Optimization of nuclei isolation for high-molecular weight DNA extraction of wild plants

<p><strong>Optimization of nuclei isolation for high-molecular weight DNA extraction from wild plants</strong></p> <p><strong>Authors</strong></p> <p>Irene Mart&iacute;nez-Garc&iacute;a¹; Martina Degli Alberti²; Aureliano, Bombarely²; Mario Xavier Ruiz-Gonz&aacute;lez¹; Santiago Vilanova¹; Silvia Manrique¹</p> <p>&nbsp;</p> <p><strong>Affiliations</strong></p> <p>1 Instituto Universitario para la Conservaci&oacute;n y Mejora de la Agrodiversidad Valenciana (COMAV), Universidad Polit&eacute;cnica de Valencia (UPV), Camino de Vera s/n 46022, Valencia, Spain) 2 Instituto de Biolog&iacute;a Molecular y Celular de Plantas (IBMCP) Primo-Yufera, Consejo Superior de Investigaciones Cient&iacute;ficas (CSIC)- Universidad Polit&eacute;cnica de Valencia (UPV), Camino de Vera s/n 46022, Valencia, Spain)</p> <p>&nbsp;</p> <p><strong>Abstract</strong></p> <p>Obtaining high-quality, high-molecular-weight (HMW) DNA is mandatory for constructing reference genomes, yet it remains a significant challenge, particularly for non-model plant species. Many plants contain polysaccharides and secondary metabolites such as polyphenols and tannins, which hinder DNA extraction. Exposure to multiple environmental stresses along their lives exacerbate this issue in wild plants collected from nature, as they may synthesize additional compounds that impair DNA extraction.</p> <p>This study investigates the efficacy of various DNA extraction protocols on four recalcitrant plant species&mdash;<em>Pistacia lentiscus</em>,&nbsp;<em>Phyllirea angustifolia</em>,&nbsp;<em>Sarcocornia fruticosa</em>, and&nbsp;<em>Limbarda crithmoides</em>&mdash;each possessing unique traits complicating DNA extraction (e.g., succulence, lignification, coloration). Samples were collected from El Saler (Valencia), and multiple protocols for nuclei isolation and DNA extraction were tested. The results were systematically evaluated to compile a comprehensive best practices guide, aiding researchers in selecting optimal methods tailored to their species of interest. This guide serves as a valuable resource for future endeavours in genome research, facilitating advancements in understanding and conservation efforts for diverse plant species.</p> <p>Funding:&nbsp;</p> <p>This work was supported by Erasmus+ Program (to MD), Programa Mar&iacute;a Zambrano 2021 (to SM and MXRG, UPV Ministerio de Universidades, Plan de Recuperaci&oacute;n, Transformaci&oacute;n y Resiliencia - Financiado por la Uni&oacute;n Europea &ndash; NextGenerationEU) and Catalan Initiative For the Earth Biogenome Project (Call1A-2023 to SM and call 2-2023 to AB and SM) and Primeros Proyectos UPV (PAID-06-22 to MXRG).</p>