Utilization of Next Generation Sequecing for Analyzing Transgenic Insertions in Plum: A Risk Assessment Study

When utilizing transgenic plants it is useful to know how many copies of the genes were inserted and the locations of these insertions in the genome.  This information can provide important insights for the interpretation of transgene expression and the resulting phenotype.  Traditionally, these questions were answered with DNA blot analyses for the copy number, and progeny analyses for both the copy number and for physical mapping.  Other techniques have been used such as inverted PCR and sequencing of specific fragments of DNA.  Next Generation sequencing presents a powerful tool for transgene insertion analysis. Transgene characterization of ’HoneySweet’ plum (Prunus domestica)had been previously obtained from DNA blot analyses, PCR approaches, small and large fragment sequencing and progeny segregation ratios. We are using ‘HoneySweet’ to compare Next Generation sequencing with traditional analytical methods already used for the detection of transgene copy number and insertion location in ‘HoneySweet’.    We are also incorporating Next Generation sequencing for marker development and gene identification for use with traditional breeding, genetic engineering and for accelerated ‘FasTrack’ breeding, a method that utilizes transgenic early flowering plums for reducing the generation time.