2 2. MEDICAGO TRUNCATULA AS A MODEL LEGUME Natural attributes of M. truncatula that make it a valuable ge- netic model include its annual habit and rapid life cycle, its diploid (2n = 16) and autogamous nature, its proliﬁc seed production, and a relatively small genome of about 550 Mb. Jemalong A17 has been selected by the research community as a reference line for most genetic and genomic approaches and is derived from the major commercial cultivar. M. trun- catula is native to the Mediterranean basin and is found in a wide range of habitats. It is therefore not surprising to ﬁnd a high level of variation among and within natural populations [29]. Using microsatellite markers, a publicly available core- collection of 346 inbred lines was developed and thus repre- sents the breadth of this natural diversity [30]. M. truncatula is used as a fodder crop in ley-farming systems in Australia, and a large and diverse collection is housed at the South Aus- tralian Research and Development Institute (SARDI) [8]. Like many higher plants, M. truncatula forms symbi- otic associations with a wide array of arbuscular mycorrhizal (AM) fungi. As a legume, M. truncatula is also able to develop root nodules with Sinorhizobium meliloti, which is one of the best-characterized rhizobium species at the genetic level [31]. Cultivation-independent techniques have been used to sam- ple the diversity of microbes associated with M. truncatula roots at various developmental stages and they reveal an ex- tremely dynamic genetic structure of its rhizosphere [32]. Mutagenesis approaches using ethyl methane sulfonate (EMS), gamma rays, and fast neutron bombardment (FNB) have generated large mutant populations of M. truncatula from which mutants aﬀected in symbiotic as well as develop- mental pathways have been identiﬁed [33–37]. T-DNA and Tnt1 mutagenesis have been developed recently to generate tagged mutants for forward and reverse genetics purposes [38–40]. Several protocols have been optimized to transform M. truncatula using Agrobacterium tumefaciens [41–45]. These protocols are particularly eﬃcient for the R108 and Jema- long 2HA lines but the regeneration eﬃciency still needs to be improved for Jemalong A17. This moderate eﬃciency as well as the time required for the regeneration steps is driv- ing the preference of the Medicago community towards Tnt1 versus T-DNA for gene tagging approaches [46] as well as the search for alternative transformation systems. Hairy root transformation via Agrobacterium rhizogenes proved to be a rapid and eﬃcient transformation system al- lowing the generation of transgenic roots in 2-3 weeks. Such “hairy roots” can be infected by rhizobia or AM fungi with symbiotic phenotypes indistinguishable from nontransgenic roots and are therefore an ideal system for plant-microbe symbiosis studies [47]. The development of DsRed as a vi- sual reporter reduced the need for Kanamycin or Basta se- lection systems which were signiﬁcantly decreasing nodula- tion eﬃciency. This hairy root transformation system is now used routinely to express protein fusions or RNA interfer- ence (RNAi) constructs [48–50]. The possibility to regener- ate transgenic plants from hairy roots of the R108 line has been reported recently. This ﬂexible approach should allow a rapid initial screening of phenotypes on hairy roots and International Journal of Plant Genomics a subsequent regeneration of transgenic plants if necessary [41]. An interesting ex vitro procedure that eliminates the need for labor-intensive in vitro culture will undoubtedly in- crease the throughput of hairy root transformations to a level compatible with genomic studies [51]. The Medicago community has therefore identiﬁed many ecotypes and developed a wide range of mutants and trans- genic lines. A current goal of the International Medicago truncatula steering committee is to address the need for a stock center able to maintain, amplify, and distribute these lines to an ever growing community. 3. MAPPING THE GENOME OF MEDICAGO TRUNCATULA Genetic and cytogenetic tools have been instrumental to the development of a “gene rich” genome sequence for M. truncatula. This project also required several bacterial artiﬁ- cial chromosomes (BAC) libraries that were developed using HindIII and EcoRI partial digests as well as a robust physical map (Figure 1). Genetic maps have been developed from F2 popula- tions and a wide array of genetic markers such as CAPS, AFLPs, RAPDs, and microsatellites (SSRs) [52–54]. One of them, based on a Jemalong A17 A17 × A20 F2 population, is currently used as a reference for the genome sequencing project (http://www.medicago.org/genome/map.php). Un- fortunately, these F2 populations are either based on a lim- ited amount of genomic DNA or require a labor-intensive vegetative propagation of F2 individuals. In order to pro- vide sustainable tools to the community, genetic maps based on recombinant inbred lines (RILs) and highly polymorphic microsatellite markers are developed and will undoubtedly represent the future reference for M. truncatula genetics (T. Huguet, personal communication). Cytogenetic maps based on ﬂuorescence in situ hybrid- ization (FISH) with interphase or metaphase chromosomes provide a quick access to the chromosomal location of BAC clones and repeated sequences [55–57]. Obtaining pachytene chromosomes is more labor intensive than metaphase chro- mosomes but provides an unequalled resolution all along the chromosome and particularly in euchromatic regions [58]. Information from such cytogenetic tools was instrumental for comparative genomics and map-based cloning projects but also allowed the determination that M. truncatula het- erochromatin was mostly localized in pericentromeric re- gions. Genetic and cytogenetic markers corresponding to the borders of these regions have been developed [57, 59, 60]. Based on this unique chromosomal structure, it is therefore possible to predict through the genetic map if a BAC clone belongs to a euchromatic or a heterochromatic region. This observation as well as the possibility to select EST-rich BAC clones led the M. truncatula community to initiate the se- quencing of euchromatic (gene-rich) regions via a BAC-by- BAC strategy (http://www.medicago.org/genome/). Four centers share the sequencing eﬀort of the 8 chro- mosomes: Bruce Roe et al. at the University of Oklahoma, Chris Town et al. at The Institute for Genomic Research (TIGR), Jane Rogers et al. at the Sanger Centre, and Francis