The genus Viburnum includes more than 160 species of shrubs and small trees distributed widely throughout the Northern hemisphere and into the Southern hemisphere (Winkworth and Donoghue, 2004). Collectively, Viburnum species provide many ideal year-round ornamental qualities. The attractive flowers and glossy green leaves of spring and summer give way to an array of bright berries and vibrant fall foliage with colors ranging from yellow to red to dark purple. The multiseasonal allure of plants in this genus makes Viburnum an important ornamental plant crop to the U.S. nursery industry. Total sales of Viburnum were $19,500,000 in 1998 (U.S. Dept. of Agr., 1998), and sales increased to $24,647,000 in 2007 (USDA-NASS, 2010). Growing awareness and ongoing breeding efforts have led to increasing consumer demand for viburnum shrubs. Viburnum dilatatum, a common and versatile species that is native to Asia, was introduced to the United States in 1846 and is valued for its vibrant fall color, leaf color retention, and outstanding cherry-red fruit production that persists into winter. For these reasons, it has become increasingly prevalent in horticultural commerce (Dirr, 2007). At maturity, the shrub reaches 1.5 to 3.0 m (5 to 10 feet) tall and exhibits lustrous dark green leaves.
There are striking diversity among horticultural characteristics in Viburnum species, including leaf shape, fruit and flower color, fertility, and plant growth habit (Dirr, 2007; Winkworth and Donoghue, 2004). Erected by Linnaeus in 1753, the genus has undergone more than 10 taxonomic revisions, and despite those classification efforts, evolutionary relationships and geographical relatedness of the species have been largely unaddressed (Winkworth and Donoghue, 2005). Winkworth and Donoghue (2005) combined molecular data using two chloroplast genes (trnK intron and psbA-trnH IGS) and three nuclear loci (nrIts, WAXY1, and WAXY2) to provide a biogeographical analysis of the genus. Although 12 well-supported species groups, or clades, were identified using the molecular data, issues within this eclectic genus still linger. For example, the relationship between the section Pseudotinus and the species V. clemensiae and V. urceolatum is not well supported. In addition, the rates of diversification remain unclear, and there is a persistent lack of resolution at the base of the phylogenetic tree (Winkworth and Donoghue, 2004, 2005). Not surprisingly, a plethora of cultivars and hybrids further complicate the organization of this vast genus (Dirr, 2007).
Although many ornamental plants, including V. dilatatum, make important contributions to regional environmental health and diversity, certain non-native species have escaped cultivation and are causing economic damage. During a period of 85 years (1906 to 1991), 79 exotic plant species have been deemed responsible for almost $97 billion in damages in the United States (Office of Technology Assessment, 1993). Many of the plants listed on invasive species lists such as Celastrus orbiculatus, Euonymus alatus, and Pyrus calleryana remain economically important and popular nursery plants (The University of Georgia, 2010). Regardless, invasive plants cause significant economic damage and can lead to considerable reductions in populations of native flora and fauna, often in response to competition for limited resources (Anonymous, 2008). Unfortunately, current methods of non-native plant management, which include physical removal, herbicidal and biological control methods, have not been practical or effective in controlling extensive proliferation of many invasive ornamental plants (Li et al., 2004). Indeed, the increase in commercial availability of potentially problematic plants makes early characterization of potential pest plants and their cultivars a high priority. The New Jersey Invasive Species Strike Team placed V. dilatatum on a 2010 Watch List of invasive species that identifies plants that may pose a threat to native plant communities (Anonymous, 2010). Viburnum dilatatum can form thickets after escaping cultivation that may displace or prevent growth of less competitive native herbs, shrubs, and trees. Shrubs yield an abundance of viable seed from a typically heavy seasonal fruit set (Dirr, 2007). In turn, viable seeds aid in the rapid colonization of land previously not colonized with V. dilatatum.
Some invasive species, including V. dilatatum, may be difficult to discern from native vegetation (Anonymous, 2008). Dirr (2007) stated that V. dilatatum is difficult to identify based on foliage, because some specimens may have branches with two differently shaped leaves comingling. Indeed, V. dilatatum has been observed along the woodland edges of the National Arboretum, often growing undetected until old enough to display their unmistakable red fruits (J. Feely, personal communication). Moreover, there is anecdotal evidence to suggest that V. dilatatum may be escaping cultivation and hybridizing with the natively occurring V. dentatum (J. Feely, personal communication). This caveat necessitates the availability of a reliable method for identification of this popular ornamental shrub and its hybrids.
A group identified as the Invasive Plant Research and Partnerships with Ornamental Horticulture and Natural Resource Management convened for a workshop in 2008 to discuss the problem of invasive plant species. Research areas were identified and included the following priorities: develop scientific means to evaluate the persistence and spread of invasive plants; enhance detection methods for invasive taxa; and acquire a means to identify invasive species, cultivars, and hybrids of cultivars such as genetic markers (Anonymous, 2008). Molecular markers provide a means of meeting these criteria.
Molecular tools have become a superior method for interspecific and intraspecific hybrid analysis and cultivar identification of ornamental plants (Pounders et al., 2007; Wadl et al., 2008, 2009; Wang et al., 2010). Microsatellites are ubiquitous throughout genomes and codominant markers, often used in both interspecific and intraspecific diversity studies (Gupta and Varshney, 2000). These genetic markers exhibit hypervariabilty and are easily detected using polymerase chain reaction (PCR) and two unique primers (Powell et al., 1996). Microsatellite markers are ideal for identification and genetic fingerprinting of plants because of the high levels of polymorphism they possess (Datta et al., 2010). Based on searches of the literature and GenBank, there is a dearth of microsatellite markers that can be applied to the genus Viburnum. In this study, we created a small insert genomic library from V. dilatatum ‘Asian Beauty’ using a biotin enrichment protocol (Wang et al., 2007). From the library, 11 polymorphic markers were developed and used to characterize a group of V. dilatatum accessions and cultivars. It is envisioned that these markers will be useful in discerning taxonomic relationships and will be used in future efforts to track and identify the origins of V. dilatatum plants within populations that have escaped cultivation.
Amos, W., Hoffmann, J.I., Frodsham, A., Zhang, L., Best, S. & Hill, A.V.S. 2007 Automated binning of microsatellite alleles: Problems and solutions Mol. Ecol. Notes 7 10 14
Anonymous 2008 Invasive Plant Research and Partnerships with Ornamental Horticulture and Natural Resource Management Workshop report U.S. National Arboretum Washington, DC <http://usda.mannlib.cornell.edu/usda/current/NursProd/NursProd-09-26-2007.pdfpage 9>.
Bassil, N.V., Muminova, M. & Njuguna, W. 2010 Microsatellite-based fingerprinting of western blackberries from plants, IQF berries and puree Acta Hort. 859 73 80
Botstein, D., White, R.L., Skolnick, M. & Davis, R.W. 1980 Construction of a genetic linkage map in man using restriction fragment length polymorphisms Amer. J. Hum. Genet. 32 314 331
Datta, J., Lal, N., Kaashyap, M. & Gupta, P.P. 2010 Efficiency of three PCR based marker systems for detecting DNA polymorphism in Cicer arietinum L. and Cajanus cajan L. Millspaugh Genet. Eng. Biotechnol. J. 2010 1 15
Gupta, P.K. & Varshney, R.K. 2000 The development and use of microsatellite markers for genetics and plant breeding with emphasis on bread wheat Euphytica 113 163 185
Hamilton, M.B., Pincus, E.L., Di Fiore, A. & Fleischer, R.C. 1999 Universal linker and ligation procedures for construction of genomic DNA libraries enriched for microsatellites Biotechniques 27 500 507
Kowarik, I. 1995 Time lags in biological invasions with regard to the success and failure of alien species 15 38 Pysek P., Prach K., Rjmanke, M. & Wade M. Plant invasions: General aspects and special problems SPB Academic Publishing Amsterdam, The Netherlands
Li, Y., Cheng, Z., Smith, W.A., Ellis, D.R., Chen, Y., Zheng, X., Pei, Y., Luo, K., Zhao, D., Yao, Q. & Duan, H. 2004 Invasive ornamental plants: Problems, challenges, and molecular tools to neutralize their invasiveness Crit. Rev. Plant Sci. 23 381 389
Office of Technology Assessment 1993 Harmful non-indigenous species in the United States Office of Technology Assessment, U.S. Congress Washington, DC
Pashley, C.H., Ellis, J.R., McCauley, D.E. & Burke, J.M. 2006 EST databases as a source for molecular markers: Lessons from Helianthus J. Heredity. 97 381 388
Pounders, C.T., Rinehart, T. & Sakhanokho, H. 2007 Evaluation of interspecific hybrids between Lagerstroemia indica and L. speciosa HortScience 42 1317 1322
Pryer, K.M., Schuettpelz, E., Huiet, L., Grusz, A.L., Rothfels, C.J., Avent, T., Schwartz, D. & Windham, M.D. 2010 DNA barcoding exposes a case of mistaken identity in the fern horticultural trade Mol. Ecol. Resources. 10 979 985
Romero, G., Adeva, C. & Battad, Z. 2009 Genetic fingerprinting: Advancing the frontiers of crop biology research Philippine Sci. Letters. 2 8 13
Rousset, F. 2008 Genepop'007: A complete reimplementation of the Genepop software for Windows and Linux Mol. Ecol. Resources. 8 103 106
Rozen, S. & Skaletsky, H.J. 1998 Primer3 1 Nov. 2010. <http://www.genome.wi.mit.edu/genome_software/other/primer3.html>.
Sambrook, J., Fritsch, E.F. & Maniatis, T. 1989 Molecular cloning: A laboratory manual Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY
The University of Georgia 2010 Center for Invasive Species and Ecosystem Health and National Park Service. Invasive plant atlas of the United States 19 Oct. 2010. <http://www.invasiveplantatlas.org/about.html>.
U.S. Dept. of Agr 1998 1998 Census of horticultural specialties Washington, DC 3 Dec. 2010. <http://www.agcensus.usda.gov/Publications/1997/Horticulture_Specialties/indexintro.aspU.S>.
USDA NASS 2010 7 Jan. 2011. <http://www.agcensus.usda.gov/Publications/2007/Online_Highlights/Census_of_Horticulture/index.asp>.
Wadl, P.A., Skinner, J.A., Dunlap, J.R., Reed, S.M., Rinehart, T.A., Pantalone, V.R. & Trigiano, R.N. 2009 Honeybee-mediated controlled pollination in Cornus florida and C. kousa intra-and interspecific crosses HortScience 44 1527 1533
Wadl, P.A., Wang, X.W., Trigiano, A.N., Skinner, J.A., Windham, M.T. & Trigiano, R.N. 2008b Molecular identification keys for cultivars and lines of Cornus florida and C. kousa based on simple sequence repeat loci J. Amer. Soc. Hort. Sci. 133 783 793
Wang, X.W., Dean, D., Wadl, P.A., Hadziabdic, D., Scheffler, B.E., Rinehart, T., Cabrera, R. & Trigiano, R.N. 2010 Development of microsatellite markers from crape myrtle (Lagerstroemia L.) HortScience 45 842 844
Wang, X.W., Kaga, A., Tomooka, N. & Vaughan, D.A. 2004 The development of SSR markers by an new method in plants and their application to gene flow studies in azuki bean. [Vigna angularis (Willd.) Ohwi & Ohashi] Theor. Appl. Genet. 109 352 360
Wang, X.W., Trigiano, R.N., Windham, M.T., DeVries, R.E., Scheffler, B.E., Rinehart, T.A. & Spiers, J.M. 2007 A simple PCR procedure for discovering microsatellites from small insert libraries Mol. Ecol. Notes 7 558 561
Winkworth, R.C. & Donoghue, M.J. 2004 Viburnum phylogeny: Evidence from the duplicated nuclear gene GBSSI Mol. Phylogenet. Evol. 33 109 126
Winkworth, R.C. & Donoghue, M.J. 2005 Viburnum phylogeny based on combined molecular data: Implications for taxonomy and biogeography Amer. J. Bot. 92 653 666