ITA-NOA   24624
INSTITUTO DE TECNOLOGIA AGROINDUSTRIAL DEL NOROESTE ARGENTINO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Identification and Analysis of flanking sequences from transgenic sugarcane with multiple inserts by use of targeted sequence capture and NGS technologies.
Autor/es:
RAMÓN ENRIQUE; ENRIQUE IBARRA-LACLETTE; VERENICE RAMIREZ; ALDO NOGUERA; BJÖRN WELIN; ATILIO PEDRO CASTAGNARO
Reunión:
Workshop; 11th Germoplasm and breeding- 8th molecular biology ISSCT workshop; 2015
Resumen:
A prerequisite for commercial release of a transgenic crop in Argentina and other countries in the world is the identification and sequence analysis of the transgene(s) insertion site in the plant genome. When analyzing a glyphosate-tolerant transgenic sugarcane variety RA 87-3 pre-selected for commercial release, by Southern blot assays a high number of transgenes inserts (nine) was found in the transgenic line. Using an approach of targeted sequence enrichment coupled with next generation sequencing (NGS) technology we were able to identify and subsequently analyze all flanking sequences to these multiple transgene insertions in the sugarcane genome. First, a library of genomic regions containing foreign DNA was isolated from the complex sugarcane genome using the Targeted Sequence Enrichment system by NimbleGen (Roche). The aforementioned library was thereafter sequenced using the Roche 454 GS FLX Titanium XL+ platform. Finally bioinformatics analyses were carried out to assess the putative flanking sequences to the corresponding insert. Out of 29 candidate contigs, 18 were confirmed as junction sequences by means of PCR reactions and Sanger sequencing. The outlined methodology using targeted sequence enrichment coupled with NGS and bioinformatic analyses, represents a novel and highly efficient strategy for the characterization of flanking sequences corresponding to inserts of a transgene in a large and complex genome such as sugarcane.