IDENTIFICATION AND ANALYSIS OF FLANKING SEQUENCES FROM TRANSGENIC SUGARCANE WITH MULTIPLE INSERTS BY USE OF TARGETED SEQUENCE CAPTURE AND NGS TECHNOLOGIES.

ENRIQUE, RAMÓN; IBARRA LACLETTE, E.; NOGUERA, A.; WELIN, B.; CASTAGNARO, ATILIO

Workshop; ISSCT 11th GERMPLASM AND BREEDING WORKSHOP/ 8th MOLECULAR BIOLOGY WORKSHOP 2015; 2015

ISSCT

A prerequisite for commercial release of a transgeniccrop in Argentina and other countries in the world is the identification andsequence analysis of the transgene(s) insertion site in the plant genome. Whenanalyzing a glyphosate-tolerant transgenic sugarcane variety RA 87-3 pre-selectedfor commercial release, by Southern blot assays a high number of transgenes inserts(nine) was found in the transgenic line. Using an approach of targeted sequenceenrichment coupled with next generation sequencing (NGS) technology we wereable to identify and subsequently analyze all flanking sequences to these multipletransgene insertions in the sugarcane genome. First, a library of genomicregions containing foreign DNA was isolated from the complex sugarcane genomeusing the Targeted Sequence Enrichment system by NimbleGen (Roche). The aforementionedlibrary was thereafter sequenced using the Roche 454 GS FLX Titanium XL+platform. Finally bioinformatics analyses were carried out to assess theputative flanking sequences to the corresponding insert. Out of 29 candidatecontigs, 18 were confirmed as junction sequences by means of PCR reactions andSanger sequencing. The outlined methodology using targeted sequence enrichmentcoupled with NGS and bioinformatic analyses, represents a novel and highlyefficient strategy for the characterization of flanking sequences correspondingto inserts of a transgene in a large and complex genome such as sugarcane.