Submitted to: Rice Technical Working Group Meeting Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 2/29/2004
Publication Date: 2/1/2005
Citation: Johnson, V.A., Redus, M., Gibbons, J.W., Moldenhauer, K.A., Jiang, J., Jia, Y. 2005. Marker assisted selection for the rice blast resistance gene pi-ta: development and use of an improved co-dominant analysis method [abstract]. Rice Technical Working Group Meeting Proceedings. Abstract p. 62. Interpretive Summary:
Technical Abstract: Fragment analysis with a dominant molecular marker has been in use in the UA RREC Rice Breeding and Genetics Program for DNA Marker Assisted Selection (MAS) to accelerate the development of cultivars with improved rice blast (Magnaporthe grisea) disease resistance by monitoring the incorporation of the resistance gene, Pi-ta. As the breeders gain confidence in the use of MAS as a breeding tool and begin to analyze ever increasing numbers of samples, it has become necessary to develop and optimize new methods that are faster, more efficient, more accurate, and cost less per sample. A new automated analysis method of a co-dominant Pi-ta marker was developed for population screening and to verify the results of rice blast pathogenicity studies. This PCR-based method capitalizes on the conserved nucleotide length polymorphism of the Pi-ta gene within indica-derived alleles. The PCR is performed using a three-primer system, two "forward" primers and one "reverse" primer, all obtained from Integrated DNA Technologies (Coralville, Iowa). One "forward" primer specifically amplifies the resistant allele, and is modified at the 5' end with a fluorescent blue 6-FAM label. The other "forward" primer specifically amplifies the susceptible allele, and is modified at the 5' end with a fluorescent green HEX label. The "reverse" primer, which is common to both "forward" primers, is unlabeled. Resulting amplification products are approximately 181 bp for the resistant allele and 182 bp for the susceptible allele. Genomic DNA is extracted from seedling leaf tissue using a modified PEX/CTAB extraction method. The DNA samples are then quantified in a 96-well format. PCR DNA template blocks are prepared containing 86 unknown samples and 10 samples of resistant and susceptible controls and parental lines. All DNA samples are diluted to a 20-ng/µl concentration. PCR is performed and the resulting amplification products are prepared for analysis. Fluorescent fragment separation is performed on an ABI 3700, and subsequent data is analyzed using GeneScan and Genotyper software, all from Applied Bio-systems (Foster City, CA). Over 2,300 individual samples have been analyzed using this method, enabling the breeders to classify the genotype of advanced breeding lines, confirm pseudo-hybrids in segregating populations, and identify homozygous susceptible materials from the first generation of triple cross (TC1) and F3 populations. On average, the breeders have been able to eliminate over a third of the individuals in segregating populations in the first round of MAS, saving valuable time and resources.