Genet Resour Crop Evol DOI 10.1007/s10722-016-0436-6 RESEARCH ARTICLE Genetic relationships of boxwood (Buxus L.) accessions based on genic simple sequence repeat markers Chandra S. Thammina . Richard T. Olsen . Matthew Kramer . Margaret R. Pooler Received: 15 March 2016 / Accepted: 16 August 2016 Ó Springer Science+Business Media Dordrecht (outside the USA) 2016 Abstract Boxwoods (Buxus L., Buxaceae) are pop- analyzed with a distance matrix based on Jaccard’s ular woody landscape shrubs grown for their diverse similarity index, followed by Unweighted Pair Group forms and broad-leaved evergreen foliage. We used Method with Arithmetic Mean clustering. Two major genic simple sequence repeat (genic-SSR) markers to clusters were identified, with four subclusters consist- assess genetic diversity and relatedness of 275 acces- ing of individual accessions from B. balearica Lam., sions from the National Boxwood Collection at the B. bodinieri Le´vl., B. harlandii Hance, B. microphylla U.S. National Arboretum. Flow cytometry was con- Siebold et Zuccarini, B. sempervirens L., B. sinica ducted to determine the relative ploidy of each (Rehd. et Wils.) M. Cheng, and their putative inter- accession. Genic-SSR loci were highly variable specific hybrids. The accessions generally clustered by among the accessions, detecting an average of 6.7 cultivar, provenance, or species. Clustering within alleles per locus based on 17 primer pairs. Data were each group typically reflected breeding pedigrees, when known, and the clusters were supported by bootstrap results. This information will be used for Electronic supplementary material The online version of breeding programs and collection management, and this article (doi:10.1007/s10722-016-0436-6) contains supple- for identifying possible sources of disease tolerance mentary material, which is available to authorized users. for boxwood blight and other diseases and pests. C. S. Thammina Á M. R. Pooler (&) Floral and Nursery Plants Research Unit, USDA-ARS Keywords Buxus Á Calonectria pseudonaviculata Á U.S. National Arboretum, 10300 Baltimore Avenue, Cultivar diversity Á Flow cytometry Á Germplasm Á Building 010A, Beltsville, MD 20705, USA Microsatellite markers Á Polymorphism e-mail: [email protected] C. S. Thammina Department of Plant Biology and Pathology, Rutgers Introduction University, 59 Dudley Road, New Brunswick, NJ 08901, USA Boxwoods (Buxus L., Buxaceae) are popular land- R. T. Olsen scape shrubs or small trees grown for their diverse USDA-ARS U.S. National Arboretum, 3501 New York forms and broad-leaved evergreen foliage. They are Ave NE, Washington, DC 20002, USA used in the landscape as specimen plants, hedges, mass plantings, or as topiary (Larson 1999; Batdorf M. Kramer USDA-ARS, Statistics Group, 10300 Baltimore Avenue, 2004, 2005; Van Trier and Hermans 2005). Each Building 005, Beltsville, MD 20705, USA year, more than 13 million boxwood plants are sold in 123 Genet Resour Crop Evol the United States, with an annual value of over $100 origins, and genetic divergence (Harris-Shultz et al. million (USDA-NASS 2010). The genus Buxus grows 2015). Van Laere et al. (2011) used amplified in a range of ecological conditions from dry scrub to fragment length polymorphism (AFLP) markers to montane rain and cloud forests (Van Laere et al. 2015). characterize and differentiate between European and The genus contains 95-100 species originating in Asian boxwood taxa, and von Balthazar et al. (2000) Africa, Eurasia, the Caribbean, and Central America used internal transcribed spacer regions and plastid (Larson 1999). The most economically important ndhF sequences to describe phylogenetic relation- species, B. sempervirens L., has approximately 400 ships. Our objective was to show the utility of named cultivars (Niemiera 2012). Boxwood plants previously developed genic SSR markers (Thammina grown in temperate zones are increasingly threatened et al. 2014) and ploidy analysis to characterize the by a destructive blight disease caused by the diversity and, in some cases, the identity of Buxus ascomycete fungus Calonectria pseudonaviculata accessions in the National Boxwood Collection at the Henricot (syn. Cylindrocladium pseudonaviculatum, USNA. Cy. buxicola). This disease causes necrotic stem and leaf lesions, defoliation, and usually plant death (Henricot and Culham 2002; Douglas 2012). First Materials and methods identified in the United Kingdom in 1994, the disease has spread throughout continental Europe, parts of Plant materials western Asia, and into North America (Ivors et al. 2012; Elmhirst et al. 2013; Gehesquie`re et al. 2013; Young leaves were collected in May/June from 275 Malapi-Wight et al. 2014). In the United States, the Buxus accessions (Table 1 and Supplemental Table 1) first confirmed reports of the disease were made from maintained in the USNA National Boxwood Collec- Connecticut and North Carolina in November 2011 tion in Washington, DC and Beltsville, MD. These (Ivors et al. 2012), followed by diagnosis in 12 were selected from the entire boxwood collection, additional states and three Canadian provinces (Elm- which includes approximately 700 individual plants, hirst et al. 2013; Hagan and Conner 2013; Malapi- to include reference taxa, economically important Wight et al. 2014; Williams-Woodward 2014). To cultivars, wild-collected species, and unknown or date, all tested cultivated Buxus taxa are susceptible to questionable accessions for verification and identifi- boxwood blight, although some taxa seem to be more cation. Taxa included B. balearica Lam., B. bodinieri vulnerable to fungal infection than others (Henricot Le´vl., B. harlandii Hance, B. microphylla Siebold et et al. 2008; Douglas 2012; LaMondia 2014, 2015). Zuccarini, B. sinica (Rehd. et Wils.) M. Cheng, B. Published reports indicate that boxwood blight can be sempervirens L., hybrids, cultivars, and unknown effectively managed with fungicides (Henricot et al. species. Leaf material was stored at -20 °C until use. 2008; LaMondia 2015); however, developing blight- tolerant boxwood cultivars is a favored, long-term DNA extraction and PCR amplification management strategy for this disease (Guo et al. 2015). Genomic DNA was extracted from frozen leaf tissue The National Boxwood Collection at the U.S. using a DNeasy Plant Mini Kit in a QIAcube (Qiagen, National Arboretum (USNA) contains more than 700 Valencia, CA, USA) according to the manufacturer’s Buxus accessions, making it one of the most complete recommendations, and quantified with a NanoDrop collections in the world and a valuable genetic 1000 Spectrophotometer (Thermo Fisher Scientific, resource for developing blight-tolerant varieties. Wilmington, DE, USA). Twenty-three trinucleotide However, the genetic diversity and relatedness of genic-SSR primer pairs (Supplemental Table 2) these accessions has not been determined. Although developed in our lab to amplify polymorphic loci in morphological features can be useful in determining previous tests (Thammina et al. 2014) were used to genetic relationships in Buxaceae (Carlquist 1982; assess genetic diversity. Polymerase chain reactions Ko¨hler and Bru¨ckner 1990), molecular markers are (PCR) and analysis of PCR products were carried out needed to differentiate among closely related acces- as previously published (Thammina et al. 2014). To sions, identify plants, and help determine provenance, avoid false negatives, primers that resulted in null 123 Genet Resour Crop Evol Table 1 Summary of the number of accessions of each Buxus taxon included in this study Taxon Number of accessions included in this study Buxus balearica Lam. 5 Buxus bodinieri Le´vl. 2 Buxus harlandii Hance 18 Buxus microphylla Siebold et Zucc. 26 Buxus microphylla var. japonica (Mu¨ll. Arg. ex Miq.) Rehder et E.H. Wilson 23 Buxus sempervirens L. 139 Buxus sinica (Rehder et E.H. Wilson) M. Cheng 8 Buxus sinica var. aemulans (Rehder et E.H. Wilson) M. Cheng 1 Buxus sinica var. insularis (Nakai) M. Cheng 31 Buxus sp. 7 Cultivars of undesignated species 15 Detailed accession information can be found in Supplemental Table 1 alleles in some of the samples were tested at least STRUCTURE was run with a range of groups (two twice. Non-amplified loci were scored as missing data. to seven) and results for the most parsimonious Allele sizes and number of alleles per locus were number of groups were examined in detail. Following determined with GeneMarker version 2.6.3 (SoftGe- the first split, this methodology was repeated on each netics, State College, PA, USA). subset, and the probabilities of individuals belonging to particular groups were examined. Data analysis Flow cytometry The amplified allele data were converted to a binary matrix (presence/absence) for each allele by using Boxwood samples were processed and analyzed on a R-polysat package, version 1.1 (Clark and Jasieniuk CyFlow Space flow cytometer (Partec, Munster, 2011). Because it was unknown whether our acces- Germany) with ploidy determination based on the sions were autopolyploids or allopolyploids, each total DNA content per nucleus (linear fluorescence genic-SSR allele was treated as independent in this intensity). Nuclei were extracted with a Partec CyStain study. A genetic distance matrix was generated based UV Precise P kit according to the manufacturer’s on Jaccard’s similarity index implemented in the instructions. Approximately 0.5 cm2 fresh leaf tissue ‘pvclust’ package version 1.3-2 of R software (Suzuki harvested in May/June was chopped with a razor blade and Shimodaira 2014). The Jaccard distance was in a 55-mm-diameter Petri dish containing 400 lL chosen since it does not use the absence of an allele as nucleus extraction buffer (Partec). The resulting slurry a shared characteristic (Legendre and Legendre 1998). was incubated for 2 min at room temperature and Accessions were then clustered by using the UPGMA filtered through a 50-lm CellTrics disposable filter algorithm implemented in ‘pvclust.’ The UPGMA (Partec) placed on top of a plastic 12 9 75-mm culture algorithm was chosen because in a phenetic analysis, it tube. 1.6 ml of staining buffer was added to each tube makes no assumptions about evolutionary rate or and incubated at room temperature for 60 s.