Rice Genetics IV - IRRI books - International Rice Research Institute

hypervariability make them valuable as genetic markers (Weber and May 1989). Inrice, more than 500 microsatellite markers have been developed and used to constructgenetic maps (Wu and Tanksley 1993, Akagi et al 1996, Panaud et al 1995, 1996,Chen et al 1997, Temnykh et al 2000, 2001). These markers provide important codominantlandmarks that are well distributed throughout the rice genome. SSRs havebeen widely used in rice genetics to fingerprint accessions, analyze diversity, identifyintrogressions in interspecific crosses, trace pedigrees, locate genes and QTLs on ricechromosomes, and in marker-assisted selection. In sequencing and genomics applications,SSRs are increasingly useful as sequence tag connectors (STCs) linking thephysical and genetic maps of rice.SSR marker developmentIn the past, the advantages of microsatellite markers were partially offset by the difficultyinherent in marker development, as laborious iterations of genomic DNA libraryscreening with radio-labeled probes were required to isolate SSR-containingsequences. In rice, the growing pool of DNA sequence information being generatedby the International Rice Genome Sequencing Project (IRGSP) and by independentorganizations (see, for example, www.rice-research.org and www.syngenta.com) providesthe basis for efficient, high-throughput in silico identification of SSR loci. Thepublic availability of genomic sequences has dramatically altered the strategies usedfor marker development and has largely removed the technical and economic limitationsthat previously limited the number of SSR loci that were available for rice.To facilitate the development of new microsatellite markers using publicly availableDNA sequence information, a set of computer programs has been developed forsemiautomated identification of nonredundant SSR loci and for primer design. Thescript consists of two parts and is publicly available at http://ars-genome.cornell.edu/rice/tools.html. The searching script identifies all microsatellites of a user-definedminimum repeat length in a set of downloaded sequences; records the database ID(i.e., GenBank accession number), motif type, number of perfect repeats, and sequencecoordinates for each SSR; does a redundancy search based on flanking sequenceidentity; and reports the results in an output file. Primers are then designedusing the program Primer 0.5 (Daly et al 1991). The search can be used to identifySSRs in different types and lengths of DNA sequence, including relatively short sequences(300–700 bp) derived from the ends of cDNA clones (known as expressedsequence tags, or ESTs), or from the ends of rice genomic DNA cloned into bacterialartificial chromosomes (BACs), or long contigs assembled from fully sequenced BACand P1-derived artificial chromosome (PAC) clones that may be up to 1 Mb in length(Temnykh et al 2001).Based on information about the frequency and average length of specific SSRmotifs in rice, classes of SSRs most likely to provide good genome coverage and highlevels of polymorphism can be selected for primer design and used as DNA markers.Akagi et al (1996) first noted that AT-rich microsatellites tended to show more lengthvariation than tracts consisting of GC-rich motifs and suggested that these would118 McCouch et al