Increasing efficiency of SSR marker genotyping for rice molecular breeding

Simple sequence repeat (SSR) markers or microsatellites' have been widely-used DNA markers in rice breeding and genetics research for more than 15 years. After the completion of the rice genome sequence in 2005, more than 18,000 SSRS have been identified, which is by far the largest number of publicly available SSRS for any crop species. This number continues to increase due to progress in rice genomics research. Currently SSRS are widely-used within rice breeding programs at the International Rice Research Institute (IRRI). SSRS are used for DNA finger printing and genetic diversity analysis, construction of linkage maps for quantitative trait loci (QTL) analysis and several applications of marker assisted selection. The cost of marker genotyping is still the most important factor limiting the greater use of markers in rice breeding. Therefore, the authors have investigated ways to improve genotyping efficiency or reducing the cost of SSR data points per sample. In this poster, the authors report on preliminary empirical research findings involving: 1) establishing a 'genotyping' set of highly polymorphic SSRS, primarily for indica rice genotypes; 2) high throughput DNA extraction methods; 3) used of reduced reaction volumes for PCR; 4) using shorter PCR programs; 5) multiple gel loading; 6) multiplex PCR; and 7) data management. Preliminary screening of 326 SSRS indicated a wide range of polymorphic information content (PIC) values and 'quality' of PCR amplicons. Some primers consistently produced clearly-scorable SSR marker alleles whereas others consistently failed. A pilot experiment testing 3 PCR cycles involving 39 SSRS indicated that shorter PCR cycles (1.5 hr compared to the standard 3.5 m) produced clearly scorable marker alleles with considerably less background amplification. Preliminary tests concerning multiple gel loading indicated potential for increasing genotyping efficiency however data management is critical to ensure that appropriate SSR markers can be combined together during gel electrophoresis.