Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Summary

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Abstract

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

Introduction

Genome sequencing is a powerful tool to dissect the underlying genetic basis of important plant traits. Most genome-sequencing studies focus on the nuclear genome content, as the majority of genes are located in the nucleus. However, organellar genomes, including the mitochondria (across eukaryotes) and plastids (in plants; the specialized form, the chloroplast, works in photosynthesis) contribute significant genetic information essential to organismal development, stress response, and overall fitness¹. Organellar genomes are typically included in total DNA extractions intended for nuclear genome sequencing, although methods to reduce organelle....

Protocol

1. Generation of Plant Materials for Organellar Isolation and DNA Extraction

1. Standard growth of wheat seedlings
   1. Plant seeds in vermiculite in small, square pots with 4 - 6 seeds per corner. Transfer to a greenhouse or growth chamber with a 16 h light cycle, 23 ºC day/18 ºC night.
   2. Water the plants each day. Fertilize the plants with ¼ teaspoon of granular 20-20-20 N-P-K fertilizer upon germination and at 7 days post-germination.
2. Alternative etiolation of wheat seedlings
   1. Follow step 1.1, but place the pots in a dark growth chamber, 23 °C for 16 h/....

Results

The protocols presented in this manuscript describe two distinct methods to enrich for organellar DNA from plant tissue. The conditions presented here reflect optimization for wheat tissue. A comparison of key steps in the protocols, required tissue input, and DNA output are described in Figure 1. The steps of the DC protocol we tested follow similar conditions to those described previously (Figure 1A). Harvested tissue must be p.......

Discussion

To date, most organellar sequencing studies center on traditional DC methods to enrich for specific DNA. Methods to isolate organelles from diverse plants have been described, including moss⁴⁰; monocots such as wheat¹⁵ and oats¹¹; and dicots such as arabidopsis¹¹, sunflower¹⁷, and rapeseed¹⁴. Most protocols focus on leaf tissue^13,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-mercaptoethanol (beta-mercaptoethanol; BME)	Sigma Aldrich	M3148-100ml
2-propanol (Isopropyl alcohol/isopropanol), bioreagent	Sigma Aldrich	I9516
agarose, Bio-Rad Cetified Megabase agarose	Bio-Rad	1613108
analytical balance	Mettler Toledo	AB54-S
balance	Mettler Toledo	PB1502-S
bovine serum albumin (BSA)	Sigma Aldrich	B4287-25G
Ceramic grinding cylinders, 3/8in x 7/8in	SPEX SamplePrep	2183
Cryogenic Blocks compatible with tissue homogenizer for holding 50 mL tubes	SPEX SamplePrep	2664
DNaseI	Sigma	DN25
ethanol, absolute	Decon Laboratories	2716
Ethylenediamine Tetraacetic Acid (EDTA), 0.5 M Solution, pH 8.0	Fisher	BP2482-500
gel imaging system
gel stain			Such as GelRed or Ethidium Bromide
grinding pestle, wide tip for 2 mL conical tubes
Guanidine-HCl, 8 M solution	ThermoFisher	24115
LightCycler 480 SYBR Green I Master	Roche	4707516001
liquid nitrogen
Lysing enzymes from Trichoderma harzianum	Sigma	L1412
Magnesium Chloride	G Bioscience	24115
magnetic rack	ThermoFisher	A13346
microcentrifuge tubes, LoBind 1.5 mL	Eppendorf	22431021
microcentrifuge tubes, standard nuclease-free 1.5 mL	Eppendorf
microcentrifuge, refrigerated	Sorvall	Legend X1R	Or equivalent product, must be capable of reaching at least 18,000 x g with rotors for 50 mL tubes, Oak Ridge tubes, and 1.5 mL tubes
microcentrifuge, room temperature	Eppendorf	5424	Or equivalent product, must be capable of reaching at least 18,000 x g with rotor for 1.5 mL and 2 mL microcentrifuge tubes
Microcon DNA Fast Flow Centrifugal Filter Units	EMD Millipore	MRCFOR100
Miracloth, 1 square per sample cut to fit funnel	EMD Millipore	475855
NEBNext Microbiome DNA Enrichment Kit	New England Biolabs	E2612L
parafilm	Parafilm M	PM992
plastic pots and trays
polyvinylpyrrolidone (PVP)	Fisher	BP431-100
Proteinase K	Qiagen	19131
Pulsed-Field Gel Electrophoresis rig (e.g. CHEF DR III)	Bio-Rad	1703697
purification beads, Agencourt AMpureXP beads	Beckman Coulter	A63881
QIAamp DNA Mini Kit	Qiagen	51304
Qiagen 20/g Genomic Tip DNA Extraction Kit	Qiagen	10223
Qiagen Buffer EB (elution buffer)	Qiagen	19086
Qiagen DNA Extraction Buffer Set	Qiagen	19060
QiaRack	Qiagen	19015
qPCR machine (e.g. Roche Light Cycler 480)	Roche
qPCR plate sealing film	Roche	4729757001
qPCR plate, 96 well plate	Roche	4729692001
Qubit assay tubes	Life Technologies	Q32856
Qubit Broad Spectrum assay kit	Life Technologies	Q32850
Qubit High Sensitivity assay kit	Life Technologies	Q32851
RNaseA	Qiagen	19101
Serological pipettes (20 mL) and pipet-aid	Fisher	13-678-11
Small funnels, 1 per sample
Sodium Chloride	Ambion	AM9759
Soft paintbrush, 2 per sample
SPEX SamplePrep 2010 Geno/Grinder or another type of tissue homogenizer	SPEX SamplePrep		Or another comparable tissue homogenizer. If you do not have access to a tissue homogenizer, then grinding in a pre-chilled mortar and pestle will suffice (see protocol for details). However, a homogenizer will give more consistent results and total homogenization time is reduced.
Sucrose	Omnipure	8550
TBE
thermomixer
Tris	Sigma	T2819-100ml
Triton X-100	Promega	H5142
tube rotater
tubes, 50 mL conical polypropylene	Corning	352070
tubes, 50 mL high-speed polypropylene	ThermoScientific/Nalgene	3119-0050	e.g. Nalgene Oakridge tubes or equivalent
vermiculite
water bath
water, sterile and certified Nuclease-free	Fisher	1481
water, sterile milliQ