An efficient and rapid protocol for plant nuclear DNA preparation suitable for next generation sequencing methods

Gregory Carrier (1, 2) , Sylvain Santoni (3) , Marguerite Rodier-Goud (4) , Aurelie Canaguier (5) , Alexandre de Kochko (3) , Christine Dubreuil-Tranchand (3) , Patrice This (3) , Jean-Michel Boursiquot (3) , Loic Le Cunff (1, 2)

1 UMR AGAP - Amélioration génétique et adaptation des plantes méditerranéennes et tropicales
2 UMT Géno-vigne® - Géno-vigne®
3 UMR DIAPC - Diversité et adaptation des plantes cultivées
4 UMR DAP - Développement et amélioration des plantes
5 URGV - Unité de recherche en génomique végétale

Premise of the study : In this study, we developed a nuclear DNA extraction protocol for Next Generation Sequencers (NGS). Methods and Results : We applied this extraction method to grapevines and coffee trees, which are known to contain many secondary metabolites. The nuclear DNA obtained was sequenced by the 454/GS-FLX method. We obtained excellent results, with less than 4% cytoplasmic DNA, in a similar way to a BAC (Bacterial Artificial Chromosome)-building protocol. We also compared our protocol with a classic DNA extraction using specific cytoplasmic DNA amplification. Results showed a lower cytoplasmic DNA contamination with the new protocol. Conclusions : The method presented here is fast and economical. The DNA obtained is of high quality, with a low level of cytoplasmic DNA contamination, and very efficient for the construction of sequencing libraries.

Mots clés: séquenceurs de nouvelle génération; dna; extraction; fruit arboriculture; fruit tree; coffee tree; grapevine; extraction de l'ADN nucléaire des plantes; next generation sequencers; nuclear plant DNA extraction; nuclei isolation; isolement des noyaux

Mots clés: vigne; vitis vinifera; caféier; adn; arboriculture fruitière; viticulture; gène; arbre fruitier; séquençage; coffea

Domaines: Botanique

Gregory Carrier, Sylvain Santoni, Marguerite Rodier-Goud, Aurelie Canaguier, Alexandre de Kochko, et al.. An efficient and rapid protocol for plant nuclear DNA preparation suitable for next generation sequencing methods. American Journal of Botany, 2011, 98 (1), pp.e13-e15. ⟨10.3732/ajb.1000371⟩. ⟨hal-01506007⟩