Molecular Ecology Resources (2008) 8, 643–646 doi: 10.1111/j.1471-8286.2007.02030.x © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES Thirteen nuclear microsatellite loci for butternut (Juglans cinerea L.) SEAN HOBAN,* ROBERT ANDERSON,† TIM MCCLEARY,* SCOTT SCHLARBAUM‡ and JEANNE ROMERO-SEVERSON* * University of Notre Dame, Department of Biological Sciences, Notre Dame, IN 46556, USA, †7899 County Road 3950, West Plains, Missouri 65775, USA, ‡Department of Forestry, Wildlife & Fisheries Institute of Agriculture, The University of Tennessee, Knoxville, Knoxville, TN 37996-4563, USA Abstract Butternut (Juglans cinerea L.) is an eastern North American forest tree severely threatened by an exotic fungal pathogen, Sirococcus clavigignenti-juglandacearum. We report here 13 nuclear microsatellites for genetic evaluation of the remaining natural populations. Summary statistics are reported for individuals from a population of butternuts in central Kentucky (N = 63). All markers were polymorphic, with an average of 13.7 alleles per locus observed. Four loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P< 0.05). Keywords: butternut canker, conservation genetics, forest restoration, tree improvement, walnut culture Received 27 August 2007; revision accepted 11 September 2007 Butternut ( Juglans cinerea L.) is a medium-size hardwood tree native to eastern North American forest ecosystems, most often occurring in riparian zones (Fleguel 1996). A lethal and aggressive fungus, Sirococcus clavigignenti-juglandacearum V.M.G. Nair, Kostichka, & Kuntz, which causes butternut canker disease, has eliminated native butternut populations in several southern states and greatly reduced numbers throughout the natural range (Rink 1990). Although previous reports suggest that butternut may have low genetic diversity, thus limiting the species’ ability to resist exotic pests and pathogens (Fjellstrom & Parfitt 1994; Morin et al. 2000), range-wide investigation of the genetic diversity and population structure of butternut is lacking (Ostry et al. 2002). Also, there is evidence of varying tolerance to the disease in wild butternut populations (Schlarbaum et al. 2004), but the genetic basis of tolerance remains unknown. Genetic investigations of the Juglans genus have used several DNA marker systems, including restriction frag- ment length polymorphisms (Fjellstrom & Parfitt 1994), random amplified polymorphic DNA markers (Nicese et al. 1998), isozyme loci (Morin et al. 2000), and chloroplast sequence data (Aradhya et al. 2004). A recent study of black walnut ( Juglans nigra L.), a valuable hardwood that occurs sympatrically with butternut in most of its range, used 12 microsatellites to reveal a remarkable lack of population substructure in the central USA (Victory et al. 2006). We present here the first group of microsatellites tailored to J. cinerea investigations. Microsatellite sequences have been published for J. nigra. However, J. nigra and J. cinerea are not within the same section of Juglans (Manning 1978) and cannot hybridize, so there may be significant genetic divergence between the two species. Primers based on the J. nigra clones may amplify some alleles in J. cinerea, but some alleles may not amplify because of mutations in the primer binding regions. These unobserved or null alleles can result in misleading departures from expected allele distributions ( Jones et al. 1998). We reduced this risk by using a library of cloned J. cinerea microsatellite sequences for marker development. We isolated DNA from freshly cut twigs of a single wild J. cinerea individual from Shannon County, Missouri, using the DNeasy Plant Mini kit (QIAGEN). Genetic Identification Systems (Chatsworth, CA, USA) used this DNA for construc- tion of genomic libraries enriched for CA, GA, and TAG repeats as described previously (Jones et al. 2002). They identified 146 microsatellite-containing sequences and designed primer pairs for 95 of them using designerpcr version 1.03 (Research Correspondence: Jeanne Romero-Severson, Fax: 574.631.7413; E-mail: jromeros@nd.edu