Heredity (2004) 93, 631–638 & 2004 Nature Publishing Group All rights reserved 0018-067X/04 $30.00 www.nature.com/hdy Genetic relationships between diploid and allotetraploid cherry species (Prunus avium, Prunus  gondouinii and Prunus cerasus) M Tavaud1,3, A Zanetto1, JL David2, F Laigret1 and E Dirlewanger1 1INRA, UREFV, BP81, 71, Avenue E Bourleaux, 33883 Villenave d’Ornon, France; 2INRA, UMR DGPC, Domaine de Melgueil, 34130 Mauguio, France Prunus avium L. (diploid, AA, 2n ¼ 2x ¼ 16), Prunus cerasus correspondence analysis allowed us to clearly distinguish L. (allotetraploı¨d, AAFF, 2n ¼ 4x ¼ 32) species, and their each species, to identify misclassifications and to assign hybrid Prunus  gondouinii Rehd., constitute the most widely unknown genotypes to one species. We showed that there cultivated cherry tree species. P. cerasus is supposed to be are specific alleles in P. cerasus, which are not present in the an hybrid species produced by the union of unreduced A genome of P. avium and which probably come from the F P. avium gametes and normal P. fruticosa gametes. A genome of P. cerasus. The frequencies of each marker in the continuum of morphological traits between these three A and the F genomes were estimated in order to identify A species makes their assignation difficult. The aim of this and F specific markers. We discuss the utility of these paper is to study the genetic relationships between tetraploid specific markers for finding the origin of the A and F genomes and diploid cherry species. In all, 114 genotypes belonging to in the allopolyploid species. these species were analyzed using 75 AFLP markers. Heredity (2004) 93, 631–638. doi:10.1038/sj.hdy.6800589 The coordinates of these genotypes on the first axis of a Published online 8 September 2004 Keywords: Prunus avium; Prunus cerasus; AFLP markers; species identification; genetic diversity Introduction tion between P. avium (producing unreduced gametes) and P. fruticosa (Figure 1). This origin was first suggested The most widely cultivated cherry trees belong to Prunus by Olden and Nybom (1968), who observed that artificial avium L. and P. cerasus L. species. Along with P. fruticosa hybrids between tetraploid P. avium and P. fruticosa were Pall., these species and their interspecific hybrids very similar to P. cerasus. Isozyme analyses, genomic constitute the Eucerasus section of the Cerasus subgenus, in situ hybridization and karyotype analysis further based on morphological criteria (Rehder, 1947; Kruss- confirmed the hybrid origin of P. cerasus (Hancock and mann, 1978). This classification and the monophyletic Iezzoni, 1988; Santi and Lemoine, 1990a; Schuster and origin of the Eucerasus clade have been confirmed by Schreiber, 2000). The patterns of inheritance of seven chloroplastDNA variation analysis (Badenes and Parfitt, isozymes in different crosses of sour cherry indicated 1995). that P. cerasus may be a segmental allopolyploid (Beaver P. avium includes sweet cherries, cultivated for human and Iezzoni, 1993). Recent studies based on cpDNA consumption and wild cherry trees grown for their wood markers detected two distinct chlorotypes in P. cerasus, (Webster, 1996). This species is diploid (AA, 2n ¼ 2x ¼ 16) which strongly suggest that crosses between P. avium and and its natural range covers the temperate regions P. fruticosa have occurred at least twice to produce sour of Europe, from the Northern part of Spain to the cherry (Badenes and Parfitt, 1995; Iezzoni and Hancock, Southeastern part of Russia (Hedrick, 1915). P. fruticosa, 1996; Brettin et al, 2000). Moreover, these results suggest the ground cherry tree, is a tetraploid wild species that, most of the time, P. fruticosa was the female (2n ¼ 4x ¼ 32) thought to be autotetraploid (FFFF), some- progenitor of P. cerasus, but in few cases P. avium was times used as rootstocks for other Prunus species. This the female parent, via the formation of unreduced species is widespread over the major part of central ovules. Triploid hybrids through the fusion of normal Europe, Siberia and Northern Asia (Hedrick, 1915). P. gametes of P. avium and P. fruticosa occur naturally but cerasus, the sour cherry tree, is cultivated for fruit, used in remain sterile. Moreover, they are not clonally propa- jam or liquor. It is an allotetraploid species (AAFF, gated by man since they exhibit many unfavorable P. 2n ¼ 4x ¼ 32), thought to result from natural hybridiza- fruticosa traits (Olden and Nybom, 1968). The duke cherries, which result from crosses between Correspondence: Dr M Tavaud, INRA, UREFV, BP81, 71, Avenue E P. avium and P. cerasus, are cultivated at a much smaller Bourleaux, 33883 Villenave d’Ornon, France. scale. Different names have been given to this species E-mail: [email protected] including P. acida Dum, Cerasus regalis, P. avium ssp 3Current address: INRA, UMR DGPC, Domaine de Melgueil, 34130 Mauguio, France. regalis, but the name used currently is P.  gondouinii Received 27 November 2003; accepted 16 July 2004; published Rehd. (Faust and Suranyi, 1997; Saunier and Claverie, online 8 September 2004 2001). This species is allotetraploid (AAAF, 2n ¼ 4x ¼ 32), Genetic relationships between three cherry species M Tavaud et al 632 cerasus and to identify the origin of the A genome in both Prunus fruticosa Prunus avium tetraploid species (P. cerasus and P.  gondouinii). 2n=4x=32 2n=2x=16 FFFF AA * Materials and methods Plant material Prunus cerasus In all, 114 genotypes were studied: 68 P. avium genotypes 2n=4x=32 * (33 cultivated and 35 wild) sampled throughout the AAFF natural range of the species, 25 P. cerasus genotypes and 9 P.  gondouinii genotypes according to morphological characters (Table 1). In total, 12 French genotypes which had not been previously studied were not assigned to a species. The 33 cultivated sweet cherry trees were old Prunus x gondouinii cultivars, from 13 different countries. The 35 wild cherry =Prunus acida Dum trees were from several European forests. Sour and duke 2n=4x=32 cherry trees and unassigned genotypes were from the AAAF French germplasm, mainly maintained in INRA orchards, at the ‘Unite´ Expe´rimentale d’Arboriculture de Bordeaux’. Figure 1 Hypothesis for the relationships between the four species belonging to the Eucerasus section. *P. avium is thought to produce diploid gametes. A and F are haploid genomes coming from P. AFLP analyses avium and P. fruticosa respectively. Genomic DNA was extracted from young leaves, follow- ingthemethoddescribedbyViruelet al (1995). We stemming from the fertilization of sour cherry by essentially followed the AFLP protocol developed by Vos unreduced gametes of sweet cherry (Iezzoni et al, 1990). et al (1995) with minor modifications. DNA was digested These hybrids are often sterile, due to disturbances with EcoRI and MseI restriction enzymes. Digestion was during meiosis, but they are clonally propagated by man. performed for 4 h at 371C in a final volume of 17.5 ml The duke cherry trees present tree and fruit character- containing 10 mM Tris-Ac, 10 mM MgAc, 50 mM KAc, istics intermediate between their progenitors. 5mM DTT, 25U EcoRI (Pharmacia), 4 U MseI(New Great morphological variation exists among P. avium, England Biolabs) and 250 ng of genomic DNA. Two P. fruticosa and P. cerasus species. Multivariate analysis on linkers, one for the EcoRI sticky ends and the other for sour cherry revealed continuous variation between the the MseI sticky ends, were ligated for 3 h at 371Ctothe P. avium, P. cerasus and P. fruticosa traits throughout the digested DNA by adding 2.5 ml of a mix containing geographic distribution of the species. In Western 2.5 pmol EcoRI linker, 25 pmol MseI linker, 8 mM ATP, Europe, P. cerasus trees look like P. avium, whereas in 10 mM Tris-HAc, 10 mM MgAc, 50 mM KAc, 5 mM DTT Eastern Europe P. cerasus are closer to P. fruticosa (Hillig and 0.85 U T4 DNA ligase (Pharmacia). This ligation and Iezzoni, 1988; Krahl et al, 1991). This continuum of product was diluted five-fold. A first preselective PCR morphological characteristics makes species assignation amplification was performed using EcoRI þ AandMseI þ difficult when considering only phenotypic traits. C primers in a 50 ml mix of 20 mM Tris–HCl (pH 8.4), Genetic resource conservation, essential for future 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 30 ng of breeding programs, requires a good characterization of each primer, 1 U Taq DNA polymerase (GibcoBRL) and 5 ml germplasm genetic diversity and a proper assignation of of diluted ligation product. The reaction was carried out in individual genotypes to species. Molecular markers a Perkin-Elmer 9600 thermocycler and the samples were supply tools to analyze genetic diversity in Prunus subjected to 28 amplification cycles with three steps of 30 s species (Baird et al, 1996). They could be helpful by at 941C, 60 s at 561C and 60 s at 721C. The preamplification giving an accurate and unambiguous assignation of each products were diluted 10-fold and used as starting genotype to a particular species. Isozymes have been material for the selective radioactive amplification. already used to characterize genetic diversity in P. avium For selective amplification, EcoRI and MseI primers and P. cerasus, but the number of available isozymes is with three and two additional nucleotides, respectively, limited and their polymorphisms are low (Santi and were used (Table 2), the first one being 33P-labelled using Lemoine, 1990b). AFLP markers could be useful because T4 polynucleotide kinase. The PCR reaction was per- they are reproducible, they need no previous information formed in a 20 ml volume of 20 mM Tris–HCl (pH 8.4), 33 about the genome and they allow the detection of many 50 mM KCl, 1.5 mM MgCl2, 5 ng [ P] EcoRI primer, 30 ng markers in few analysis (Vos et al, 1995; Jones et al, 1997).