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

By Mun, Jeong-Hwan; Kim, Dong-Jin et al. | Genetics, April 2006 | Go to article overview

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


Mun, Jeong-Hwan, Kim, Dong-Jin, Choi, Hong-Kyu, Gish, John, et al., Genetics


ABSTRACT

Microsatellites are tandemly repeated short DNA sequences that are favored as molecular-genetic markers due to their high polymorphism index. Plant genomes characterized to date exhibit taxon-specific differences in frequency, genomic location, and motif structure of microsatellites, indicating that extant microsatellites originated recently and turn over 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-rich BAC sequences, 27 Mbp of nonredundant transcript sequences, 20 Mbp of random whole genome shotgun sequences, 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., ≥20 nt) microsatellite every 12 kbp, while the frequency of individual motifs varied according to the genome fraction under analysis. A total of 1,236 microsatellites were analyzed for polymorphism between parents of our reference intraspecific mapping population, revealing that motifs (AT)^sub n^, (AG)^sub n^, (AC)^sub n^, and (AAT)^sub n^ exhibit the highest allelic diversity. A total of 378 genetic markers could be integrated with sequenced BAC clones, anchoring 274 physical contigs that represent 174 Mbp of the genome and composing an estimated 70% of the euchromatic gene space.

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 symbiotic nitrogen fixation underlies the value of legumes as a source of dietary protein, while the diversity of their metabolic output provides a wide range of pharmacologically valuable secondary natural products, including isoflavonoids and triterpene saponins. Although Arabidopsis and rice serve as models for dicot and monocot species, respectively, they cannot serve as models for identifying the 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), combined with its relatively small (466 Mb; BENNETT and LEITCH 1995) and efficiently organized genome (KULIKOVA et al. 2001, 2004), have motivated an international effort to develop and apply the tools of genomics in M. truncatula to key questions in legume biology. One aspect of this effort has been the development of enabling methodologies, such as efficient transformation methods (TRINH et al. 1998; ÊÁÌÁÔÅ et al. 2000; ZHOU et al. 2004), high-throughput systems for forward and reverse genetics, including insertional mutagenesis (D'ERFURTH et al. 2003), RNAi (LiMPENS et al. 2003, 2004), and TILLING (VANDENBOSCH and STACEY 2003), and an effective network among research groups (http://www.medicago.org). In parallel to these activities, national and international programs are collaborating to characterize the genome of M. truncatula at the transcript (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 (YouNGrf al. 2005).

Cytogenetic and genetic data predict that the genome of M. truncatula is organized into separate gene-rich euchromatic arms and gene-poor heterochromatic pericentromeric regions (KULIKOVA et al. 2001, 2004; CHOI et al. 2004a). These results underlie a strategy for sequencing the M. truncatula genome wherein the euchromatic chromosome arms are first delimited within a physical map and then subjected to a BAC-by-BAC sequencing approach. As of March 2004, 44,292 BACs (~11X coverage) had been fingerprinted by HindlII digestion and agarose gel electrophoresis. An initial stringent build of the map yielded 1370 con tigs with an average length of 340 kbp, covering an estimated 466 Mbp or 93% of the genome. …

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