Mol Breeding (2015) 35:59 DOI 10.1007/s11032-015-0217-5 Single nucleotide polymorphism-based parentage analysis and population structure in garden asparagus, a worldwide genetic stock classification Francesco Mercati • Paolo Riccardi • Alex Harkess • Tea Sala • Maria Rosa Abenavoli • Jim Leebens-Mack Agostino Falavigna • Francesco Sunseri • Received: 5 May 2014 / Accepted: 27 November 2014 Ó Springer Science+Business Media Dordrecht 2015 Abstract Knowledge of genetic diversity and population structure in breeding material could be of great importance for crop improvement. Inheritance of molecular markers has been proved to be a powerful tool for verifying or discovering the parentage of cultivars in several crops. The present study aimed to undertake an extended parentage analysis using a large sample of garden asparagus (Asparagus officinalis L.) cultivars based on single nucleotide polymorphism (SNP) markers. In the past, asparagus cultivars began to be classified according to the countries and towns where they were grown. Among them, Violet Dutch is one of the oldest asparagus cultivars, considered to be Francesco Mercati and Paolo Riccardi contributed equally to this study and should be considered co-first authors. Electronic supplementary material The online version of this article (doi:10.1007/s11032-015-0217-5) contains supplementary material, which is available to authorized users. F. Mercati
M. R. Abenavoli
F. Sunseri (&) Dipartimento AGRARIA, Università ‘‘Mediterranea’’ di Reggio Calabria, Salita Melissari, 89124 Reggio Calabria, RC, Italy e-mail: francesco.sunseri@unirc.it P. Riccardi
T. Sala
A. Falavigna CRA-ORL, Unità di Ricerca per l’Orticoltura, via Paullese 28, 26836 Montanaso Lombardo, LO, Italy A. Harkess
J. Leebens-Mack Department of Plant Biology, University of Georgia, Athens, GA 30602, USA the genetic stock from which several modern cultivars were derived. Starting from Violet Dutch, the breeding programs branched in two directions, yielding Argenteuil and Braunschweiger varieties in France and Germany, respectively. These lines became very important in all breeding programs, replacing older populations and landraces. This could account for the narrow genetic basis of cultivated asparagus, but in fact very few molecular marker studies have confirmed this hypothesis to date. In the present paper, using a new set of 144 SNPs, genetic relationships were investigated within an important collection of anther donor asparagus genotypes and a large panel of Italian double haploids (DHs) extensively used in the Italian and international breeding programs over the last 30 years. The results were useful for confirming the narrow variability of modern asparagus germplasm and for comparing the pedigree notes with genetic analyses. The results of this work showed that the DH collection includes two main and distinct genetic backgrounds, likely derived from Argenteuil and Braunschweiger genetic stocks, as expected. Moreover, the genetic analyses showed that the cv. Mary Washington, previously indicated as being derived only from Argenteuil, has a mixed origin from the two main genetic stocks. In addition, the present study underlines how this cultivar plays a central role in the pedigree of many modern cultivars/hybrids, giving new impact to the pedigree notes already described taking into account the large number of DHs derived from Mary Washington. 123 59 Page 2 of 12 Keywords Asparagus officinalis L.
Pedigree notes
Single nucleotide polymorphism
Genetic diversity Introduction Asparagus is a large genus, comprising about 150 herbaceous perennials, tender woody shrubs and vine species distributed throughout Asia, Africa and Europe. Garden asparagus (A. officinalis L.) is a high-value, widely cultivated vegetable crop (Bailey 1942), and other species are commonly used as medicinals or ornamentals (Stajner et al. 2002). Asparagus species are classified into the subgenera Asparagus, Protasparagus and Myrsiphyllum (Ellison and Kinelski 1986; Clifford and Conran 1987). Species included in the subgenus Asparagus are dioecious, while those of the other subgenera are hermaphroditic. The center of diversity for subgenus Asparagus and the suspected region of domestication includes Eastern Europe, Caucasus and Siberia (Sturtevant 1980). Greeks and then Romans imported the culture of cultivating asparagus from the east, and, in the process, the old-Iranian word ‘sparega’, meaning shoot, rod, spray, was transformed to ‘asparagos’ and ‘asparagus’ in Greek and Latin, respectively. The ancient Romans spread the asparagus crop throughout Europe, and already in 79 B.C. Plinio described how to grow it in his famous Naturalis Historia. There is also evidence that crusading troops brought asparagus seeds from Arabian countries to the Rhine valley around 1212 C.E. (Reuther 1984). After the Roman Empire declined, asparagus cultivation was confined to feudal lands and monastery gardens as a medicinal plant. During the Renaissance asparagus and several other neglected species were rediscovered as worthwhile vegetables, and in the sixteenth century garden asparagus cultivation spread through Germany, France, England and The Netherlands. Asparagus cultivars began to be identified and classified according to the countries and towns where they were grown, giving rise to provenences such as Riga, Ivancice, Ghent, Ulm, Vendome, Besancon and Violet Dutch (Kidner 1947; Lužný 1979; Knaflewski 1996). Violet Dutch is considered to be one of the 123 Mol Breeding (2015) 35:59 oldest asparagus lines, and it is thought to be the genetic stock from which several modern cultivars were derived (Kidner 1947; Knaflewski 1996; Fig. S1). The breeding programs including Violet Dutch historically moved in two directions, yielding the Argenteuil and Braunschweiger varieties in France and Germany, respectively. Together these two lines gained international importance, replacing older populations and landraces (Knaflewski 1996). Molecular markers have been used to assess old and often incomplete pedigrees and inform breeding programs in many crops such as beans (Kwak and Gepts 2009). Thus, molecular markers are useful not only for selecting genotypes with favourable traits, but also for assessing genetic diversity, genetic structure and parental relationships among breeding stocks (Sim et al. 2012). In Asparagus, the utilization of genomic molecular markers (RAPD, RFLP and AFLP) has already been employed for surveys of variation (Khandka et al. 1996; Caruso et al. 2008), assessment of gender (Jiang and Sink 1997; Reamon-Büttner and Jung 2000; Nakayama et al. 2006; Kanno et al. 2014) and development of genetic (Jiang et al. 1997; Spada et al. 1998) and physical maps (Telgmann-Rauber et al. 2007). Furthermore, expressed sequence tags (ESTs) and more recently RNA Seq data have been utilized to develop gene-linked and often more informative molecular markers in several crops (Haseneyer et al. 2011). Next-generation sequencing (NGS) is facilitating the development of very large marker panels, including simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) (Harismendy et al. 2009), and the identification of diagnostic alleles in plant germplasm collections (Qiu et al. 2003; Kota et al. 2003; Hamilton et al. 2012). In garden asparagus, EST data have recently been mined to develop large SSR and SNP marker panels for analyses of ancestry and marker-assisted breeding (Caruso et al. 2008; Mercati et al. 2013). In this paper, we used recently developed SNP markers to investigate relationships within a collection of anther donor lines and a large panel of Italian double haploids (DHs) that have been used in the Italian and international breeding programs over the last 30 years (Riccardi et al. 2011). The results of this work indicated that the DH collection includes two main and distinct genetic backgrounds, likely derived from Argenteuil and Braunschweiger genetic stocks. Mol Breeding (2015) 35:59 Page 3 of 12 Materials and methods microcentrifuge tubes. Two hundred mg of frozen leaf sample were ground with liquid nitrogen and genomic DNA was resuspended in 100 ll of distilled water. DNA concentrations and quality were assessed with the ND-1000 NanoDrop spectrophotometer (Thermo Scientific). Plant material Sixty-six doubled haploid (DH) clones and their 22 anther donor genotypes were included in our genetic assays (Tables 1, 2). The anther donors comprised a representative sampling of genetic backgrounds utilized both in the Italian and international breeding programs (Riccardi et al. 2011), including lines thought to be derived from Argenteuil, Braunschweiger, crosses between these two ancestral lines, and cv. Mary Washington, which has been hypothesized as part of the Argenteuil lineage (Fig. S1). DH clones were developed following the protocol by Qiao and Falavigna (1990). Tissue samples were collected from plants grown either in open field or in greenhouse and stored at -80°C until DNA analysis. DNA extraction Total DNA was extracted following the CTAB method (Murray and Thompson 1980), scaled down for Table 1 List of anther donors used in population structure analysis together with their putative gene pool origin and variety type 59 Genetic diversity assessment based on SNP genotyping The Illumina BeadXpress was used for SNP genotyping at the Georgia Genomics Center (University of Georgia, http://dna.uga.edu/). One hundred and forty-four previously identified gene-linked SNPs (Riccardi et al. 2012; Mercati et al. 2013) were chosen for the analysis. Briefly, more than 200,000 transcripts derived from male and female asparagus accessions were sequenced (NCBI SRA Project ID PRJNA184373) on the GSFLX 454 pyrosequencing platform, assembled and compared with other data for garden asparagus available in NCBI’s EST database (http://www.ncbi.nlm.nih.gov/ nucest?term=txid4686) for SNP calling. Of more than 1,700 putative SNPs initially identified, 284 were Anther donor Origina (putative gene pool) Variety type 1 921-1 Argenteuil Heterozygous clone 2 Eros Argenteuil All-male hybrid 3 Gladio Argenteuil All-male hybrid 4 Andreas Argenteuil All male hybrid 5 Bassano Argenteuil Heterozygous clone 6 Marte Argenteuil (M. Washington) All-male hybrid 7 H553 Argenteuil All-male hybrid 8 9 Lucullus 709-1 Braunschweiger Braunschweiger All-male hybrid Heterozygous clone 10 UC157 Argenteuil (M. Washington) Simple hybridb 11 Giant Argenteuil (M. Washington) All-male hybrid 12 H465 Argenteuil 9 Braunschweiger All-male hybrid 13 H499 Argenteuil 9 Braunschweiger All-male hybrid 14 H524 Argenteuil (M. Washington) All-male hybrid 15 Zeno Argenteuil 9 Braunschweiger All-male hybrid The putative origin of each anther donor was attributed based on the published and unpublished historical pedigree notes (see Knaflewski 1996) 16 Apollo Argenteuil (M. Washington) Simple hybridb 17 AM841 Argenteuil All-male hybrid 18 Grande Argenteuil (M. Washington) Simple hybridb 19 Gronlim Braunschweiger All-male hybrid 20 H500 Argenteuil (M. Washington) All-male hybrid b 21 Italo Argenteuil (M. Washington) All-male hybrid 22 Ariane Braunschweiger Simple hybridb a Hybrid derived from two heterozygous clones/ varieties 123 59 Page 4 of 12 Table 2 The 66 doubled haploid (DH) clones analyzed. The accession number is from CRA-ORL asparagus germplasm collection, the name indicates the anther donor genotype Mol Breeding (2015) 35:59 Accession Name DH clone sex Accession Name DH clone sex 3584 Andreas_a M 5400 H500_a F 3658 Bassano_a M 5407 H500_b M 3305 Eros_a M 5447 H500_c F 3401 Eros_b M 5448 H500_d M 3559 Gladio_a M 5510 H500_e M 3594 Gladio_b F 5514 H500_f F 3657 Gladio_c M 5470 Italo_a M 3684 3806 Gladio_d Gladio_e M M 5478 5480 Italo_b Italo_c F M 5500 H553_a F 5491 Italo_d F 4966 Marte_a M 3014 UC157_a F 4967 Marte_b M 3570 H465_a M 4987 Marte_c M 3688 H465_b F 4991 Marte_d M 3693 H465_c M 4999 Marte_e M 3731 H465_d M 5184 Marte_f M 3840 H465_e F 5191 Marte_g M 3873 H465_f M 1770 921-1_a M 3905 H465_g M 1800 921-1_b F 4058 H465_h M 4881 AM841_a M 4119 H465_i F 4880 AM841_b F 4309 H499_a F 5391 Gronlim_a M 4307 H499_b F 5390 4746 Gronlim_b H524_a F M 4357 4408 H499_c H499_d F M 1559 Lucullus_a M 4406 H499_e F 1666 709-1_a M 4450 H499_f M 4806 Apollo_a F 4500 H499_g F 5555 Ariane_a M 4545 H499_h F 3015 Giant_a F 4650 H499_i F 3069 Giant_b M 4678 H499_l F 3315 Giant_c M 4750 Zeno_a F 3307 Giant_d F 4790 Zeno_b M 5045 Grande_a F 4790 Zeno_c F chosen to be assayed on the Illumina BeadXpress platform after stringent filtering of possible paralogs and sequencing errors (see Riccardi et al. 2012 for details). As described by Mercati et al. (2013), a genetic map with 13 linkage groups (LG), corresponding to n = x = 10 haploid chromosomes, was obtained using 144 of these markers which exhibited Mendelian segregation patterns. Raw BeadXpress data were converted to genotype calls using the BeadStudio software with the standard normalization procedure including removal of outliers, background correction and scaling of raw 123 hybridization intensity data. Estimated genotype clusters and genotype calls were visualized on two-dimensional Cartesian plots using the GenCall (GC) function of the BeadStudio package (Akhunov et al. 2009). Data analysis DH clone anther donors were placed in genotypic clusters using STRUCTURE version 2.3.4 (Pritchard et al. 2000), which employs a Bayesian clustering approach to identify distinct gene pools and to assign Mol Breeding (2015) 35:59 individuals to K populations based on the allele frequencies at each locus. Evaluation of the appropriate number of genetic clusters (Ks) was performed following guidelines published by Pritchard and Wen (2003) and simulation analyses (Evanno et al. 2005). Program settings used the admixture ancestry and correlated marker frequency models. Alpha was inferred from the data and lambda was set to 1 (Pritchard and Wen 2003; Evanno et al. 2005). For each K (ranging from 1 to 10), 20 independent runs (500,000 burn-in, 1,000,000 Marchov chain Monte Carlo) were carried out. CLUMPP (CLUster Matching and Permutation Program; Jakobsson and Rosenberg 2007) was utilized to average the 20 runs and histograms were generated using the program DISTRUCT (Rosenberg 2004). While garden asparagus is a dioecious outcross species, breeding history may have resulted in deviations from Hardy–Weinberg equilibrium. Instruct software version #1 (http:// cbsuapps.tc.cornell.edu/InStruct.aspx; Gao et al. 2007) was used to assess whether cluster assignments inferred by STRUCTURE may have been confounded by non-random mating within garden asparagus. Genetic distances among asparagus DHs were calculated by MEGA5 (Tamura et al. 2011) from the SNP calls and the neighbor-joining (NJ) method (Saitou and Nei 1987) was then used to construct the dendrogram from the distance matrix (Nei 1978). The level of support for nodes in the NJ tree was evaluated by bootstrap analysis with 1,000 replicates. Finally, genetic similarities among all DH clones were assessed through principal coordinate analysis (PCoA) using the GenAlEx 6 program (Peakall and Smouse 2012). Page 5 of 12 59 Results We used SNP-based genetic analysis to test the hypothesis that modern asparagus cultivars and hybrids are derived from an ancestral Violet Dutch background that was channeled into two distinctive lineages (Fig. S1). Population structure analysis showed the assignment of each anther donor genotype to clusters, starting from K = 1–10 (Fig. 1). The DK evaluation statistic of Evanno et al. (2005) revealed a clear optimum for K = 2. Cluster assignments using STRUCTURE and InStruct were nearly identical, suggesting that the STRUCTURE results were not confounded by non-random mating. These results appear to be in agreement with Knaflewski’s (1996) hypothesis that germplasm included in the international breeding programs is derived from two Network reconstruction To evaluate the number of haplotypes and their relatedness, the median-joining (MJ) network reconstruction method (Bandelt et al. 1999) was used. This method allows multi-state characters and it has been shown to be robust and effective even when internal node haplotypes are not sampled (Cassens et al. 2005). The MJ network method followed by the MP option to clean up the network (Polzin and Daneschmand 2003) was performed by using NETWORK 4.51.0 software (Fluxus Technology, www.fluxus-engineering.com). All characters were weighted equally (default setting) and the indels considered as single events. Fig. 1 Hierarchical organization of genetic relatedness on 22 anther donor genotypes based on 124 SNP markers and analyzed by the STRUCTURE program as described in ‘‘Materials and methods’’ for K = 2–10. Bar graphs were developed with the program DISTRUCT 123 59 Page 6 of 12 main genetic backgrounds (Argenteuil and Braunschweiger), but many anther donor lines that were hypothesized as derived from cv. Mary Washington were groups with putatively Braunschweiger lines (Fig. 1). Furthermore, AM841, which was hypothesized to be derived from Argenteuil, also grouped with the Braunschweiger and Mary Washington lines. With K set at 2, the remaining six Argenteuil anther donors clustered with Marte, a Mary Washington-derived line, and the fifteen other donors formed a cluster including all Braunschweiger lines, the other Mary Washington lines AM841. This grouping was also seen when K was set between 3 and 10, although two genotypes, UC157 and Giant (both derived from cv. Mary Washington on the basis of pedigree data), were moved from the Braunschweiger cluster to a third distinct cluster when K was set to be greater than 2 (Fig. 1). Indeed, these two genotypes exhibited a distinct multi-locus genetic profile when compared to lines in the Braunschweiger and Argenteuil groups. Results from analyses of the 144 SNPs in 66 DHs were largely consistent with the STRUCTURE results for their anther donors (Fig. 2). Most of the DH lines with Argenteuil pedigree clustered together, with the exception of AM841 DH lines which clustered firmly with the Mary Washington and Braunschweigerderived lines, and 921-1b and Gladio_e which clustered together with some Mary Washington and hybrid-derived DHs closer to the larger Argenteuil cluster. DH lines derived from Mary Washington, Braunschweiger and Argenteuil 9 Braunschweiger anther donors were distributed across the NJ tree. Interestingly, DHs derived from Argenteuil 9 Braunschweiger hybrid H499 were placed in two distinct clusters, one close to the Argenteuil cluster and another with the Mary Washington and Braunschweiger-derived lines. Although anther donors UC157 and Giant formed a distinct cluster in the STRUCTURE analyses with K C 3, their DH lines were distributed across the NJ tree. The PCoA was consistent with the NJ tree and explained about 56 % of the variability among the collection analyzed; the first axis accounted for 40 % of variability, while the second one for 16 % (Fig. S2). Most of the Argenteuil-derived DHs had high values on coordinate 1 and clustered together with the exception of those derived from AM841. The DH lines with Braunschweiger pedigree showed affinity with Mary Washington-derived DHs, having higher 123 Mol Breeding (2015) 35:59 values for coordinate 2. One group of DHs with Argenteuil 9 Braunschweiger hybrid anther donors formed a cluster with low values on both coordinates. Finally, the haplotype network analysis appeared largely in agreement to cluster and structure analyses showing two principal groups (a) and (b), where the DH clones belonging to Argenteuil and Braunschweiger were grouped, respectively (Fig. S3). Both samples derived from crosses between Argenteuil and Braunschweiger and from cv. Mary Washington were placed between the two principal groups, confirming our results. Discussion Modern asparagus cultivars and hybrids derived from an ancestral Violet Dutch background were channeled through two distinctive lineages (Fig. S1). In France, breeders aimed to obtain more pointed spear tips, whereas more rounded tips were bred in German and English programs (Knaflewski 1996). These efforts yielded Argenteuil and Braunschweiger genetic stocks in France and Germany, respectively. These two genetic stocks quickly replaced old populations and landraces and spread worldwide. In many countries, breeding programs were carried out starting from Argenteuil yielding, among others, Early and Late Argenteuil in France, Reading Giant in England and Palmetto in the USA. At the same time, Braunschweiger is thought to have been developed into German cultivars including Ruhm von Braunschweig first and later Schwetzinger Meisterschuss and Hucheĺs Leistungsauslese hybrids (Greiner 1990; Knaflewski 1996). Improved genetic materials were developed from Argenteuil and Braunschweiger using a combination of conventional and modern techniques. Traditional selfing and full-sib inbreeding (Sneep 1953; Marks 1979; Scholten and Boonen 1996) have been employed along with the development of polyembryonic seed stock (Corriols et al. 1990; Denis and Rameau 1994), double hybrids (Corriols-Thévenin 1979), clonal hybrids (Benson and Takatori 1978), in vitro anther culture (Doré 1974; Falavigna et al. 1999) and genetic transformation (Delbreil et al. 1993). In this study, we aimed to investigate genetic similarities among genotypes included in a large Italian germplasm collection, representing the worldwide genetic diversity available in garden asparagus Mol Breeding (2015) 35:59 Page 7 of 12 59 Fig. 2 Neighbor-joining (NJ) tree based on 144 SNPs showing allele diversity among 66 cultivated asparagus DHs. Samples marked with diamond and square represent the DH clones belonging to Argenteuil and Braunschweiger STRUCTURE clusters, respectively (see also Table 1). The samples marked with circle derived from crosses between Argenteuil 9 Braunschweiger, while the samples marked with triangle are those derived from cv. Mary Washington (A. officinalis L.). STRUCTURE analysis of SNP data from 22 anther donors sheds new light on this germplasm. These donors were used by Qiao and Falavigna (1990) to generate DH lines that have been used extensively in the Italian and international breeding programs, and inferred relationships among 66 of these are consistent with the STRUCTURE clustering of their parents and suggest that some of the anther donors harbor considerable genetic variation. The genetic analyses also indicate some inconsistences with pedigree information and previously hypothesized relationships among major lines used in asparagus breeding programs (Knaflewski 1996; Fig. S1). Perhaps the most striking result is the finding that lines derived from cv. Mary Washington show more genetic similarity to Braunschweiger than to Argenteuil lines. The results of STRUCTURE analysis showed that anther donor genotypes grouped into two different genetic stocks, one dominated by Argenteuil and the other by Braunschweiger and Mary Washington lines (Fig. 1). This result is largely reinforced by NJ 123 59 Page 8 of 12 clustering (Fig. 2) and PCoA (Fig. S2) analyses of DHs derived from the anther donor collection. Figure 2 also suggests that some of the anther donors harbor genetic variation derived from diverse germplasm in their parentage. This was to be expected for the Argenteuil 9 Braunschweiger hybrid anther donors (Table 1), but the Mary Washington-derived lines H500 and Giant also have DHs in diverse clusters in the NJ tree. More generally, Mary Washingtonderived lines are well distributed in the NJ tree. This result suggests that Mary Washington may have been derived from a mix of Argenteuil and Braunschweiger genetic stock. DHs derived from UK–American genotypes At the beginning of the twentieth century, in the USA extensive breeding efforts were carried out by J.B. Norton, who developed the Martha Washington and Mary Washington lines selected for rust (Puccinia asparagi) tolerance (Fig. 3). The female parent of Mary Washington derived from Reading Giant and the male one from an unknown population (Ellison 1986; Knaflewski 1996), probably related to the Braunschweiger gene pool (this hypothesis appeared to be confirmed by SNP analysis). In California, breeding programs based on Mary Washington gave rise to UC500 and UC309 cultivars (Hanna 1952); subsequent selections from UC500 resulted in the UC157 hybrid (Benson and Takatori 1978) widely cultivated in warm climatic conditions worldwide. In New Jersey, breeding selection was also based on Mary Washington and some andromonoecious plants derived from Mary Washington gave rise to several all-male hybrids including Greenwich and the Jersey group (Fig. S2) (Ellison and Kinelski 1986). In California, male plants belonging to New Jersey genetic stocks and three female Mary Washingtonderived plants were utilized as parents of the hybrids Apollo, Atlas and Grande (Fig. 3). Clustering and PCoA analyses (Fig. 2 and S2) confirmed the presence of DHs from UK–American breeding genetic stocks in both the branches related to Argenteuil and Braunschweiger populations (DHs from UC, J. Giant, H500, Apollo, Grande, H524 and Italo) (Fig. 3). At a glance, these results can be explained by hypothesizing that one of the Mary Washington parents derived from the Braunschweiger background, as already supposed by Knaflewski (1996). The same author proposed also 123 Mol Breeding (2015) 35:59 that the male hybrids obtained from Jersey and UC backgrounds derived from Mary Washington and its andromonoecious derived lines (Knaflewski 1996). Hence, it is possible to hypothesize that downstream genetic stocks derived from Mary Washington could harbor large genomic regions already introgressed from the Braunschweiger background. Moreover, the distribution of DH groups derived from Argenteuil 9 Braunschweiger crosses (H465, H499, Zeno; yellow circle genotypes) in the NJ tree seem to confirm the mixed origin hypothesis for USA genetic stocks (Fig. 2). In contrast, the DHs obtained from Marte clustered in the Argenteuil branch (Fig. 2); however, this appears in agreement with Marte’s history, derived from a cross between a Mary Washington parent and an Italian parent belonging to the Early Argenteuil group. DHs from German varieties In Germany, the popular open pollinated cultivars, firstly Ruhm von Braunschweig and later Schwetzinger Meisterschuss and Hucheĺs Leistungsauslese, were obtained from cv. Braunschweiger. They were present in the German field up to the end of the twentieth century (Knaflewski 1996); moreover the tetraploid cultivar Helios was also obtained from Hucheĺs Leistungsauslese (Skiebe et al. 1991) (Fig. 3). From the cv. Schwetzinger Meisterschuss, supermale plants were obtained by self-pollination of andromonoecious plants (Sneep 1953), frequently utilized as parents of German and Dutch all-male hybrids. Cluster analysis revealed that DHs obtained from German genetic stocks (Ariane and Lucullus) grouped in the Braunschweiger background branch (Fig. 2). Indeed, Lucullus was bred in Sud-westdeutsche Saatzucht in Rastatt and it was the first German all-male asparagus cultivar (Bohne 1977). Starting from Lucullus, a large part of German and Dutch allmale hybrids were then obtained (Fig. 3). DHs from Dutch varieties In The Netherlands, formal breeding programs started in the middle of the twentieth century from Ruhm von Braunschweig genetic stock. Following extensive fullsib crosses, a great number of inbred families were obtained, giving rise to Limbras (a cross between Mol Breeding (2015) 35:59 Page 9 of 12 59 Fig. 3 A revised pedigree of the major asparagus cultivars worldwide, indicating their geographical breeding origin and their most valuable varieties obtained during historical European and American breeding Ruhm von Braunschweig and Argenteuil) (Astrego 1951; Boonen 1987; Knaflewski 1996) (Fig. 3). A supermale plant was obtained by self-pollinating a Ruhm von Braunschweig andromonoecious plant, and utilized for breeding the first all-male Dutch hybrid, named Franklim (Fig. 3). Later, all-male Dutch hybrids were bred using supermale plants obtained from the German hybrid Lucullus (derived from Schwetzinger Meisterschuss). From Lucullus were derived Gronlim, Geynlim, Venlim, Backlim, Thielim, Boonlim, Horlim and Gijnlim hybrids; the first four hybrids had the same male parent while two different supermale plants were used for breeding cvs. Boonlim and Horlim. As mother plants, a Mary Washington-derived plant for the first two hybrids (Geynlim and Backlim) and a Limburgia-derived inbred line were used for Backlim and Thielim, while an Early Argenteuil-derived inbred line was utilized for Boonlim and Horlim. In particular, Limburgia was a cultivar derived from the first attempts to cross Mary Washington-derived plants with those from Ruhm von Braunschweig; moreover, the world-famous cultivar Limbras was later obtained from Limburgia (Boonen 1987). In recent decades, UC157 (from California) was included in the Dutch breeding programs to select for earliness (Van den Broek and Boonen 1990; Scholten and Boonen 1996). The cluster analysis showed that DHs from Dutch-derived genetic stocks grouped in both main branches (Fig. 2). In particular, Gronlim-derived DHs cluster in the Braunschweiger branch, H499derived DHs in the Argenteuil branch, while Zenoderived DHs are present in both branches. These results can be explained by considering that 5391 (Gronlim_a) and 5390 (Gronlim_b), DHs from Gronlim, are expected to group in the Braunschweiger branch. H499 is an Italian all-male hybrid obtained from a cross between a Boonlim and an Eros-derived DH (both belong to the Early Argenteuil group), so it is not surprising to find its DHs grouped in the Argenteuil branch. Finally, Zeno is an Italian all-male hybrid obtained from the cross between a Gijnlim-derived DH with an Italian Early Argenteuil-derived DH (Figs. 2, 3). DHs from French varieties In France, the breeding programs were mainly based on the Argenteuil cultivar, adopting double cross and clonal hybrid breeding techniques (Corriols-Thévenin 1979). French DHs include the famous Minerva, Junion, Larac and Mira (Thévenin 1967; Thévenin and Doré 1976; Corriols-Thévenin 1979), while Andreas 123 59 Page 10 of 12 hybrid was the first French asparagus cultivar derived from homozygous parents; the male parent was obtained from anther culture, the female one from polyembryonic seed (Corriols et al. 1990) (Fig. 3). Cluster and PCoA analyses revealed that 3584 (Andreas_a) DH from French genetic stock grouped, as expected, in the Argenteuil branch (Fig. 2 and S2). DHs from Italian varieties In Italy, asparagus breeding started in the last two decades of the twentieth century at the Council Research Agriculture (CRA) station of Montanaso Lombardo (Lodi, Italy). The breeding program started from the in vitro anther culture technique to obtain DHs from Early Argenteuil-derived male plants, a cultivar traditionally cultivated in the Po valley (Falavigna et al. 1999). The Early Argenteuil pool joined to Californian genetic stock led to the development of several ‘‘twoway hybrids’’ (H) crossing male and female DH clones, and different ‘‘three-way hybrids’’ (AM) crossing heterozygous female and DH male clones. The best Italian hybrids developed at CRA research station were, among others, Eros, Gladio, Marte, Ringo, Sirio, H524 and AM841 (Falavigna et al. 1999) (Fig. 3). As expected, cluster and PCoA analyses showed several DHs (from H553, Bassano, Gladio, Eros and Marte) from Italian genetic stocks grouped in the branch related to Argenteuil (Fig. 2 and Fig. S2). Apollo and Grande grouped with the Braunschweiger line, while the DHs from the hybrid H500 were included in both clusters (Fig. 2). In retrospect, these latter results make sense given the American (J. Giant and UC157) origin of this large DH group. Indeed, as previously discussed, J. Giant and UC157 hybrids were spawned from Mary Washington and its andromonoecious-derived selection plants. Inbreeding likely increased in these andromonoecious lines and the introgression of Braunschweiger genetic background. In any event, our analysis of the Italian germplasm supports the hypothesis that the unknown population male parent of Mary Washington (Ellison 1986; Knaflewski 1996) was derived from Braunschweiger or a related gene pool. The clustering also showed that the DHs from the Italian H465 hybrid were included into the Braunschweiger/Mary Washington cluster (Fig. 2). This result is consistent with H465 breeding history; in fact it was obtained from a cross between two DHs in the 123 Mol Breeding (2015) 35:59 Italian collection, both derived from plants collected in German fields including both Argenteuil and Braunschweiger genetic stocks. Finally, the Italian ‘‘three-way hybrid’’ AM841 was unexpectedly included in the Braunschweiger cluster (see Fig. 1). This result was confirmed in the cluster analysis, where 4881 (AM841_a) and 4880 (AM841_b) DHs clustered in the large cluster including Braunschweiger and Mary Washington-derived DHs (Fig. 2). PCoA analysis showed similar results, in fact 4881 (AM841_a) and 4880 (AM841_b) DHs were included in the left-bottom quadrant (red color) together with Braunschweiger 9 Argenteuil genotypes (Fig. S2). These results are surprising because AM841 is the product of a cross between an Argo hybrid-derived DH and the French variety Dariane, both belonging to the Argenteuil background (Fig. 3). Conclusion In conclusion, whereas all current asparagus cultivars may be derived from Violet Dutch, our results indicate that many DHs from USA, France and Italy seem to be classifiable as derived from the Argenteuil gene-pool, whilst those from Germany and the Netherlands (also partially from the USA) originated from the Braunschweiger gene-pool. These results are widely in accordance with the genetic variation resulting from a germplasm asparagus collection reported by Geoffriau et al. (1992), showing that accessions from the Netherlands and Germany tended to group together, and were distinguished from USA, Italy, Spain, Taiwan and France genetic stocks. The present study, focusing on a wide Italian DH collection and based on a substantial panel of SNPs, gave rise to a new insight into the parentage relationships of the most important genetic stocks utilized over the last century (and nowadays) in worldwide breeding programs. 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