Plant Mol Biol Rep DOI 10.1007/s11105-014-0754-9
ORIGINAL PAPER
Genetic Diversity, Population Structure and Construction of a Core Collection of Apple Cultivars from Italian Germplasm Wei Liang & Luca Dondini & Paolo De Franceschi & Roberta Paris & Silviero Sansavini & Stefano Tartarini
# Springer Science+Business Media New York 2014
Abstract Apple germplasm collections are increasingly appreciated as a repository for the genetic improvement of species, and their evaluation is an essential prerequisite for their utilization in apple breeding. A set of 418 apple genotypes, including 383 accessions from the Italian germplasm and 35 International cultivars as reference, was analyzed using 15 SSRs with the aim of assessing the genetic diversity within this panel of varieties, evaluating relationships among them and determining their genetic structure. Genetic analyses performed by Bayesian model-based clustering revealed a clear differentiation of two major groups (G1 and G2). Local Italian accessions were grouped mainly in G2 while all except one of the reference cultivars were found in G1. Each of these two clusters has been further divided into two subgroups by a nested approach. These results were confirmed by factorial correspondence (FCA) and molecular variance (AMOVA) analyses. A core collection of 55 accessions, representative of the Italian apple germplasm and capable of retaining all the 238 SSR alleles detected on 192 unique genotypes, was established by the M-strategy method. The Italian apple germplasm represents an important source of genetic diversity which can be used, in addition to other characterized European germplasm collections, to optimize the efficiency of
Electronic supplementary material The online version of this article (doi:10.1007/s11105-014-0754-9) contains supplementary material, which is available to authorized users. W. Liang : L. Dondini : P. De Franceschi : R. Paris : S. Sansavini : S. Tartarini (*) Department of Agricultural Sciences, University of Bologna, Bologna, Italy e-mail:
[email protected] Present Address: R. Paris Centro di Ricerca per le Colture Industriali, Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Bologna, Italy
genome-wide association studies aimed at identifying the genomic regions controlling major horticultural traits. Keywords Malus × domestica . SSR markers . Genetic structure . Apple biodiversity
Introduction The domesticated apple (Malus × domestica Borkh.) is one of the most cultivated fruit trees worldwide and the fourth most economically important, following citrus, grape and banana (Hummer and Janick 2009). There are more than 10,000 documented apple cultivars, but only a few of them dominate the world fruit production (Janick and Moore 1996). Most commercial cultivars have an ancient origin and a long propagation history, such as ‘McIntosh’ (1800s), ‘Jonathan’ (1820s), ‘Cox’s Orange Pippin’ (1830s), ‘Granny Smith’ (1860s), ‘Delicious’ (1870s) and ‘Golden Delicious’ (1890s). In the last century, a few well-adapted genotypes (e.g., ‘Red Delicious’, ‘Golden Delicious’ and ‘Jonathan’) have been extensively used in apple breeding to select new varieties with desirable traits, but, as a consequence, the genetic base of the species was narrowed (Hokanson et al. 2001; Noiton and Alspach 1996). Moreover, the wide use of clonal selection (e.g., the polyclonal cultivars ‘Red Delicious’, ‘Gala’ and ‘Fuji’) further reduced the genetic variability of commercial apple cultivars (Brooks and Olmo 1997). Old and local accessions have been almost excluded from orchards because of their low productivity and the quality of their fruits, which in most cases do not meet the standard of modern varieties. However, to prevent the loss of genetic diversity, these old and local accessions should be adequately preserved (Cohen et al. 1991). In spite of the generally low fruit quality of most of the old germplasm accessions, apple repositories still remain a valuable source of allelic variability for many traits which is not
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exploited by current breeding programs; a proper molecular characterization is essential for their efficient management, by identifying clonal relationships, synonyms, homonyms and propagation or labeling errors (true-to-type correspondence of accessions). Microsatellites are considered the marker of choice for exploring the genetic diversity for several reasons: (1) they are abundant and relatively evenly distributed in the genome (Buschiazzo and Gemmell 2006; Kelkar et al. 2008); (2) they are codominant and multi-allelic (Kelkar et al. 2008); and (3) they can be easily analyzed by PCR-based methods, including fluorescent automated genotyping and multiplexing (Csencsics et al. 2010; Lepais and Bacles 2011; Dutta et al. 2011). Approximately 300 SSR markers have been developed in apple (Guilford et al. 1997; Liebhard et al. 2002; SilfverbergDilworth et al. 2006; Han and Korban 2008; Celton et al. 2009) and successfully used to assess genetic diversity and relationships among different apple germplasm collections from Spain (Pereira-Lorenzo et al. 2007, 2008; Urrestarazu et al. 2012), Czech Republic (Patzak et al. 2012), and Sweden (Garkava-Gustavsson et al. 2008), as well as wild apple species (Coart et al. 2003; Larsen et al. 2006; Richards et al. 2009; Volk et al. 2009; Cornille et al. 2012; Reim et al. 2013). On the basis of SSR allele frequencies, it is also possible to investigate population structure among groups of individuals. Such analyses generally rely on the model-based Bayesian approach implemented in the software STRUCTURE (Pritchard et al. 2000), that does not require any prior information to assign individuals to different populations and reveal introgression even if the parental population cannot be sampled (Pritchard et al. 2000; Iketani et al. 2010). This approach has been widely used to assess the genetic structure in several fruit tree species such as pear (Volk et al. 2006; Miranda et al. 2010; Iketani et al. 2010; Ferreira dos Santos et al. 2011), peach (Aranzana et al. 2010), plum (Horvath et al. 2011) and sweet cherry (Mariette et al. 2010). In apple, it has been mainly used in wild apple species, like M. sieversii (Richards et al. 2009), M. orientalis (Volk et al. 2009) and M. sylvestris (Coart et al. 2003; Larsen et al. 2006; Reim et al. 2013). Moreover, the recent contribution of various wild Malus species to the Malus × domestica gene pool has also been investigated by structure analysis (Cornille et al. 2012). Regarding M. × domestica, three ancestral groups were reconstructed for local apple cultivars from La Palma in Spain (Pereira-Lorenzo et al. 2008), and six subgroups for local apple cultivars from northeastern Spain were identified by a nested structure approach (Urrestarazu et al. 2012). In the Scandinavian apple collections, the same approach identified three populations mainly reflecting their geographic origin: Swedish, Finnish or European (Garkava-Gustavsson et al. 2008). The main drawback of germplasm collections is the high management cost of the large number of accessions. A possible solution is the establishment of core collections, defined as the smallest group of accessions that is representative of the whole
genetic diversity within the collection (Frankel 1984; Brown 1989 and 1995). The strategies for the construction of core collections can be based on maximizing the variability, as in the M-strategy method (Schoen and Brown 1995), or on similarity clustering in stratified methods (Escribano et al. 2008). The M-strategy method has been reported as the most efficient one in the development of a core collection in cherimoya (Escribano et al. 2008). Among numerous potential applications, such as SNP discovery or capturing the maximum amount of allelic diversity for quantitative genetic analyses, core collections can be useful when available resources do not allow the assay of a larger number of plants and as a first step in genetic association studies (Barnaud et al. 2006; Le Cunff et al. 2008; Aranzana et al. 2010; Dunemann et al. 2012). The primary goal of our study is to evaluate the genetic diversity and relationships within a large collection of Italian apple accessions by SSR markers. Through this molecular characterization it will be possible to: (1) identify synonymous and homonymous genotypes that are difficult to distinguish using standard morphological descriptors; (2) identify putative triploid accessions that are useless in breeding programs; (3) identify a collection of unique diploid genotypes for the genetic diversity analysis; (4) evaluate population structure within the collection using the model-based Bayesian method; and (5) select the best subset of genotypes that define an Italian “apple core collection” that can be compared to other collections or further genotyped for association studies.
Materials and Methods Plant Material and DNA Extraction A collection of 418 apple accessions was investigated, including 383 accessions of the Italian germplasm and 35 international reference cultivars maintained at the Cadriano Experimental Station of the Department of Agricultural Sciences (University of Bologna, Italy; Online Resource 1). For each accession, genomic DNAwas extracted from 50 mg of young freeze-dried leaves following the standard CTAB protocol (Maguire et al. 1994). Genomic DNA was quantified by Nanodrop™ ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and diluted to 10 ng/μl. SSR Analysis A set of 15 SSRs was chosen from the HiDRAS website (http://users.unimi.it/hidras/), mainly on the basis of their distribution across the apple genome (Online Resource 2). All PCR primers were synthesized with generic non complementary nucleotide tails (Hayden et al. 2008). The tail-specific forward primer (tag-F) was labeled at its 5’-end with one of the following fluorescent dyes: VIC, FAM, NED,
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and PET (Applied Biosystems, Warrington, UK; Online Resource 2). Single SSR amplifications were performed in a 10μl reaction mixture containing 1× reaction buffer (Applied Biosystems, Foster City, CA, USA), 1.5 mM MgCl2, 0.2 mM dNTPs (Fermentas, Lithuania), 5 nM each SSR locus-specific primer, 76 nM dye-labeled tag-F and unlabeled tag-R primers, 10 ng genomic DNA and 0.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The PCR reactions were carried out in a 2720 thermal cycler (Applied Biosystems) with the following amplification protocol: an initial denaturation step of 10 min at 95 °C, followed by 20 cycles of 30 s at 92 °C, 90 s at 60 °C, and 60 s at 72 °C, and then 40 cycles of 15 s at 92 °C, 30 s at 45 °C, and 60 s at 72 °C, with a final extension step of 10 min at 72 °C. Multi-pooling groups (MPG) of SSRs labeled with the four different fluorescent dyes were designed for SSR genotyping on an ABI PRISM 3730 DNA analyzer (Online Resource 2). SSRs were pooled by mixing PCR products labeled with different dyes in a ratio of 1:1:1:2 for VIC:FAM:NED:PET; 3 μl of the PCR products mixture were added to 7 μl of formamide containing 0.2 μl of GeneScan500 LIZ size standard (Applied Biosystems). Fragments were visually analyzed and scored by using Peak Scanner v.1.0 (Applied Biosystems). To monitor the reproducibility in different amplifications, two reference cultivars, “Fuji” and “Gala”, were included in each single run. Genetic Diversity Analysis Considering that apple accessions can be polyploid, the software SPAGeDi v.1.4 (Hardy and Vekemans 2002) was used to compute genetic information statistics, as this software supports analyses of datasets containing individuals with different ploidy levels. Genetic information statistics included number of alleles per locus (A), effective number of alleles [Ae = (∑pi2)−1, where pi is the frequency of the ith allele], number of rare alleles per locus (B, number of alleles with frequency
