Sequence-tagged microsatellite profiling (STMP): a rapid technique for developing SSR markers.
We describe a technique, sequence-tagged microsatellite profiling (STMP), to rapidly generate large numbers of simple sequence repeat (SSR) markers from genomic or cDNA. This technique eliminates the need for library screening to identify SSR-containing clones and provides an approximately 25-fold increase in sequencing throughput compared to traditional methods. STMP generates short but characteristic nucleotide sequence tags for fragments that are present within a pool of SSR amplicons. These tags are then ligated together to form concatemers for cloning and sequencing. The analysis of thousands of tags gives rise to a representational profile of the abundance and frequency of SSRs within the DNA pool, from which low copy sequences can be identified. As each tag contains sufficient nucleotide sequence for primer design, their conversion into PCR primers allows the amplification of corresponding full-length fragments from the pool of SSR amplicons. These fragments permit the full characterisation of a SSR locus and provide flanking sequence for the development of a microsatellite marker. Alternatively, sequence tag primers can be used to directly amplify corresponding SSR loci from genomic DNA, thereby reducing the cost of developing a microsatellite marker to the synthesis of just one sequence-specific primer. We demonstrate the utility of STMP by the development of SSR markers in bread wheat.
Hayden
M J
MJ
Plant Breeding Institute, University of Sydney, PMB 11, Camden, NSW 2570, Australia and Quality Wheat CRC, Locked Bag 1345, PO North Ryde, NSW 2113, Australia. matthewh@camden.usyd.edu.au
Sharp
P J
PJ
D001315:Australia; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism; D018628:Gene Dosage; D020869:Gene Expression Profiling; D005819:Genetic Markers / Q000235:genetics*; D018745:Genome, Plant; D018895:Microsatellite Repeats / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics; D016324:Sequence Tagged Sites; D014908:Triticum / Q000235:genetics*
D017931:DNA Primers; D016373:DNA, Antisense; D018744:DNA, Plant; D005819:Genetic Markers; D004706:Endodeoxyribonucleases