft Yellow White Oud Aouicha V71 Grafting Testour S-I Late Medium Medium Medium Soft Light Orange Light Orange Oud El Haj Tahar V70 Grafting Testour S-I Very Late Medium Very Light Soft Yellow Green Yellow Oud Gnaa V27B Grafting Testour S-I Late Medium Medium Medium Soft Yellow Light Orange Oud H’mida V21D Grafting Testour S-I Late Medium Very Light Firm Yellow Yellow Oud Rhayem V18A Grafting Testour S-C Medium Early Medium Medium Soft Yellow Green White Thani V59B Grafting Gabes S-I Early Early Light Soft Yellow Green Yellow Zbidi V7A Grafting Kairouan S-C Medium Medium Light Medium Soft Yellow Green Yellow [1] S-C, self-compatible; S-I, self-incompatible and skin and flesh colour) in Tunisian (Table 1) and genitors, although self-compatibility has been intro- Spanish (Table 2) apricot germplasm (Fig. 2). Regard- duced in the new cultivars (Table 2). ing the self-incompatibility expressed by most Tunisian All cultivars showed a wide range of flowering dates cultivars (90%) (Table 1), this trait has the advantage from early to very late. In the case of the traditional of maintaining genetic variability within populations cultivars from Tunisia, early or late flowering were not even though it is a negative trait from the agronomic key traits for selection (Table 1). In the case of the point of view. Self-compatibility is synonymous with Spanish germplasm, traditional cultivars like ‘Currot’ higher production. In the case of the Spanish breeding were used in the breeding programme for introducing programme, self-incompatibility is present in the early flowering, although late flowering is present in North-American cultivars used as PPV resistance pro- the North-American cultivars used as PPV resistance Spanish Journal of Agricultural Research September 2016 • Volume 14 • Issue 3 • e0706 Comparative analysis of traditional and modern apricot breeding programs 5 Table 2. Progenitors and new releases from the Spanish breeding programme at CEBAS-CSIC with pedigree, propagation mode and origin. The following seven agronomic traits were studied: self-compatibility, flowering and ripening time, fruit weight and firmness, and skin and flesh colour. Cultivar Pedigree[1] Propagation Origin Compatibility[2] Flowering Ripening Weight Firmness Skin colour Flesh colour Progenitors Búlida Unknown Grafting Spain S-C Late Medium Medium Medium Firm Light Orange Light Orange Currot Unknown Grafting Spain S-C Early Early Light Medium Soft White White Mauricio Unknown Grafting Spain S-C Medium Medium Medium Medium Soft Yellow Yellow Bergeron Unknown Grafting France S-C Very Late Very Late Heavy Medium Firm Light Orange Orange Goldrich Sunglo × Perfection Grafting USA S-I Late Late Very Heavy Firm Orange Orange Orange Red Lasg. Mashad × Grafting USA S-I Very Late Medium Medium Firm Orange Orange NJA2 Palsteyn Blenhein × Canino Grafting South Africa S-C Early Medium Heavy Medium Firm Light Orange Orange New releases Dorada Bergeron × Moniquí Grafting Spain S-C Very Late Very Late Heavy Medium Firm Yellow Yellow Estrella OR × Z211-18 Grafting Spain S-I Medium Medium Very Heavy Firm Orange Orange Maravilla OR × Z211-18 Grafting Spain S-I Medium Medium Very Heavy Firm Light Orange Light Orange Mirlo Blanco Rojo Pasión × BP Grafting Spain S-C Early Early Heavy Medium Firm Light Orange Light Orange Mirlo Naranja Rojo Pasión × BP Grafting Spain S-C Early Early Heavy Medium Firm Orange Light Orange Mirlo Rojo Rojo Pasión × BP Grafting Spain S-C Early Early Heavy Firm Light Orange Light Orange Murciana OR × Currot Grafting Spain S-C Late Medium Heavy Firm Light Orange Orange Rojo Pasión OR × Currot Grafting Spain S-C Medium Medium Heavy Medium Soft Light Orange Light Orange Rosa OR × Palsteyn Grafting Spain S-I Medium Medium Very Heavy Firm Light Orange Orange Selene Goldrich × A2564 Grafting Spain S-C Late Late Heavy Firm Orange Orange Sublime OR × Z211-18 Grafting Spain S-I Medium Medium Very Heavy Firm Orange Orange Toñi OR × Palsteyn Grafting Spain S-C Medium Early Very Heavy Medium Soft Light Orange Orange Valorange OR × Currot Grafting Spain S-C Medium Medium Very Heavy Firm Orange Orange 5-57 (OR × Currot) × BP Grafting Spain S-I Early Early Heavy Medium Firm Orange Light Orange 7-41 Rojo Pasión × BP Grafting Spain S-C Early Early Heavy Firm Orange Light Orange 8-50 OR × TB Grafting Spain S-I Very Late Very Late Heavy Medium Firm Orange Orange 8-61 OR × TB Grafting Spain S-C Late Late Heavy Medium Firm Orange Orange 9-5 OR × TB Grafting Spain S-C Very Late Very Late Heavy Medium Firm Orange Orange 9-55 OR × TB Grafting Spain S-I Very Late Late Heavy Medium Firm Orange Orange 10-18 OR × TB Grafting Spain S-I Very Late Very Late Very Heavy Firm Orange Orange 10-20 OR × TB Grafting Spain S-C Very Late Late Heavy Medium Firm Orange Orange 10-57 (Goldrich × TB) Grafting Spain S-I Very Late Very Late Heavy Medium Firm Orange Orange × OP 11-1 (Goldrich × TB) Grafting Spain S-I Very Late Very Late Heavy Medium Firm Orange Orange × OP [1]OR, Orange Red; BP, Búlida Precoz; TB, Tardif de Bordaneil; OP, open pollination. [2]S-C, self-compatible; S-I, self-incompatible. progenitors (‘Orange Red’, ‘Goldrich’). In addition, average weight of the other progenitors from France, most of the Tunisian apricot accessions showed early USA and South Africa was 72.85 g. Finally, in the new or medium ripening time (Table 1), whereas the Span- releases from the CEBAS-CSIC breeding programme, ish genotypes showed a wide range of maturity dates, the average weight was 72.82 g. Most of the Tunisian from early to very late (Table 2). In the Spanish breed- apricot cultivars showed soft or medium-soft fruits ing programme, both early and late ripening progenitors (Table 1). In the case of the Spanish collection, tradi- were used in the crosses in order to release new varie- tional cultivars used as progenitors were mainly char- ties covering the entire production time from the begin- acterised by medium-soft fruits. The North American ning of May to late June. cultivars used as progenitors in the Spanish breeding The fruit weight average (20.15 g) observed in the programmes, however, had firm fruit (Table 2). Most Tunisian cultivars was lower than that of the germplasm of the new releases resulting from the crosses per- evaluated in Spain. In the case of traditional Spanish formed at CEBAS-CSIC had medium-firm or firm cultivars used as progenitors (‘Currot’, ‘Mauricio’, fruits. On the other hand, most Tunisian apricot culti- ‘Búlida’), the average weight was 47.12 g, whereas the vars showed yellow or yellow green colouring in the Spanish Journal of Agricultural Research September 2016 • Volume 14 • Issue 3 • e0706 6 Mohamed A. Batnini, Lamia Krichen, Hedia Bourguiba, Neila Trifi-Farah, David Ruiz et al. Table 3. Origin of the 20 SSR markers tested and relationships with the phenotypic traits. Linkage group (LG), genetic position in centimorgans (cM), physical location in the Prunus reference genome (Peach v1.0), trait linkage and references. Marker Species Reference LG Position (cM) Location Peach v1.0 Trait References BPPCT025 Peach Dirlewanger et al. (2002) 6 56.41 Scaffold_6:21129947 Skin colour Salazar et al. (2013) CPPCT022 Peach Aranzana et al. (2002) 7 18.61 Scaffold_7:10225365 Flowering time Fan et al. (2010) CPSCT005 Plum Mnejja et al. (2004) 4 93.62 Scaffold_4:29887942 Flowering time Salazar et al. (2013) Ripening time Salazar et al. (2013) PaCITA7 Apricot Lopes et al. (2002) 1 25.92 Scaffold_1: -- Flowering time Salazar et al. (2013) Fruit weight Salazar et al. (2013) pchcms5 Peach Sosinski et al. (2000) 6 44.71 Scaffold_6:19166407 Self-compatibility Vilanova et al. (2003) PGS.121 Apricot Soriano et al. (2012) 1 23.92 Scaffold_1:8078385 Flesh colour Salazar et al. (2013) UDA027 Almond Testolin et al. (2004) 4 74.71 Scaffold_4:18639695 Ripening time Salazar et al. (2013) UDAp404 Apricot Messina et al. (2004) 4 9.3 Scaffold_4: -- Ripening time Salazar et al. (2013) UDAp407 Apricot Messina et al. (2004) 7 41.3 Scaffold_7: -- Flowering time Salazar et al. (2013) UDAp439 Apricot Messina et al. (2004) 4 49.6 Scaffold_4: -- Ripening time Salazar et al. (2013) UDAp449 Apricot Messina et al. (2004) 3 42.52 Scaffold_3: -- Skin colour Ruiz et al. (2010) UDAp460 Apricot Messina et al. (2004) 7 28.4 Scaffold_7: -- Flowering time Fan et al. (2010) UDAp471 Apricot Messina et al. (2004) 7 51.32 Scaffold_7: -- Nut weight Fernández et al. (2011) Ripening time Fernández et al. (2011) UDP96001 Peach Cipriani et al. (1999) 6 17.51 Scaffold_6:7040757 Skin colour Verde et al. (2002) UDP96003 Peach Cipriani et al. (1999) 4 28.31 Scaffold_4:8757450 Ripening time Salazar et al. (2013) Fruit weight Salazar et al. (2013) UDP98021 Peach Cipriani et al. (1999) 6 9.82 Scaffold_6: -- Fruit weight Eduardo et al. (2011) Skin colour Eduardo et al. (2011) UDP98025 Peach Testolin et al. (2000) 2 9.61 Scaffold_2:10872102 Fruit weight Eduardo et al. (2011) UDP98406 Peach Testolin et al. (2000) 2 80.22 Scaffold_1: -- Ripening time Eduardo et al. (2011) UDP98412 Peach Testolin et al. (2000) 6 72.001 Scaffold_6:24753353 Ripening time Salazar et al. (2013) UDP98416 Peach Testolin et al. (2000) 6 10.62 Scaffold_6: -- Ripening time Verde et al. (2002) 1In Prunus reference Map Texas × Early Gold. 2In the linkage map of the study skin (Table 1). Regarding the genotypes from the Span- ‘7-41’). Group II contained 29 accessions, including 15 ish collection, the cultivars used as progenitors are accessions from Tunisia (5 grafted and 10 propagated characterised by light orange or orange skin colour with by seed) and the rest of the CEBAS-CSIC collection (14 the exception of ‘Currot’ (white) and ‘Mauricio’ (yel- accessions). This second group could be divided into low). Most of the new releases included in this study three subgroups: II A (9 Tunisian accessions); II B (4 resulting from the CEBAS-CSIC breeding program Tunisian and 4 Spanish accessions); and II C [10 Span- showed orange fruits (Table 2). ish and 2 Tunisian accessions (‘Oud Aouicha V71’ and Finally, all Tunisian apricot cultivars showed white, ‘Oud Gnaa V27B’)]. The subgroups II B and II C con- yellow or light orange flesh colour with only one excep- tained accessions from the north and the centre of Tuni- tion (‘Amor El Euch V5A’), which showed orange flesh sia, but none from the south. Finally, Group III included (Table 1). The traditional Spanish cultivars used as 27 accessions from Tunisia. This group could be di- progenitors (‘Currot’, ‘Mauricio’ and ‘Búlida’) were vided into the following three subgroups: III A, which characterised by white, yellow and light orange flesh, exclusively contained ‘Bargougs’ accessions [44 (B, C, respectively, whereas the foreign cultivars used as pro- D, E); 46 (C); and 42 (A, C, G)]; III B, which mainly genitors in the Spanish breeding programmes produced included accessions from the south of Tunisia in addition orange fleshed fruits. Most of the new releases included to ‘Oud El Haj Tahar V70’ from Testour in the north in this study resulting from the crosses performed at (Fig. 1); and subgroup III C, which included all the re- CEBAS-CSIC had orange-fleshed fruits (Table 2). maining accessions from Tunisia (Fig. 3A). Results of the cluster analysis revealed clear differ- In addition, the FCA analysis showed that the acces- ences between the cultivars studied, which pooled into sions studied were highly diverse in terms of pheno- three main groups. Group I contained 16 accessions from type. There were clear, closely-related groups of ac- the Spanish collection, including seven cultivars (‘Cur- cessions, and these groupings were related to rot’, ‘Goldrich’, ‘Mirlo Rojo’, ‘Valorange’, ‘Murciana’, geographic origin: Group I is made up of Tunisian ‘Selene’, and ‘Orange Red’) and nine selections (‘8-50’, accessions and Group II consists of the CEBAS-CSIC ‘11-1’, ‘5-57’, ‘9-5’, ‘9-55’, ‘10-18’, ‘8-61’, ‘10-2’ and collection. There were two main subgroups among the Spanish Journal of Agricultural Research September 2016 • Volume 14 • Issue 3 • e0706 Comparative analysis of traditional and modern apricot breeding programs 7 Murciana Maravilla Currot Bedri Thani V48G Chéchi Bazza Selene Rojo Pasión Mirlo Anaranjado Amor El Euch V5A Khad Hlima V2A Orange Red Mirlo Blanco Mirlo Rojo Oud Aouicha V71 Bayoudhi V11A Figure 2. Fruits from some of the apricot cultivars and accessions evaluated. Scale bar of 5 cm. Tunisian accessions: accessions from the north and the most polymorphic locus was PSG1.21 (0.833) and the centre of Tunisia in the first subgroup (indicated in least polymorphic was UDP98025 (0.040) (Table 4). green and brown respectively in Fig. 3B), and acces- The 20 SSR markers assayed for genetic diversity sions from the south and the oases of Tunisia in the studies generated a total of 110 genotypes with an av- second subgroup (indicated in blue). There was no clear erage of 6 genotypes per marker. Mean expected het- grouping among varieties in the Spanish collection. erozygosity (He) was about 0.48 with maximum He FCA results also showed that apricot material in Tuni- values recorded by SSR markers for PSG1.21 (0.851) sia was less diversified than the Spanish apricot mate- and minimum He values recorded by SSR markers for rial (in black in Fig. 3B). UDP98025 (0.041). Observed heterozygosis (Ho) ranged from 0.000 (UDP96001) to 0.831 (UDAp471), with a mean value of 0.359. A significant heterozygo- Molecular diversity and association genetic sity deficit was observed for four loci (UDP96001, studies UDP98025, UDP98416 and UDAp449), however, for the rest of the SSR markers studied, no significant dif- Amplification of SSR loci was successful with the 20 ference was found between the expected and the ob- tested primers. Polymorphic bands were generated for served heterozygosity (Table 4). On the other hand, for all the primers used, with a total of 60 scored polymor- the Wright’s fixation index, the calculated Fst values phic bands. The number of presumed alleles revealed by ranged from 0.002 (UDA027) to 0.518 (UDAp407), the 20 SSRs ranged from two (BPPCT025, PaCITA7, with an average of 0.141. Fis values ranged from -0.22 pchcms5, UDA027, UDAp407, UDAp449, UDAp460, (UDAp471) to 1 (UDP96001), with an average of 0.20 UDP96001, UDP98025 and UDP98416) to ten (Table 4). (PSG1.21), with a mean of three alleles per locus (Table Table 5 shows the SSR alleles obtained with a high 4). The average PIC value for the 20 loci was 0.410. The degree of co-segregation within the phenotypes of the Spanish Journal of Agricultural Research September 2016 • Volume 14 • Issue 3 • e0706 8 Mohamed A. Batnini, Lamia Krichen, Hedia Bourguiba, Neila Trifi-Farah, David Ruiz et al. A) III II I Factorial analysis: Axes 1/3 B) Figure 3. Unrooted NJ (Neighbour Joining) dendogram based on the mean character difference distances, Spanish apricot germplasm is indicated in black colour and Tunisian apricot germplasm in red (A). Genetic structure based on FCA analysis (B) among apricot cultivars evaluated using phenotypic traits; clearly identified subgroups of Tunisian apricot germplasm indicated in green, brown, blue; and Spanish accessions (black). Spanish Journal of Agricultural Research September 2016 • Volume 14 • Issue 3 • e0706 Comparative analysis of traditional and modern apricot breeding programs 9 Table 4. Molecular diversity parameters of the SSR loci used in the apricot genetic analysis. Number of samples (N), number of samples that amplify (n), number of genotypes found (Genotypes), number of alleles amplified (Alleles), major allele frequency (MAF), expected heterozygosity (He), observed heterozygosity (Ho), polymorphism information content (PIC), fixation index (Fst), mean value of gene flow (Nm), and inbreeding coefficient (Fis). Marker N n Genotypes Alleles MAF He Ho PIC Fst Nm Fis BPPCT025 72 67 3 2 0.619 0.471 0.433 0.360 0.005 48.77 0.09 CPPCT022 72 71 9 6 0.437 0.686 0.479 0.631 0.163 1.29 0.24 CPSCT005 72 72 6 3 0.451 0.646 0.472 0.573 0.252 0.74 0.15 PaCITA7 72 72 3 2 0.785 0.338 0.264 0.281 0.466 0.29 -0.11 pchcms5 72 69 3 2 0.681 0.434 0.261 0.340 0.021 11.71 0.41 PSG1.21 72 72 25 10 0.188 0.851 0.764 0.833 0.091 2.49 0.07 UDA027 72 72 3 2 0.715 0.407 0.403 0.324 0.002 103.92 0.02 UDAp404 72 70 6 3 0.721 0.440 0.257 0.398 0.214 0.92 0.34 UDAp407 72 71 3 2 0.521 0.499 0.338 0.375 0.518 0.23 -0.03 UDAp439 72 67 6 3 0.552 0.586 0.597 0.515 0.048 5.01 -0.04 UDAp449 72 72 2 2 0.972 0.054 0.056 0.053 0.013 19.59 -0.02 UDAp460 72 70 3 2 0.771 0.353 0.286 0.290 0.385 0.40 -0.05 UDAp471 72 71 9 4 0.458 0.684 0.831 0.633 0.022 11.06 -0.22 UDP96001 72 72 2 2 0.528 0.498 0.000 0.374 0.023 10.48 1.00 UDP96003 72 69 6 3 0.645 0.517 0.449 0.459 0.124 1.77 0.08 UDP98021 72 72 6 3 0.639 0.508 0.458 0.438 0.073 3.17 0.07 UDP98025 72 72 2 2 0.979 0.041 0.042 0.040 0.046 5.15 -0.04 UDP98406 72 71 5 3 0.493 0.571 0.451 0.479 0.054 4.40 0.20 UDP98412 72 72 5 3 0.479 0.556 0.292 0.457 0.049 4.89 0.47 UDP98416 72 71 3 2 0.634 0.464 0.056 0.356 0.014 18.13 0.88 Mean 72 71 6 3 0.613 0.480 0.359 0.410 0.141 1.53 0.20 different apricot genotypes assayed. Only UDP98406 The genetic heterozygosity ranged between 0.050 (Tu- (Eduardo et al., 2011) showed a good degree of linkage nisian cultivar ‘BedriV48A’) and 0.650 (CEBAS-CSIC with ripening time. A total of 86% of the germplasm stud- selections ‘7-41’ and ‘8-50’), with an average value of ied with the allele 118 had an early or a medium ripening 0.55 for all the studied genotypes (Table 6). date and only 14% had a late or very late ripening date. The 72 genotypes were clearly classified into three Only the two markers showed good linkage with fruit major groups based on their molecular characterisation weight. When the genotypes were separated into two using SSRs, A, B and C (Fig. 4). Group A contained groups of fruit weight (> or < 60 g), we could observe 27 Spanish and 5 Tunisian genotypes; group B mainly that the allele 434 (PaCITA7) was present in all geno- types with fruits weighing less than 60 g (a total of 25 genotypes). The alleles 391 (PaCITA7) and 114 Table 5. SSR alleles obtained with the highest level of co- (UDP96003) were present in most genotypes with fruits segregation within phenotypes of the different apricot geno- weighing over 60 g (Table 5). The presence of the allele types assayed of 449 nucleotides (nt) from the marker UDAp407 has SSR allele Percentage of a given been correlated with fruit firmness (Table 5). About 90% Marker (nt) [1] phenotype of accessions with the allele 449 present were firm or Ripening time medium-firm, and only 6% were soft. Late/very late Early/medium UDP98406 118 14 86 Fruit weight Molecular heterozygosity, relationships Small (<60 g) Big (>60 g) and structure analysis PaCITA7 391 6 94 434 100 0 The genetic heterozygosity of apricot cultivars eval- UDP96003 114 6 94 uated using SSR markers ranged from 0.050 (‘Bedri Fruit firmness V48A’) to 0.600 (‘9-5’ and ‘Valorange’), with an average Firm/medium Soft value of 0.39 (Table 6). The group of traditional cultivars UDAp407 449 90 6 from Tunisia and Spain showed lower genetic heterozy- [1] Band scoring was analysed using SYNGENE® GeneTools gel gosity, which was independent of the propagation mode. analysis software. Spanish Journal of Agricultural Research September 2016 • Volume 14 • Issue 3 • e0706 10 Mohamed A. Batnini, Lamia Krichen, Hedia Bourguiba, Neila Trifi-Farah, David Ruiz et al. Table 6. Genetic heterozygosity (Ho) observed in the different apricot genotypes assayed from the Tunisian and Spanish (CEBAS-CSIC) breeding programmes using 20 SSR markers. Genotype Ho Genotype Ho Genotype Ho Cultivars selected from the Tunisian breeding programme Addadi Ahmar V15 0.368 Bargoug B42G 0.250 Bedri Louzi V57B 0.150 Amor El Euch V5A 0.368 Bargoug B44B 0.400 Bouk H’med V13A 0.350 Amor El EuchV51A 0.400 Bargoug B44C 0.368 Bouthani V20B 0.350 Amor El Euch V51C 0.350 Bargoug B44D 0.250 Chéchi Bazza V28D 0.400 Baccour V41C 0.316 Bargoug B44E 0.350 Fourati V38C 0.350 Bargoug B40F 0.316 Bargoug B44F 0.316 Khad Hlima V2A 0.211 Bargoug B40H 0.389 Bargoug B44H 0.300 Khad Hlima V2C 0.222 Bargoug B40K 0.400 Bargoug B46C 0.400 Oud Aouicha V71 0.200 Bargoug B40L 0.200 Bargoug B46D 0.350 Oud El Haj Tahar V70 0.300 Bargoug B40M 0.250 Bargoug B46E 0.300 Oud Gnaa V27B 0.350 Bargoug B43A 0.200 Bayoudhi V11A 0.250 Oud H’mida V21D 0.100 Bargoug B43B 0.350 Bedri V1A 0.053 Oud Rhayem V18A 0.400 Bargoug B42A 0.263 Bedri V48A 0.050 Thani V59B 0.158 Bargoug B42C 0.450 Bedri Thani V48G 0.053 Zbidi V7A 0.222 Mean 0.286 Spanish progenitors from the Spanish breeding programme (CEBAS-CSIC) Búlida 0.250 Currot 0.368 Mauricio 0.300 Mean 0.306 Foreign progenitors from the Spanish breeding programme (CEBAS-CSIC) Bergeron 0.421 Goldrich 0.550 Orange Red 0.300 Palsteyn 0.550 Mean 0.455 New releases from the Spanish breeding programme (CEBAS-CSIC) Dorada 0.474 Rosa 0.500 8-61 0.474 Estrella 0.474 Selene 0.400 9-5 0.600 Maravilla 0.450 Sublime 0.450 9-55 0.500 Mirlo Blanco 0.526 Toñi 0.450 10-18 0.450 Mirlo Naranja 0.450 Valorange 0.600 10-20 0.550 Mirlo Rojo 0.400 5-57 0.526 10-57 0.350 Murciana 0.450 7-41 0.650 11-1 0.250 Rojo Pasión 0.500 8-50 0.650 Mean 0.550 contained Tunisian genotypes; and group C contained given the assumed number of ancestral populations K, nearly all the remaining Tunisian genotypes in addition was highest at K=2. As defined by Evanno et al. to three of the traditional Spanish cultivars. Group A (2005), an ad hoc quantity based on the second order could be divided into two subgroups, with the first rate of change of the likelihood function with respect subgroup containing all the Spanish genotypes. The to K (∆K) showed that the best model is at K=2 (Fig- second subgroup included five ‘Bargougs’ propagated ure 5). According to the model at K=2, apricot geno- by seeds and originating from the oasis of Gafsa in the types were assigned to two genetically different clus- south-west of Tunisia. In Group B, the distribution of ters identified by STRUCTURE analysis: cluster 1 Tunisian cultivars in secondary subgroups was related grouped together all the Tunisian cultivars (42 culti- to geographic origin, so that genotypes from the north vars) with three accessions from Spain (‘Currot’, and the south clustered together and ‘Bargougs’ clus- ‘Búlida’ and ‘Mauricio’). The second cluster was made tered separately. Group C included the rest of the Tu- up of the Spanish collection (27 cultivars). The model nisian cultivars and three traditional cultivars from under K=3 classified the 72 apricot genotypes into the Spain (‘Currot’, ‘Búlida’ and ‘Mauricio’), which were following three clusters: the first cluster included 27 related to the cultivars ‘Baccour V41C’ and ‘Amor El Spanish cultivars; the second cluster included six cul- Euch V51C’ from the south of Tunisia as well as to one tivars from Tunisia; and the third cluster included three accession of ‘Bargoug B46D’ from the oasis of Midess. cultivars from the Spanish breeding programme (‘Cur- Finally, using model-based Bayesian clustering, the rot’, ‘Búlida’ and ‘Mauricio’) and 36 Tunisian cultivars estimated log probability of the data (ln Pr (X|K)), (Fig. 5). Spanish Journal of Agricultural Research September 2016 • Volume 14 • Issue 3 • e0706