Submitted to: American Society of Sugar Cane Technologists
Publication Type: Abstract Only
Publication Acceptance Date: May 9, 2005
Publication Date: July 20, 2005
Citation: Pan, Y., Scheffler, B.E., Richard Jr, E.P. 2005. Application of High-Throughput DNA Marker Technology in Sugarcane Breeding: Phase II - PCR Amplification and Capillary Electrophoresis. Journal of the American Society of Sugar Cane Technologists. 25:117. Available: http://www.assct.org/journal/journal.htm Technical Abstract: Each year, only a small portion of the seedlings (approximately 120,000 seedlings from 300 commercial and basic crosses) are transplanted to the field at the SRU’s Ardoyne Farm due to space limitations in the greenhouse and field. We are continuing to explore the utilization of high-throughput (HT) DNA marker technology in an effort to maximize the number of hybrid seedlings being transplanted to the greenhouse and field. The HT-DNA extraction method described at this meeting in 2004 and currently being used at our lab, allows the preparation of DNA templates from 600 seedlings per day per technician using 96-well DNA sample microplates and as such is no longer a limiting step. Our goal in further developing this process was to be able to assess cross quality (percentage of seedlings with DNA fragments from both parents) using 22 randomly picked seedlings from each cross. In order to process a minimum of 6,600 HT-DNA samples beginning in early February, we automated the PCR reaction preparation step by using a robot station that set up four 384-well PCR reaction plates in 30 minutes for sixteen 96-well HT-DNA sample plates (1,536 samples in total). PCR amplification reactions were then conducted on a thermal cycler equipped with four 384-well heating blocks for durations not to exceed two hours. Using the same robot system, we automated the capillary electrophoresis sample plate preparation for the same number of samples in 30 minutes by mixing 1 ul of amplified DNA products with 9 ul HiDye formamide solution containing the RoxTM 500 size standards. Amplified DNA fragments were separated through capillary electrophoresis on ABI3730 Genetic Analyzer for duration of about 22 hours. The separation process was recorded automatically into run files that could be analyzed by the ABI software GeneMapper. Based on the single marker-PCR and single marker capillary electrophoresis just described, we were able to process 6,624 HT DNA samples from sixty-nine 96-well sample plates with two microsatellite markers in 8 days. The breeders were able to obtain microsatellite genotyping data for 1,646 individuals from the Saccharum spontaneum cytoplasm project where spont clones were used as the female parents and for 2,304 individuals representing 93 commercial crosses before beginning the transplanting season on April 11, 2005. The output of ABI3730 Genetic Analyzer can be maximized to 9,216 HT-DNA samples per day if up to six PCR amplified microsatellite DNA products with three fluorescence labels were pool-plexed and separated through a single capillary electrophoresis run.