Detection of genomic changes in transgenic Bt rice populations through genetic fingerprinting using amplified fragment length polymorphism (AFLP)

Abstract

A highly precise molecular marker technique, Amplified Fragment Length Polymorphism (AFLP) was used to characterize the changes in genomic structure occurred due to introduction of foreign gene and/ or due to the induced tissue culture stress during the development of various transgenic Bt rice populations. The transgenic rice plants having complex to simple Bt gene and selectable marker gene organizations were selected from the six insect resistant Bt rice lines. Our results clearly demonstrate that integration pattern, extent of rearrangement of foreign DNA and method of transformation may influence the genomic changes in transgenic rice populations. Eleven of fourteen AFLP primer combinations tested, generated distinct scorable banding pattern, which were further used in this study. Three primer combinations E-TA / M-CTT (IRRI-NPT), E-AG / M-CAC (Tulasi) and E-AA / M-CAG (IR 68899B) produced only monomorphic bands in all the transgenic and control rice plants .A percentage of 0.61% of 430 million bases of haploid rice genome were examined by the use of 11 AFLP primer combinations employed in this study. The DNA fingerprints generated by AFLP analysis, of each Bt rice population was compared with their respective non-transgenic control and was found to be > 97% similar. This indicated that the introduction of Bt gene into the genome of six rice varieties showed few genomic changes. The comparison shows that fewer changes were observed among the transgenic plants developed by Agrobacterium infection than that of developed through particle bombardment. Transgene integration pattern and their copy number were associated with the extent of genomic changes observed in the transgenic Bt rice varieties. AFLP fingerprints of the six transgenic rice varieties evidenced few, but consistent polymorphic bands between the transgenic individuals with different PI values. Relationships among the transgenic populations with their control rice plants were expressed in the form of dendrograms.