Reference "Design and application of microsatellite marker panels for semiautomated genotyping of rice (Oryza sativa L.)"

Reference ID

7844

Title

Design and application of microsatellite marker panels for semiautomated genotyping of rice (Oryza sativa L.)

Source

Crop Science, 2002, vol. 42, pp. 2092-2099

Authors (4)

Coburn-J-R	Temnykh-S-V
Paul-E-M	McCouch-S-R

Abstract

The objective of this study was to develop a systematic and flexible method for
assembling multiplex simple sequence repeat (SSR) marker panels for high
throughput genome analysis in rice, Oryza sativa, and to test these panels on a
set of cultivated rice germplasm. To do this, 159 microsatellite markers were
fluorescently labeled and assembled into 21 multiplex panels for semiautomated
genotyping, providing genome-wide coverage of the 12 rice chromosomes. Panels
are comprised of an average of eight markers each, occurring at approximately
11-centimorgan (cM) intervals throughout the genome. On a standard set of 13
genetically diverse cultivars of Oryza, these markers detected an average of
five alleles per locus and had a mean polymorphism information content (P.I.C.
value) of 0.67. Polymerase chain reactions (PCR) were optimized on a per marker
basis to generate a uniform amount of PCR product and each primer pair was
assessed in replicated trials for reliability of allele size estimates. T4 DNA
polymerase was used to treat PCR products where the standard deviation of allele
molecular weight was greater than 0.5 base pairs (bp). This treatment minimized
the variance so that, in the multiplex set reported here, the average std.
dev./marker was 0.24 bp, allowing accurate discrimination of alleles that
differed by a single nucleotide. The resulting data on allele sizes were then
entered into GeneFlow analysis software for the evaluation of polymorphism
patterns among diverse rice cultivars. The use of an automated software tool for
designing multiplex panels on the basis of both highly polymorphic and more
conservative SSR markers resulted in the development of a highly informative
semiautomated genotyping system for applications in rice genetics and breeding.