Authors: Rosanne E Casu Alexandra Selivanova Jai M Perroux
Publish Date: 2011/09/28
Volume: 31, Issue: 1, Pages: 167-177
Abstract
Accurate and timely detection of transgene copy number in sugarcane is currently hampered by the requirement to use Southern blotting needing relatively large amounts of genomic DNA and therefore the continued growth and maintenance of bulky plants in containment glasshouses In addition the sugarcane genome is both polyploid and aneuploid complicating the identification of appropriate genes for use as references in the development of a highthroughput method Using bioinformatic techniques followed by in vitro testing two genes that appear to occur once per base genome of sugarcane were identified Using these genes as reference genes a highthroughput assay employing RTqPCR was developed and tested using a group of sugarcane plants that contained unknown numbers of copies of the nptII gene encoding kanamycin resistance Using this assay transgene copy numbers from 3 to more than 50 were identified In comparison Southern blotting accurately identified the number of transgene copies for one line and by inference for another but was not able to provide an accurate estimation for transgenic lines containing numerous copies of the nptII gene Using the reference genes identified in this study a highthroughput assay for the determination of transgene copy number was developed and tested for sugarcane This method requires much less input DNA can be performed much earlier in the production of transgenic sugarcane plants and allows much more efficient assessment of numerous potentially transgenic lines than Southern blottingThe authors would like to thank Janine Nielsen for expert technical advice and assistance and Drs Anne Rae Graham Bonnett Danny Llewellyn and Linda Tabe for critical reading of the manuscript AS was supported by a CSIRO Plant Industry Summer Studentship
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