Abstract Plant genome sequencing based on long reads has increasingly been applied also to tree species in recent years. A crucial step in these genome projects is the successful extraction of high-molecular-weight DNA in high quality and sufficient quantity, which is imperative for long-read sequencing. The extraction of high-molecular-weight DNA in trees has been limited and difficult to conduct. To achieve successful extraction, modification of the protocol for tree species is necessary. Here, we present a protocol for high-molecular-weight DNA extraction from the broadleaved tree species Fraxinus excelsior and from the conifer species Taxus baccata . The protocol is based on nuclei isolation and is divided into two main steps, i.e. nuclei separation based on the nuclei isolation buffer and DNA extraction using the Nanobind® plant nuclei kit by Pacific Biosciences (PacBio). The protocol can be applied to different tree species to obtain high-molecular-weight DNA in high quality, which can be used for Oxford Nanopore Technologies (ONT) and PacBio sequencing. ONT sequencing using four high-molecular-weight DNA preparations from Fraxinus excel-sior resulted in different read length N50 values (12.91-38.19 kb) and total base output (5.81-23.17 Gb), emphasizing the complex nature of the sequencing pipeline of high-molecular-weight DNA. PacBio HiFi sequencing of Taxus baccata produced circular consensus sequencing reads with an average length of 12.9 kb and an N50 of 13.59 kb. Altogether, this study presents a protocol to obtain high-molecular-weight DNA from different challenging tree species and discusses several important points that can be considered when adapting the protocol to additional species.

Melina Krautwurst

Annika Eikhof

Sylke Winkler

Daniel Bross

Birgit Kersten

Niels A. Müller

Silvae genetica/Silvae Genetica

SILVAE GENETICA 是一本国际同行评审期刊，在理论和应用森林遗传学和树木育种的所有领域拥有超过 65 年的传统和经验。它继续由 W. LANGNER 于 1951 年创立的“ZEITSCHRIFT FÜR FORSTGENETIK UND FORSTPFLANZÜCHTUNG”（森林遗传学和林木育种杂志）。

Silvae Genetica is an international peer-reviewed journal with over 65 years of tradition and experience in all areas of theoretical and applied forest genetics and tree breeding. It continues the legacy of the 'Zeitschrift für Forstgenetik und Forstpflanzenzüchtung' founded by W. Langner in 1951.

《Silvae Genetica》是一本国际同行评审期刊，拥有超过65年的历史，专注于理论和应用森林遗传学及树木育种的各个领域。该期刊延续了W. Langner于1951年创办的《Zeitschrift für Forstgenetik und Forstpflanzenzüchtung》（森林遗传学和林木育种杂志）的传统与经验。

Category	Quartile
农林科学	4
农林科学, 林学	4
农林科学, 遗传学	4

High-molecular-weight DNA extraction for broadleaved and conifer tree species

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Journal Info

Category	Quartile
GENETICS & HEREDITY	4