Extraction of High Molecular Weight DNA from Fungal Rust Spores for Long Read Sequencing

Methods Mol Biol. 2017;1659:49-57. doi: 10.1007/978-1-4939-7249-4_5.

Abstract

Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information. Current long-read sequencing technologies hold great promise to provide more contiguous and haplotype-phased genome assemblies. Long reads are able to span repetitive regions and phase structural differences between the haplomes. This increased genome resolution enables the identification of complex loci and the study of genome evolution beyond simple nucleotide polymorphisms. Long-read technologies require pure high molecular weight DNA as an input for sequencing. Here, we describe a DNA extraction protocol for rust spores that yields pure double-stranded DNA molecules with molecular weight of >50 kilo-base pairs (kbp). The isolated DNA is of sufficient purity for PacBio long-read sequencing, but may require additional purification for other sequencing technologies such as Nanopore and 10× Genomics.

Keywords: 10× Genomics; HMW DNA; Long-read sequencing; Nanopore; PacBio; Puccinia graminis f. sp. tritici; Puccinia striiformis f. sp. tritici; Rust fungi.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basidiomycota / chemistry
  • Basidiomycota / genetics*
  • Chemical Fractionation / methods*
  • DNA, Fungal / chemistry
  • DNA, Fungal / genetics
  • DNA, Fungal / isolation & purification*
  • Molecular Weight
  • Plant Diseases / microbiology*
  • Sequence Analysis, DNA / methods
  • Spores, Fungal / chemistry
  • Spores, Fungal / genetics*
  • Triticum / microbiology*

Substances

  • DNA, Fungal