An efficient micro-method of DNA isolation from mature leaves of four hardwood tree species Acer, Fraxinus, Prunus and Quercus

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An efficient micro-method of DNA isolation from

mature leaves of four hardwood tree species Acer,

Fraxinus, Prunus and Quercus

François Lefort, Gerard. C. Douglas

To cite this version:

Note

An efficient micro-method of DNA

isolation

from

mature

leaves of four hardwood

tree

_species

Acer, Fraxinus,

Prunus

and

_Quercus

François

Lefort*

Gerard. C.

_Douglas

_a

a

Teagasc,

Food and

_{Agriculture Development Authority, Kinsealy}

Research Centre, Malahide Road, Dublin 17, Ireland

b

Laboratory

of Plant

_Physiology

and

_{Biotechnology, Department}

of

_{Biology, University}

of Crete, P.O. Box 208, 71409 Heraklio, Greece

(Received 5

_January

1998;

accepted

1 October 1998)

Abstract - It is difficult to

_purify

DNA from mature tree leaves at the end of the

_growing

season, because of their thick cell wall, and

high

content in

_{polysaccharides, phenolic compounds}

and endonucleases. A

_simple,

fast and efficient method for DNA

_purification

from 100 mg fresh

weight

leaf

_samples

is described here. It has been

_developed

for

_extracting

DNA from mature leaves of _Quercus, Fraxinus Prunus and Acer. The

_protocol

is a modified CTAB

_{(hexadecyltrimethylammonium}

_bromide) method

_including

a

combina-tion of

_{β-mercaptoethanol,}

_{polyvinylpyrrolidone,}

sodium

_dodecyl

sulfate and lithium chloride

_including

short

_{centrifugation}

runs. It is very efficient

yielding

up to 950 _μg

_DNA/g

of fresh

_weight,

even _{when very} mature leaves are

_processed.

The extracted DNA was

used as

_template

to characterise oaks

_by

microsatellite

_analysis.

Its

_efficiency

has been

_compared

to four

_commercially

available kits and two other

_published.

CTAB

_protocols.

The

_protocol

is also

_{inexpensive compared}

to commercial kits. (© Inra/Elsevier, Paris.)

Acer / DNA

_purification

/ Fraxinus / Prunus /

Quercus

Résumé - Une micro-méthode d’extraction d’ADN à

partir

de feuilles matures de _quatre

_espèces

d’arbres forestiers Acer,

Fraxinus,

Prunus et

_Quercus.

Il est difficile de

_purifier

l’ADN de feuilles d’arbres à maturité et

_{spécialement}

à la fin de la

_période

de croissance c’est-à-dire en _{automne, pour}

_plusieurs

_{raisons, telles que de fortes concentrations de}

_{polysaccharides,}

de

_composés

phénoliques

et d’endonucléases ainsi que des

parois

cellulaires

_épaisses.

Nous décrivons une micro-méthode efficace et

_rapide

per-mettant de

_purifier

de l’ADN à

_partir

de 100 mg de

poids

frais de feuilles à maturité des

espèces

d’arbres suivantes :

Quercus

robur,

Q. petraea, Fraxinus excelsior, Prunus avium et Acer

_{pseudoplatanus.}

Le

_protocole

est basé sur l’utilisation de bromure

d’hexadé-cyltriméthylammonium

(CTAB) combiné a

_l’emploi

de

_{β-mercaptoéthanol,}

de

_{polyvinylpyrrolidone,}

de sodium

_dodécyl

sulfate et de chlorure de lithium. Cette micro-méthode _permet d’obtenir

_jusqu’à

950 _μg DNA _{/ g de}

_poids

frais. L’ADN, extrait d’une variété de matériels

_végétaux

(culture in vitro, matériel de serre ou

_prélevés

en _{forêt) par} cette méthode, est de la

_qualité

nécessaire aux

tech-niques

de

_biologie

moléculaire

_{(digestion enzymatique, clonage}

ou

_{amplification}

_{par la réaction de la}

_polymérase

en chaîne (PCR) de marqueurs microsatellites). Son efficacité

_comparée

à celle de _quatre

_protocoles

commercialisés et deux autres

_protocoles

basés sur

l’emploi

de CTAB est

_supérieure

en rendement et

_qualité.

Ce

_protocole

a enfin

_l’avantage

_{d’être bon marché par rapport} aux

pro-tocles commerciaux. (© Inra/Elsevier, Paris.)

Acer / ADN / Fraxinus / Prunus /

_Quercus

*

Correspondence

and

_reprints

1. INTRODUCTION

Plant

_breeding

and

_genetic

identification have until

recently

relied

_only

on

_phenotype

_analysis

_(either

_by

direct

_phenotype

assessment, or

_{by analysis}

of varied

isoenzymes

systems).

Because

_phenotypic

traits are

affected

_by

_many

_factors,

it can be a valuable method to

assess

_polymorphic

variation. The appearance of

molec-ular

_{genetic techniques}

such RFLP

_(restriction

_fragment

length polymorphism)

and then RAPD

_(random

ampli-fied

_polymorphic

_DNA)-PCR

offers a direct access to

the DNA level.

Furthermore,

microsatellites _sequences

are

_becoming

available for an

_increasing

number of

_plant

and tree

_species.

Microsatellite PCR offers a reliable method to assess DNA

_polymorphism

of numerous

indi-viduals within a

_species

and among

_species

of a same

genus.

However, all these molecular

_{techniques require}

the

availability

of DNA in sufficient

_quantity

and of

_good

quality

and

_purity.

Although

several different extraction methods have been

_published

for herbaceous

_plants

and trees

[1- 4, 8,

11-13] and

are even available as commercial

_kits,

proto-cols are either too

_long,

involve excessive volumes of extraction

buffer,

are

_only

efficient for a _range of

_species

or for one _type of

_plant

material. Last but not

least,

most

of the

_protocols

are

_simply

not efficient for difficult material.

_{Many protocols}

_may

_provide

DNA when

pro-cessing

in vitro material or _young leaves of herbaceous

plants

and trees;

however,

they

may be unsuitable for

extracting

DNA from mature or

_dry

leaves. DNA

finger-printing

means that a _great number of

_samples

have to be

processed,

thus the DNA

_purification

_protocol

must be fast and _easy to standardise in order to extract numerous

samples

in a

_workday.

After

_having

tested a number of commercial kits and

published

methods for DNA extraction on the different

trees we are

_working

_with,

we

_developed

a

_protocol

to extract DNA from mature leaves harvested in

_October,

from four hardwood tree

_species:

_{Quercus, Fraxinus,}

Prunus and Acer.

2. MATERIAL AND METHODS

2.1. Plant material

Fresh mature leaves were collected in October from

grafted

elite clones of

_{Quercus robur, Q.}

_petraea, Fraxinus

excelsior,

Prunus avium and Acer

pseudopla-tanus. Plants were _{grown in} the

_glasshouse,

outdoors

(field, arboretum)

and in some cases leaves from in vitro stocks were also used.

2.2. DNA

_purification

One hundred

_milligrams

of fresh

_plant

material

_(leaf)

was

_ground

in

_liquid

_{nitrogen using}

a ceramic mortar

and

_pestle

to

_give

a _green

_powder.

The

_powder

was

transferred to a new 1.5 mL

_{polypropylene}

tube

_using

a

spatula.

At this

_time,

1 mL of DNA extraction buffer

_[50

mM Tris-HCl

_pH

8.0,

20 mM EDTA

_pH

8.0,

0.7 M

NaCl,

0.4 M

LiCl,

1 % w/v CTAB

(hexadecyltrimethy-lammonium

bromide),

1 % w/v PVP

40,

2 % w/v

SDS]

and 10

_μL

of

_{β-mercaptoethanol}

₍₁

% final

concentra-tion)

were added. The mixture was vortexed for 5 s, mixed

_by

2-3 inversion and then incubated for 15 min at

65 °C in a water-bath.

After addition of the

_powdered

leaf material and immersion in the 65 °C water

_bath,

the mixture became clear in a few

seconds,

as soon as the different _reagents

interacted with

_{proteins, phenolic compounds}

and

poly-saccharides.

After

_incubation,

0.5 mL of

chloroform/isoamylalco-hol

_(24:1)

was added to the

tube,

the mixture was

agitat-ed

_thoroughly

until

_making

an emulsion and

_centrifuged

1-5 min in a

_microfuge

at

_{17 000}

_{g (14 000}

_{rpm in} an

ALC

_{microcentrifugette}

4214 rotor

A-12).

The aqueous

phase

was transferred to a new 1.5 mL tube and

cen-trifuged

1 min at 17

₀₀₀

_{g in}

order to

_{pellet possible}

debris. The _supernatant was then transferred to a new

tube and an

_equivalent

volume of

_isopropanol

was added

to the _aqueous solution. The tube was swirled

_gently

and

a white DNA

_precipitate

_appeared.

The tube was then

centrifuged

1 min at 17

_{000 g}

and the

_supernatant

was

withdrawn. The DNA

_pellet

was washed with 1 mL 70 %

_ethanol,

_centrifuged

for 1 min at 17 000 _g.

_Finally

the _supernatant was withdrawn and the

_pellets

allowed to

dry

on the bench for 10 min. DNA

_pellets

were

resus-pended

in 50-100

_μL

of 10 mM Tris-HCl

_pH

_8.0,

1 mM EDTA. As the solution contained RNA and

DNA,

the

protocol

was followed

_by

a RNase

_digestion

to remove

RNAs. RNA

_digestion

was

_performed

_by

_adding

2

_μL

of RNase

_(0.5

_mg

_mL

_-1

₎

_(Boehringer

_{Mannheim, UK)}

and

incubating for

30 min at 37 °C.

Although

the

_resulting

DNA mixture could be

_directly

used after RNase

_digestion

in

_{amplification}

_experiments,

it also could be

_{ultimately purified}

_through

a column

such as the Wizard

_{Clean-up System (Promega}

_Biotec,

Madison, WI, USA).

While

_developing

this

_protocol

we also tested four commercial kits (DNA

Isolator,

Genosys,

UK; Nucleon

Phytopure,

Scotlab, UK;

Snap-O-Sol

Biotexc,

USA and

Xtract,

AMS

_{Biotechnologies,}

_USA)

and two modified CTAB

_protocols

_{[4, 13].}

The Nucleon

_Phytopure

kit and the two

_{published protocols}

were

_specifically

_designed

2.3. DNA

_analysis

DNA concentration was

_given

_by

absorbance

_reading

at 260 nm and 280 nm in a UV

_{spectrophotometer.}

Purity

was estimated

_by

the OD260/OD280 ratio. DNA

quantities

were also confirmed

_by

estimation after ethidi-um bromide

_staining

viewed under UV

_light

on 1 % agarose

gels gel

in 1X Tris Borate EDTA

(TBE)

buffer.

Samples

were run

_along

with four known

_{quantities (0.1,}

0.25,

0.5 and 1

_μg)

of uncut λ DNA

_(Promega,

_USA).

DNA

_quality

was also estimated on the same

_gels.

2.4. DNA

_digestion

DNA was

_{digested by}

Hind III restriction _enzyme

(Stratagene, Cambridge,

UK)

according

to the manufac-turer’s recommendations.

2.5. Microsatellite PCR

_{amplification}

We used

_flanking

_primers

for the microsatellite locus AG110

_(EMBL

accession

_X84082)

which has been described

by

Steinkellner et al.

_[9].

The reaction volume was 50

_μL

and included 20 mM

Tris-Hcl

_pH

8.3,

50 mM

KCl,

1.5 mM

_MgCl

₂

_,

62.5

_μM

dNTPs each

_{(Biofinex, Praroman, Switzerland),}

1

_μM

forward

_primer

_{[5’-ggaggcttccttcaacctact],}

1

_μM

reverse

primer [5’-gatctcttgtgtgctgtattt],

1,5

unit

_AmpliTaq

poly-merase

_{(Perkin Elmer,}

Foster

_City,

CA,

USA)

and

approximately

50 _{ng DNA}

_template.

A 5 min initial denaturation at 94 °C was followed

_by

35

_{cycles (50}

°C for

1 min,

72 °C for 30 s, 92 °C for 1

min)

terminated

_by

an 8 min final extension at 72 °C. PCR

_products

were

checked on a 1 % _agarose

_gel

in 1xTBE and then

analysed

on a denaturant

_sequencing

_{gel (CastAway}

_gel

6 %

_{polyacrylamide)}

run in a

_{CastAway Sequencing}

System (Statagene,

La

_{Jolla, USA).}

Gels were run for 2 h at 1 500

V,

and then silver stained

_according

to a

modified silver

_staining

_protocol

_[10].

Lengths

in base

_pairs

of microsatellites PCR

_products

were estimated

_{by running}

a

_pBR

322

_{plasmid digested}

by

Hae III

_(Biofinex)

and a

_{pUC plasmid digested by}

MspI

(Biofinex)

as base

_{pair length}

ladders.

3. RESULTS AND DISCUSSION

This

_protocol

is a modification of the

_original

CTAB

protocol

of

_Doyle

and

_Doyle

_[2]

and other CTAB meth-ods

_designed

for extraction of DNA from

_plant

material

[1, 3, 4, 7, 8, 12]).

CTAB

_{(1-2 %)}

extraction buffers are

often made up in Tris-HCl

_{(0.05-0.1 mM)}

buffer in a

_pH

range

8.0-9.5,

containing

EDTA

(5-50 mM),

NaCl

(1.25-1.5 M).

They

also often include

polyvinylpyrroli-done

_(PVP

40 000 _up to 360

₀₀₀₎

and a

_variety

of

reduc-tants

_(DTT,

ascorbic

acid,

β-mercaptoethanol).

PVP and reductants are used to avoid the formation of insoluble

complexes

between

_phenolic

substances and DNA. CTAB is a cationic

_detergent

which

_disrupts

membranes

and _may also

_complex

DNA when NaCl concentration is lower than 0.7 M It is also sometimes

_{replaced by}

sodi-um

_dodecyl

_sulfate,

also a cationic

_detergent

which is known to

_complex

with

_proteins

and confer on them a

negative

charge.

We used here both

_detergents

in combi-nation with a decreased NaCl concentration at the limit

at which CTAB

_complexes

with DNA. We

_initially

used very

high

concentrations of LiCl in order obtain a

selec-tive

_{precipitation}

of RNAs. These trials did not achieve the

_expected

results but it was observed that total nucleic

acid

_yields

were increased. After different trials we

_kept

LiCl at a concentration of 0.4 M.

The

_improvement

in DNA

_yield

could

_maybe

be

explained by

the electrostatic interactions between the different

chemicals,

nucleic acids and

_proteins.

This combination of chemicals seems to _prevent more

effi-ciently

formation of insoluble

_complexes

of DNA than the classical combination of one

_detergent,

one reduc-tant, one salt offered

_by

other

_protocols.

We found a final concentration of 1 %

β-mercap-toethanol to be

_optimal

in order to

_keep

the nucleic acids in a non-oxidative environment and to denature endonu-cleases activities.

_Freezing

leaf

_samples

in

_liquid

nitro-gen facilitated cell

breakage by

grinding

since mature

leaves of trees are _very

_tough

and other methods of

homogenisation

that we tested were less successful.

Grinding

in

_{liquid nitrogen provides}

a non-oxidative

environment that _may avoid oxidation of

_phenolic

com-pounds

present in older leaves

_{during homogenisation}

of the tissue. This

_protocol

resulted in white DNA

_pellets

easily

solubilised in TE buffer.

_Figure

1 shows DNA and Hind III

_digested

DNA from mature leaves of each of the four

_species.

RNA was removed

_by

RNase

_digestion.

This

_protocol

_gave

_good

_quality

DNA of a size

some-what above 21 kb. Another

_advantage

of this

_protocol

is the small volume of extraction buffer

_enabling

all _steps

to be

_performed

in a 1.5 mL

_Eppendorf

_{type tube,}

reduc-ing

useless

_handling.

The time

_required

for a

single

extraction was about

40 min from the

_beginning

to the

_resuspension

in TE buffer and it was _easy to _process a

_large

number of

sam-ples

in a

_workday.

The most laborious _step is the

grind-ing

step and if numerous

_samples

are

extracted,

_they

_may

step could be

_optimised

with the use of automated

grinders.

This

_{protocol might}

also be used to obtain RNA if DNase

_digestion

is undertaken.

The

_protocol

described above was used to extract

DNA from 20 clones of

_Quercus,

15 clones of

Fraxinus,

16 clones of Prunus and 15 clones of Acer. For

oak,

the

yield

of DNA

_ranged

from 200 to 950 _{μg DNA per g} fresh

_weight

_{per clone} and for the three other

_species

the

yields ranged

from 350 to 950 _{μg DNA per g} fresh

weight.

OD260/OD280 ratios were 1.70-1.95.

Comparisons

between this modified CTAB

_protocol

and other tested methods are

_given

in table I.

_Among

the

four commercial kits

tested,

only

the Nucleon

_Phytopure

kit

_yielded

DNA but

_only

from in vitro culture and not

from other sources of

_plant

material. The method of Sul

and Korban

_[12]

was

_{originally designed}

for

_extracting

DNA from in vitro cultures of

_apple

tree, Italian stone

pine,

rose and tobacco.

_Applied

to

_plant

material of the four

_species

studied,

it

_{only yielded degraded}

DNA

except for in vitro material.

_Only

the method of

Graham

et al.

_[3]

_yielded

_good

_quality

DNA for all kind of

_plant

material but in very poor amounts.

Microsatellite

_{polymorphisms}

of 17 elite clones of oak obtained

_{by amplification}

of the microsatellite locus

AG 110

[9]

are shown in

_figure

2. As shown in

_figure

2,

either a two-band

_profile

was recorded when the tree was

heterozygous

at this

locus,

or a one-band

_profile

when the tree was

_homozygous

at this locus.

_Unexpectedly

one

tree Dundrum 91 _gave a three-band

_profile,

where a two

band _pattern was

_{expected (figure}

_{2, arrow).}

This could be

_{explained by}

several

_hypotheses:

this tree could be a

triploid,

a trisomican

_aneuploidy,

with one extra chromo-some, or the _pattern obtained could be an artefact of the

method.

In

conclusion,

the

_protocol

described

_provided

DNA of

_{good quality by}

a

_quick

method of extraction from

tree

_species

which have often been a

_{problem regarding}

extraction of their DNA. DNA

_yields

from a 0.1

_g

sam-ple

are sufficient for PCR and RFLP _purposes.

It gave consistent and reliable results for all sources of

plant

material,

that is to _{say from in vitro}

cultures,

from green house and in the field grown trees. DNA

_quality

is suitable for DNA

_{amplification}

as shown

_by

microsatel-lite

_{amplification}

in oak and current work on Vitis

vinifera

(unpublished

results),

and also for molecular

cloning

Lefort et al.

_[6].

This

_protocol

has also been used with

_dry

leaves of oak

[5],

dry

leaves and buds of several Fraxinus

_species

_(Dr

N.

_{Frascaria, ENGREF, Paris,}

France;

pers.

comm.),

seeds of Acacia

mangium

and Acacia

_crassicarpa,

_young and

_expanded

leaves and

chloroplast

enriched fractions of Vitis

_vinifera

from the

Acknowledgements:

Dr Francois Lefort was

support-ed

_by

_European

Union FAIR contract

_N(CT

965004. We

acknowledge

Dr

_Rejane

Streiff from the

_Laboratory

of Genetic

_Improvement

of Trees, Inra

Cestas, Bordeaux,

France,

for her

_help

in

_analysing

microsatellite

_profiles.

REFERENCES

[1] Chen D.M., de

_Filippis

L.M.,

Application

of

_genomic

DNA and RAPD-PCR in

_{genetic analysis}

and

_{fingerprinting}

of various

_species

of

_woody

_trees, Austr. For. 59 (1996) 46-55.

[2]

Doyle

J.J.,

Doyle

J.L., A

_rapid

DNA isolation

_procedure

for small

_quantities

of leaf tissue,

Phytochem.

Bull. 19 (1987)

11-15.

[3] Graham _J., Mc Nicol R.,

Greig

K., van der Ven V., Identification of red

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[4] Howland _D.E., Oliver R.P.,

Davy

A.J., A method of extraction of DNA from birch, Plant Molec. Biol.

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9

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[5] Lefort _F.,

_Douglas

_G.C., Occurrence and detection of

triploid

oaks

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microsatellite

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in:

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G.C, Lefort F. (Eds.),

Strategies

for

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of Forest

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Proceedings

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[6] Lefort F., Edwards K.,

Douglas

G.C., Identification of microsatellite

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of ash Fraxinus _excelsior, Dendrome 4

(1997) 4.

[7]

Milligan,

B.G., Plant DNA isolation, in: Hoexel A.R.,

(Ed.), Molecular Genetic

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A Practical

Approach.

The Practical

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Series, IRL Press, Oxford

University

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[8]

Murray

M.G.,

Thompson

W.F.,

Rapid

isolation of

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molecular

_{weight plant}

DNA, Nucleic Acids Res. 8 ₍₁₉₈₀₎ 4321-4325.

[9] Steinkellner H., Fluch S., Turetschek E., Lexer C.,

Streiff R., Kremer A.,

Burg

K., Glössl J., Identification and characterization of (GA/CT)n-microsatellite loci from _Quercus Petraea, Plant Molec. Biol. 33 (1997) 1093-1096.

[10] Streiff R., Lefort F., A

_protocol

for

_higher

contrasted DNA silver

_{staining, CastAway}

Times 6 (1997) 2.

[11] Stewart N., Via _L., A

_rapid

CTAB DNA isolation

tech-nique

useful for

_{fingerprinting}

and other PCR

_{applications,}

BioTechniques

14 (1993) 748-749.

[12] Sul I.W., Korban S.S., A

_highly

efficient method for

isolating genomic

DNA from

plant

tissues, Plant Tissue Cult. Biotech. 2 (1996) 113-116.

[13]

Wagner

D.B., Furnier _G.R.,

_{Saghai-Maroof}

_M.A., Williams S.M., Dancik B.P., Allard R.W.,

Chloroplast

DNA