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Distribution of Microsatellites in the Genome of Medicago truncatula: A Resource of Genetic Markers That Integrate Genetic and Physical Maps

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Distribution of Microsatellites in the Genome of Medicago truncatula: A Resource of Genetic Markers That Integrate Genetic and Physical Maps
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  1 Distribution of microsatellites in the genome of  Medicago truncatula : A resource of geneticmarkers that integrate genetic and physical maps. Jeong-Hwan Mun, *  Dong-Jin Kim, *  Hong-Kyu Choi, *  John Gish, *  Frédéric Debellé, †  JoanneMudge, +  Roxanne Denny, +  Gabriella Endré, §  Oliver Saurat, †  Anne-Marie Dudez, †  Gyorgy B.Kiss, §,#  Bruce Roe, ††  Nevin D. Young, +  and Douglas R. Cook  *,1* Department of Plant Pathology, University of California, Davis, CA, USA. † Laboratoire des Interactions Plantes-Microorganismes, INRA-CNRS, Castanet-Tolosan Cedex,France. + Department of Plant Pathology, University of Minnesota, St. Paul, MN, USA. § Biological Research Center, Institute of Genetics, Szeged, Hungary. # Institute of Genetics, Agricultural Biotechnology Center, Godollo, Hungary. †† Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA. 1 Corresponding author  : Department of Plant Pathology, University of California, 1 Shields Ave,Davis, CA 95616E-mail: drcook@ucdavis.edu   Genetics: Published Articles Ahead of Print, published on February 19, 2006 as 10.1534/genetics.105.054791  2Running head: Microsatellites in  Medicago truncatula Key words:  Medicago truncatula , gene-rich BACs, sequence-tagged site genetic marker,microsatellite, genetic mapping.Corresponding author:Douglas R. Cook Department of Plant PathologyUniversity of California1 Shields AveDavis, CA 95616drcook@ucdavis.edu530-754-6561 (office)530-754-6617 (fax)  3 ABSTRACT Microsatellites are tandemly repeated short DNA sequences that are favored as molecular-geneticmarkers due to their high polymorphism index. Plant genomes characterized to date exhibittaxon-specific differences in frequency, genomic location and motif structure of microsatellites,indicating that extant microsatellites srcinated recently and turnover quickly. With the goal of using microsatellite markers to integrate the physical and genetic maps of  Medicago   truncatula ,we surveyed the frequency and distribution of perfect microsatellites in 77 Mbp of gene-richBAC sequences, 27 Mbp of non-redundant transcript sequences, 20 Mbp of random wholegenome shotgun sequence, and 49 Mbp of BAC-end sequences. Microsatellites are predominantly located in gene-rich regions of the genome, with a density of one long (i.e., ! 20nt) microsatellite every 12 Kbp, while the frequency of individual motifs varied according to thegenome fraction under analysis. 1,236 microsatellites were analyzed for polymorphism between parents of our reference intraspecific mapping population, revealing that motifs (AT) n , (AG) n ,(AC) n , and (AAT) n  exhibit the highest allelic diversity. 378 genetic markers could be integratedwith sequenced BAC clones, anchoring 274 physical contigs that represent 174 Mbp of thegenome and comprising an estimated 70% of the euchromatic gene space.  4 INTRODUCTION Legumes are the second most important crop family, in terms of cultivated acreage,contribution to human and animal diets, and economic value. Their capacity for symbioticnitrogen fixation underlies the value of legumes as a source of dietary protein, while the diversityof their metabolic output provides a wide range of pharmacologically valuable secondary natural products, including isoflavonoids and triterpene saponins. Although  Arabidopsis  and rice serveas models for dicot and monocot species, respectively, they cannot serve as models to identifythe genetic programs responsible for legume-specific characteristics. Two legume species,namely  Medicago truncatula and  Lotus japonicus , serve as models for legume biology.The utility of  M. truncatula  as a genetic system (e.g., Penmetsa and Cook 2000), combinedwith its relatively small (466 Mb; Bennett and Leitch 1995) and efficiently organized genome(Kulikova et al. 2001 and 2004), have motivated an international effort to develop and apply thetools of genomics in  M  . truncatula  to key questions in legume biology. One aspect of this efforthas been the development of enabling methodologies, such as efficient transformation methods(Trinh et al. 1998; Kamaté et al. 2000; Zhou et al. 2004), high-throughput systems for forwardand reverse genetics including insertional mutagenesis (d’Erfurth et al. 2003), RNAi (Limpens etal. 2003 and 2004), and TILLING (Vandenbosch and Stacey 2003), and an effective network among research groups (http://www.medicago.org). In parallel to these activities, national andinternational programs are collaborating to characterize the genome of  M. truncatula  at thetranscript (Fedorova et al. 2002; Journet et al. 2002; Lamblin et al. 2003), protein (Gallardo et al.2003; Watson et al. 2003; Imin et al. 2004), and whole genome sequence levels (Young et al.2005).Cytogenetic and genetic data predict that the genome of  M. truncatula  is organized intoseparate gene-rich euchoromatic arms and gene-poor heterochromatic pericentromeric regions(Kulikova et al. 2001 and 2004; Choi et al. 2004a). These results underlie a strategy for sequencing the  M. truncatula  genome wherein the euchromatic chromosome arms are firstdelimited within a physical map and then subjected to a BAC-by-BAC sequencing approach. Asof March 2004, 44,292 BACs (~11X coverage) had been fingerprinted by  Hin dIII digestion andagarose gel electrophoresis. An initial stringent build of the map yielded 1,370 contigs with anaverage length of 340 Kbp, covering an estimated 466 Mbp or 93% of the genome. In parallel tothe development of a physical map, >800 EST-containing BAC clones were sequenced to  5 provide seed points from which to continue the whole genome sequencing effort. Sites of  potential sequence polymorphism within the initial BAC sequence data are being used tofacilitate merger of the genetic and physical maps, while the resulting chromosome assignmentsare being used to guide the distribution of BACs to sequencing centers.A major focus of the genetic mapping effort is short tandem repeats, also known as simplesequence repeats (SSRs) or microsatellites. These repetitive sequences consist of direct tandemrepeats of short (1-10 bp) nucleotide motifs. Unequal recombination between SSRs and slip-mispairing during DNA replication (Sia et al. 1997) result in polymorphism rates that tend to bemuch greater than those observed for non-repetitive DNA sequences. The high rate of mutationcombined with low selection coefficients on variant alleles result in extreme allelic diversity atmicrosatellite loci (Ross et al. 2003).Identification of SSRs in DNA sequence databases can be automated by use of publicsoftware programs, such as SSRIT (Temnykh et al. 2001). Moreover, because SSR alleles aretypically codominant and their polymorphisms can be scored either in a simple agarose gelformat or in high throughput capillary arrays, they are frequently the molecular marker of choicefor construction of genetic maps. Estimates suggest that 1% to 5% of plant ESTs contains SSRslonger than 18 nucleotides (Kantety et al. 2002). Thus, development of EST-SSR markers has become commonplace in a wide variety of plant species (Decroocq et al. 2003; Thiel et al. 2003;Sharopova et al. 2002; Kantety et al. 2002; Cordeiro et al. 2001), including  Medicago  spp.(Gutierrez et al. 2005; Sledge et al. 2005; Eujayl et al. 2004; Julier et al. 2003). SSRs are evenmore abundant in the non-coding regions of genomic sequences, providing a rich source of genetic markers to map sequenced genome regions (Cardle et al. 2000). In rice, for example,genomic-SSR markers identified from BAC sequences provided immediate links betweengenetic, physical, and sequence-based maps (Temnykh et al. 2001).In this study, we report the characteristics of perfect microsatellites within the genome of  M.truncatula . Genetic markers developed from SSRs in BAC sequences were incorporated into the  M  . truncatula  genetic map, simultaneously anchoring a predicted majority of the euchromatic portion of the physical map to chromosomal loci. In total, we analyzed 77 Mbp of genomicsequence (16.5% of the genome) obtained from gene-rich BAC clones, 27 Mbp of non-redundanttranscript sequence, 20 Mbp of low pass random whole genome shotgun data, and 49 Mb of BAC-end sequences for the presence of perfect SSRs. The resulting data set allowed comparison
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