ORIGINAL PAPER Characterisation and inheritance of nuclear microsatellite loci for use in population studies of the allotetraploid Salix alba–Salix fragilis complex Robert Andrew King & Sarah L. Harris & Angela Karp & Jacqueline H. A. Barker Received: 20 July 2009 / Revised: 28 September 2009 / Accepted: 1 October 2009 / Published online: 15 October 2009 # Springer-Verlag 2009 Abstract We present nine polymorphic di- and tri- nucleotide repeat nuclear microsatellite markers selected specifically for their use in high throughput studies concerning the dioecious allotetraploid Salix alba–Salix fragilis willow complex. These taxa and their hybrids are difficult to discriminate using morphological characters. Thus, multiplex reactions were developed for these micro- satellite loci and their effectiveness to distinguish individ- uals, especially hybrids, and their inheritance patterns in controlled crosses were determined. All loci displayed disomic–monogenic inheritance which allowed for the genotype data to be analysed as for a diploid organism. The nine loci produced a total of 67 alleles (mean, 7.4 alleles per locus; range, 3–11 alleles) in a reference panel of 57 individuals from two germplasm collections and natural populations. Gene diversity values (as measured by the expected heterozygosity) ranged from 0.000–0.820. A total of 53 distinct multilocus genotypes were observed, and ordination analysis revealed three separate clusters corresponding to S. alba, S. fragilis and hybrids. The microsatellite loci described here will be used in population genetic studies to investigate genetic variation, gene flow, levels of hybridisation and the extent of introgression in natural populations of the S. alba–S. fragilis complex. They are also useful for clonal identification, conservation and sustainable management of germplasm collections, genetic mapping and the selection of individuals and/or certifica- tion of controlled crosses for breeding programmes. Keywords Allotetraploid . Genetic diversity . Microsatellite . Hybrid . Salix alba . Salix fragilis Introduction Polyploidy, the occurrence of three or more complete sets of chromosomes in a cell nucleus, is an important mechanism in the evolution of plant species. Current estimates suggest that >70% of all angiosperm species are of polyploid origin (Masterson 1994). Two basic types of polyploidy are recognised: autopolyploids contain more than two chromosome sets of the same species while allopolyploids combine the genomes of at least two genetically distinct species (Leitch and Bennett 1997). While molecular markers such as isozymes (Soltis and Soltis 1990), random amplified polymorphic DNAs (RAPDs) (Williams et al. 1990), ISSRs (Zietkiewicz et al. 1994) and amplified fragment length polymorphisms (AFLPs) (Vos et al. 1995) are relatively cheap to develop, Communicated by W. Boerjan R. A. King : S. L. Harris : A. Karp Plant and Invertebrate Ecology Department, Rothamsted Research, Harpenden AL5 2JQ Herts, UK J. H. A. Barker (*) Plant Sciences Department, Rothamsted Research, Harpenden AL5 2JQ Herts, UK e-mail: jackie.barker@bbsrc.ac.uk Present Address: R. A. King Cardiff School of Biosciences, Biomedical Sciences Building, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK Present Address: S. L. Harris School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK Tree Genetics & Genomes (2010) 6:247–258 DOI 10.1007/s11295-009-0245-6