Single-nucleotide polymorphism discovery and diversity in the model legume Medicago truncatula KARINE LORIDON,* CONCETTA BURGARELLA,* NATHALIE CHANTRET,* FRE ´ DE ´ RIC MARTINS,†,‡ JE ´ RO ˆ ME GOUZY,§ JEAN-MARIE PROSPE ´ RI* and JOE ¨ LLE RONFORT* *INRA, UMR AGAP, 2 place Pierre Viala, F-34060 Montpellier Cedex 1, France, †INRA, Plateforme GeT (Ge ´nome et Transcriptome), Ge ´nopole Toulouse, Chemin de Borde Rouge, BP 52627, F-31326 Castanet-Tolosan, France, ‡Inserm/Universite ´ Paul Sabatier, UMR 1048, Institut des Maladies Me ´taboliques et Cardiovasculaires (I2MC), BP 84225, F-31432, Toulouse Cedex 4, France, §INRA/CNRS, UMR 441/2594, Laboratoire Interactions Plantes Micro-organismes, Chemin de Borde Rouge, F-31320 Castanet Tolosan, France Abstract Extensive genomic resources are available in the model legume Medicago truncatula. Here, we present the discovery and design of the first array of single-nucleotide polymorphism (SNP) markers in M. truncatula through large-scale Sanger resequencing of genomic fragments spanning the genome, in a diverse panel of 16 M. truncatula accessions. Both anonymous fragments and fragments targeting candidate genes for flowering phenology and symbiosis were surveyed for nucleotide variation in almost 230 kb of unique genomic regions. A set of 384 SNP markers was designed for an Illumina’s GoldenGate assay, genotyped on a collection of 192 inbred lines (CC192) representing the geographical range of the species and used to survey the diversity of two natural populations. Finally, 86% of the tested SNPs were of high quality and exhibited polymorphism in the CC192 collection. Even at the population level, we detected polymorphism for more than 50% of the selected SNPs. Analysis of the allele frequency spectrum in the CC192 showed a reduced ascertainment bias, mostly limited to very rare alleles (frequency <0.01). The substantial polymorphism detected at the species and population levels, the high marker quality and the potential to survey large samples of individuals make this set of SNP markers a valuable tool to improve our understanding of the effect of demographic and selective factors that shape the natural genetic diversity within the selfing species Medicago truncatula. Keywords: ascertainment bias, Medicago truncatula, selfing, sequence diversity, single-nucleotide polymorphism Received 11 May 2012; revision received 16 August 2012; accepted 20 August 2012 Introduction Single-nucleotide polymorphisms (SNPs) have become of standard use in human genetics (International Hap- Map Consortium 2007) and in model organisms like Dro- sophila melanogaster and Arabidopsis thaliana (Chen et al. 2009; Zhang & Borevitz 2009) and are particularly rele- vant genetic markers for evolutionary and conservation genetic analyses (Morin et al. 2004). However, their use in these areas is only slowly emerging (see for example, Fournier-Level et al. 2011; Gomaa et al. 2011; Hancock et al. 2011). Interest in SNP markers in evolutionary genetics comes primarily from their abundance and widespread distribution throughout the genome (Rafal- ski 2002; Deschamps & Campbell 2010). SNPs are usually biallelic so that more SNP markers are required compared to other molecular markers, especially micro- satellites (Hauser et al. 2011), but their binary nature makes genetic variation easy to score and comparisons of patterns of variation between markers as well as between populations more straightforward. SNPs are easily amenable to automation, and during the last dec- ade, many medium- to high-throughput SNP genotyping methods have been developed (for reviews, see Kim & Misra 2007; Gupta et al. 2008). The choice between all these technologies depends strongly on the number of samples and markers to be analysed. The Illumina’s GoldenGate assay allows genotyping large collections of samples (up to 480) for hundreds of SNP markers (up to 3072) and has been reported in several plant genetics studies as producing reliable results and displaying high proportions of successful SNP markers (Hyten et al. 2008; Akhunov et al. 2009; Deulvot et al. 2010; Grattapa- glia et al. 2011; Thomson et al. 2012). Correspondence: Karine Loridon, Fax: +33-(0)4-67-04-54-15; E-mail: karine.loridon@supagro.inra.fr © 2012 Blackwell Publishing Ltd Molecular Ecology Resources (2013) 13, 84–95 doi: 10.1111/1755-0998.12021