Molecular Ecology Notes (2007) 7, 1155–1161 doi: 10.1111/j.1471-8286.2007.01814.x © 2007 University of Århus Blackwell Publishing Ltd PRIMER NOTE Cross-species amplification of 105 Lolium perenne SSR loci in 23 species within the Poaceae LOUISE BACH JENSEN, PREBEN BACH HOLM and THOMAS LÜBBERSTEDT Aarhus University, Faculty of Agricultural Sciences, Department of Genetics and Biotechnology, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark Abstract Amplification of 105 Lolium perenne SSR markers was studied in 23 grass species representing seven tribes from three subfamilies of Poaceae. Twelve of the SSR markers are published for the first time. Between 2% and 96% of the SSR markers could be amplified within a given species. A subset of eight SSR markers was evaluated for polymorphism across nine of the 23 grass species. Four to seven of the markers were polymorphic within each species, with an average detection of 2.4 alleles per species. Keywords: cross-species amplification, grass, Lolium perenne, SSR Received 18 February 2007; revision accepted 27 March 2007 Lolium perenne, the most important forage species in temperate regions, belongs to the grass family Poaceae, subfamily Pooideae, tribe Poeae. The grass family includes our most important agricultural food and forage crops as well as several other species of commercial and practical importance. Simple sequence repeat (SSR) markers are the current markers of choice for most genetic and breeding applications due to their abundance, widespread distri- bution, high levels of polymorphism, codominant mode of inheritance, high reproducibility and ease of (polymerase chain reaction) PCR-based analysis. However, since the identification of SSR loci is a labour-intensive and costly process, the development and application of SSRs in plants has mainly been restricted to the most important crops in agriculture. In L. perenne , more than 120 SSR markers from both genomic libraries and expressed sequence tags (ESTs) are currently publicly available (Kubik et al . 1999, 2001; Jones et al . 2001; Jensen et al . 2005; Lauvergeat et al . 2005). Our objectives were to (i) examine the cross-species amplification of 105 L. perenne SSR markers in 23 grass species representing seven tribes from three subfamilies of Poaceae, and (ii) evaluate the polymorphism of a subset of the SSR markers. One to nine accessions were randomly chosen for each of the 23 species studied (Table 1). Leaf tissue was harvested from one plant per individual accession and genomic DNA was isolated according to Guidet et al . (1991). After deter- mination of DNA concentration, equal amounts of DNA from all accessions within each of the 23 species were combined, so that each DNA sample contained DNA from one to nine accessions depending on the species. Twelve of the SSR markers used in the current study have not been published before, detailed information regarding these primers have been submitted to the Molecular Ecology primer database as well as the GenBank. Seven markers [LpSSR002 (GenBank Accession no. DQ404324), LpSSR005 (DQ404325), LpSSR013 (DQ404326), LpSSR024 (DQ404327), LpSSR063 (DQ404328), LpSSR069 (DQ404329) and LpSSR087 (DQ404330)] were developed at the Danish Institute of Agricultural Sciences (DIAS) from genomic libraries (Jensen et al . 2005), two markers (DLF026 and DLF028) were devel- oped at DLF-Trifolium from ESTs (Jensen et al . 2005) and three markers [B2-E1 (GenBank Accession no. DQ404331), B2-F11 (DQ404332) and B2-H6 (DQ404333)] were developed at Institut National de Recherche Agronomique, Unité de Génétique et d’Amélioration des Plantes (France) from genomic libraries (Lauvergeat et al . 2005). The other 93 mark- ers have been described previously (Kubik et al . 1999, 2001; Jones et al . 2001; Muylle 2003; Jensen et al . 2005; Lauvergeat et al . 2005). All SSR markers were tested under the same PCR con- ditions as originally applied for amplification in L. perenne. PCR amplification reactions for each of the SSR markers were performed in a 10 μ L reaction volume containing 25 ng of genomic DNA, 1 μ L of 10 × PCR buffer, 2 m m MgCl 2 , 0.2 mm of each dNTP, 0.1 μm of each forward and reverse primer and 0.5 U Taq polymerase. PCR amplifica- tion was performed in an MJ Research thermalcycler (MJ Correspondence: Louise Bach Jensen, Email: louiseb.jensen@agrisci.dk