Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

Summary

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

Abstract

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 µg DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., > 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Introduction

Powdery mildews are a group of obligate biotrophic fungal plant pathogens that, when taken together, are the largest cause of plant disease worldwide¹. There are over 900 described species of powdery mildew, which have been taxonomically grouped into five tribes within the family Erisyphaceae². Due both to their economic importance and the intimate relationship they develop with their hosts, powdery mildew diseases have been the subject of research for > 100 years. Upon infection, powdery mildews elicit drastic changes in the cellular structure, metabolism and molecular biology of their hosts, to benefit thi....

Protocol

1. Preparation of Fungal Material

1. Growing Powdery Mildew
   1. Grow plants in growth chambers with the following conditions: 22 °C day temperature, 20 °C night temperature, 80% relative humidity, 14-h day-length and a light intensity of 125 µE/m²/s (provided by fluorescent lighting).
   2. Infect cucumber plants (Cucumis sativa variety Bush Champion) with Golovinomyces cichoracearum strain UCSC1 as described^18,29.
2. Harvesting Powdery Mildew Conidia
   1. Harvest conidia from infected plants at 7 - 10 days after inoc....

Results

A representative example of 60ng purified genomic DNA from G. cichoracearum run on an agarose gel using gel electrophoresis and using pulsed-field gel electrophoresis are shown in Figures 1 and 2, respectively. Genomic DNA preparations that pass, marginally pass and fail quality control are represented. Genomic DNA preparations that fully pass quality control are ideal for sequencing using long-read genome sequencing approaches. .......

Discussion

In order to obtain pure, high molecular weight genomic DNA from the obligate biotrophic powdery mildew fungi, a modified version of previously described methods was developed³⁰. The average yield using this optimized protocol is 7 µg DNA per 150 mg conidia, a doubling of the yield obtained with a prior protocol. Also, the average size increased from approximately 20 kbp to over 48.5 kbp. This protocol was optimized in the cucumber-G. cichoracearum system. In order to obtain the best .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MM400 Ball Mill	Retsch	20.745.0001	Or equivalent ball mill
2 mL tubes for ball milling	Sarstedt	72.694.007
5/32" stainless steel milling balls	OPS Diagnostics	GBSS 165-5000-01
Microprocessor controlled 280 Series Water Bath Preset to 65 °C	Thermo Fisher Scientific	2825	Or equivalent water bath
Microprocessor controlled 280 Series Water Bath Preset to 37 °C	Thermo Fisher Scientific	2825	Or equivalent water bath
Eppendorf 5417R refrigerated microcentrifuge	Krakeler Scientific	38-022621807	Or equivalent microcentrifuge
Chlorform:IAA (24:1 v/v)	Sigma-Aldrich	C0549	Hazardous in case of skin contact or inhalation. Wear nitrile gloves, protective eyewear, lab coat and work in chemical hood
Phenol:Chloroform:IAA (25:24:1 v/v)	Thermo Fisher Scientific	15593031	Hazardous in case of skin contact or inhalation. Wear nitrile gloves, protective eyewear, lab coat and work in chemical hood
100% Isopropanol	Sigma-Aldrich	W292907
100% Ethanol	Sigma-Aldrich	34923	Chill to -20 °C prior use
Sodium Acetate	Sigma-Aldrich	S2889
Rnase, Dnase-free (10 mg/mL)	Thermo Fisher Scientific	EN0531
Potassium metabisulfite	Sigma-Aldrich	P2522
Sodium Lauryl Sacroinate	Sigma-Aldrich	L9150
Tris base	Sigma-Aldrich	10708976001
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	EDS
Sodium chloride (NaCl)	Sigma-Aldrich	S9888
Cetyltrimethyl ammonium bromide (CTAB)	Sigma-Aldrich	H6269
Uvex by Honeywell Futura Goggles S345C, Uvextreme	Staples	423263	Or equivalent eyewear
High Five COBALT nitrile gloves, LARGE	Neta Scientific	HFG-N193	Or equivalent gloves
GelRed Nucleic Acid Gel Stain 10,000x in water	Biotium	41003
Ethidium Bromide 10 mg/mL	Biotium	40042	Hazardous in case of skin contact. Wear nitrile gloves and protective eyewear
Agarose Ultrapure Bioreagent	VWR International LLC	JT4063
GeneRuler 1 kb Plus DNA Ladder	Thermo Fisher Scientific	FERSM1332
8 - 48 kb CHEF DNA size standards	Bio-Rad	170-3707
Pippin Prep	Life Technologies Corporation	4472172	Or equivalent pulse-field gel aparatus
Whatman qualitative filter paper, Grade 1	Sigma-Aldrich	WHA1001042	Or equivalent growth chamber
Percival Growth Chamber	Percival	AR66LXC9
NanoDrop 8000 UV-Vis Spectrophotometer	Thermo Fisher Scientific	ND-8000-GL	Or equivalent spectrophotometer
Quant-iT PicoGreen dsDNA Assay Kit	Thermo Fisher Scientific	P11496
TAE Buffer (Tris-acetate-EDTA) (50x)	Thermo Fisher Scientific	B49	Dilute 1/50 with water before use (to 1x)
TE Buffer	Thermo Fisher Scientific	12090015
Tris-Borate-EDTA, 10x Solution (Electrophoresis)	Thermo Fisher Scientific	BP13334	Dilute 1/10 with water before use (to 1x)
Liquid Nitrogen			Liquid nitrogen (-196 °C) is a freezing hazard. It will also expand rapidly upon warming and should not keep in a tightly closed container