ORIGINAL PAPER DNA markers identify hybrids between butternut (Juglans cinerea L.) and Japanese walnut (Juglans ailantifolia Carr.) Peng Zhao & Keith E. Woeste Received: 5 May 2010 / Revised: 6 October 2010 / Accepted: 11 October 2010 # Springer-Verlag (outside the USA) 2010 Abstract Butternut (Juglans cinerea L.) is a temperate deciduous hardwood native to the eastern USA and southern Canada valued for its nuts and wood. Butternut’ s survival is threatened by butternut canker, a disease caused by the exotic fungus Sirococcus clavigignenti- juglandacearum Nair, Kostichka & Kuntz. Field observa- tions indicate that trees commonly called buartnut (a hybrid of butternut and its close congener Japanese walnut (Juglans ailantifolia × J. cinerea)) may be more resistant to butternut canker than is either parental species. Hybrids are difficult to distinguish morphologically from butternuts, and scientists have expressed concern over the possibility of range-wide genetic invasion by Japanese walnut via hybridization with butternut. We used pair-wise combina- tions of 40 random primers to screen bulked DNA pools of butternut, Japanese walnut, and buartnuts to identify genomic regions unique to Japanese walnut. We ultimately identified one ITS region marker, one chloroplast marker, one mitochondrial marker, and six nuclear markers. The utility of the markers for identifying hybrids was tested and verified using more than 190 genotypes. The markers will be used to identify buartnut hybrids based on the presence of introgressed genomic fragments inherited from Japanese walnut. We confirmed that hybrids have a complex genetic history and present features of the parental species in all possible combinations. These results will assist in the identification and testing of (non-hybrid) butternut for breeding and reintroduction of the species to its former habitats. Keywords Juglans × bixbyi . RAPD . SCAR . CAPS . Internal transcribed spacer . trnT-F . Hybrid invasion . Butternut canker Introduction Butternut (Juglans cinerea L.) also called white walnut, oilnut, or lemonnut is a short-lived, deciduous, cold- tolerant, tree species important for its nuts, wood, and wildlife mast (http://plant-materials.nrcs.usda.gov/). Butter- nut is native to northeastern and central USA and southern Canada eastward from Ontario (Rink 1990; Farrar 1995), its nuts and wood are of high quality, but its widely scattered distribution, relatively soft wood, medium-sized kernel, and hard shell have prevented it from becoming a commercially important timber or nut species (Williams 1990; Ostry and Pijut 2000). Butternut is threatened by butternut canker, a disease caused by the fungus Sirococcus clavigignenti- juglandacearum Nair, Kostichka & Kuntz (Nair et al. 1979). The disease has killed up to 80% of the trees in some states and is threatening its survival (Ostry et al. 1994; Ostry 1998). Japanese walnut (Juglans ailantifolia Carr.) is a native to Japan and Sakhalin (Ohwi 1965) and can also serve as a host to S. clavigignenti-juglandacearum (Nair et al. 1979; Orchard 1984). It was introduced into North America from Japan about 1870 by a nurseryman in San Jose, CA (Manning 1978; Ostry 1997). A horticultural Communicated by A. Kremer P. Zhao College of Forestry, Northwest Agriculture & Forestry University, Yangling, Shaanxi 712100, China K. E. Woeste (*) USDA Forest Service Hardwood Tree Improvement and Regeneration Center (HTIRC), Department of Forestry and Natural Resources, Purdue University, 715 West State Street, West Lafayette, IN 47907, USA e-mail: woeste@purdue.edu Tree Genetics & Genomes DOI 10.1007/s11295-010-0352-4