Rapid isolation of high molecular weight DNA from marine macroalgae

MARINE ECOLOGY PROGRESS SERIES

Mar. Ecol. Prog. Ser.

Published July 30

Rapid isolation of high molecular weight

DNA

from marine macroalgae

Shivji',

Rogers2,

Stanhope3

'School of Fisheries, University of Washington, Seattle, Washington 98195, USA,

and Tetra Tech. Inc., 11820 Northup Way. Suite 100E. Bellevue, Washington 98005, USA'

'college of Environmental Science and Forestry, State University of New York, Syracuse, New York 13210, USA

3Wayne State University. School of Medicine. MRB-422, 550 East Canfield Avenue, Detroit, Michigan 48201, USA

ABSTRACT: Application of molecular techniques to study marine rnacroalgae is in its infancy, and is

likely to be facilitated by the ability to routinely isolate high quality DNA from these plants. The

generally high polysaccharide and polyphenol content in rnacroalgae, however, often interferes with

the isolation and subsequent enzymatic manipulation of their nucleic acids. We describe the use

CTAB method for the isolation of high molecular weight DNA from marine macroalgae. The method is

rapid, simple, inexpensive, does not require density gradient ultracentrifugation, and has general

applicability to red, brown and green seaweeds. The isolated

DNA

appears sufficiently pure for appli-

cation of most commonly used molecular techniques such as restriction endonuclease digestion,

Southern blot hybridization, cloning, and ampl~fication using the polymerase chain reaction.

The

method was also tested on the marine angiosperm

Zostera

marina

(eelgrass).

INTRODUCTION

Although the application of recombinant DNA tech-

nology to study macroalgae is in its infancy, the use of

these techniques promises to yield biologically inter-

esting, and possibly commercially useful discoveries. A

requirement for the application of such techniques to

study macroalgae is the ability to isolate high mole-

cular weight nucleic acids of sufficient purity for enzy-

matic manipulations. Isolation of high quality nucleic

acids from seaweeds is, however, hampered by the fact

that these plants have cell walls, and often possess

copious amounts of mucilaginous polysaccharides,

polyphenolic compounds, diverse pigments and other

secondary metabolites (McCandless 1981, Ragan

1981). Many of these compounds CO-purify with the

nucleic acids during extraction procedures, and often

interfere with subsequent enzymatic processing of the

nucleic acids for molecular biological studies (Su

Gibor 1988, Parsons et al. 1990, Roe11

Morse 1991).

Although DNA that is sufficiently pure for enzymatic

manipulation has been isolated from some seaweeds,

Present address

Inter-Research 1992

the methods employed involve ultracentrifugation and

are time-consuming, labor-intensive and expensive

(Fain et al. 1988, Goff

Coleman 1988, Parsons et al.

1990, Shivji 1991). Research in systematics and popu-

lation biology of seaweeds often requires analysis of

large sample sizes, and would benefit from inexpen-

sive and more rapid methods of DNA isolation.

We have earlier reported on a CTAB (hexadecyltri-

methylammonium bromide) method to isolate

DNA

from very small amounts of higher plant tissue (Rogers

Bendich 1985). We now describe a modified version

of this method to extract high molecular weight

DNA

from marine macroalgae. The procedure is rapid, eco-

nomical, does not require cesium chloride ultracentri-

fugation, and yields

DNA

of sufficient purity for use in

restriction enzyme analysis, Southern blot hybridiza-

tion, cloning, and the polymerase chain reaction (PCR).

MATERIALS AND METHODS

Cladophoropsis membranaceae

(UWCC

190), Cau-

lerpa vanbosseae (UWCC 179), Acetabularia crenulata

(UWCC 672), Derbesia sp. (UWCC 274), Sphacelaria

198

Mar. Ecol. Prog. Ser.

84.

197-203,

1992

sp. (UWCC 666) and

Griffithsia pacifica

(UWCC

238)

were obtained from the University of Washington,

Seattle, Washington (USA) culture collection. All other

algae (Table 1) and the eelgrass

Zostera

marina

were

collected from intertidal or subtidal areas in either

Puget Sound, Washington, the outer coast of Wash-

ington, or areas in southern British Columbia, Canada.

DNA

isolation

methods.

Plants collected from nature

were wrapped in paper towels moistened with sea-

water and transported to the laboratory on ice. An

effort was made to collect healthy, young plants

that were free of epiphytes. In the laboratory, plants

were rinsed briefly in running tap water and gently

scrubbed with paper towels to remove most of the

surface microbial and epiphytic organisms. Excess

moisture was removed by blotting between paper

towels. The plants were then wrapped in aluminum

foil and frozen at -70

until further use.

For DNA extractions, pieces of algal tissue were

frozen in liquid nitrogen, mixed with a small amount of

dry ice, and ground to a fine powder using a mortar

and pestle. The ground tissue dry ice mixture was

quickly transferred to sterilized

1.5

m1 microcentrifuge

tubes, which were then placed at -70 "C with the tops

open. Tubes were capped after sublimination of the

dry ice, and stored at -70

until needed for

DNA

extraction, at which time an approximately equal

volume of

CTAB isolation buffer

w/v CTAB

(Sigma), 100 mM Tris-HC1 (pH 8.0), 20 mM EDTA,

(w/v)

polyvinylpyrrollidone (MW

OOO),

1.4M NaCl],

pre-heated to

"C in a water bath was added to the

ground algal sample. The tube contents were mixed

thoroughly to ensure the algal tissue was completely

hydrated, and placed at

for

min. The

Table

Susceptibility of algal DNAs to restriction endonuclease digestion. DNAs were digested overnight at

with 30 units

of each enzyme.

complete digestion;

+/-:

variable results (i.e. partial or complete digestion depending on DNA preparation).

nt: enzyme not tested

Restriction

endonuclease

EcoRI PstI Hind111 BamHI

Red

algae

Iridaea cordata (Turner) Bory

Gracilaria sp. Greville

Branchioglossum

sp.

Kylin

Bossiella sp. Silva

Gastroclonium coulteri (Harvey) Kylin

Smithora naiadum (Anderson) Hollenberg

Porphyra fhuretii Dawson

Porphyra torta Krishnamurthy

Porphyra miniata

Agardh

Porphyra nereocystis Anderson

Gigartina exasperata Harvey

Bailey

Griffithsia pacifica Kylin

Rhodyrnenia sp. Greville

Neoagardhiella bailey; Wynne

Taylor

Brown algae

Nereocystis luetkeana Postels

Ruprecht

Macrocystis integrifolia Rory

Costaria costata (C. Agardh) Saunders

Laminaria saccharina (L.) Lamouroux

Alaria rnarginata Postels

Ruprecht

Hedophyllum sessile (C. Agardh) Setchell

Agarum fim briatum Harvey

Sargassum muticum (Yendo) Fensholt

Sphacelaria

sp.

Lyngbye

Petalonia debilis (C. Agardh] Derbes

Solier

Scytoslphon lomentaria (Lyngbye)

Agardh

Focus

sp.

(L.)

Green algae

Acetabularia crenulata Lamouroux

Caulerpa vanbosseae Lamouroux

Cladophoropsis membranaceae Borgesen

Derbesia sp. Solier

Shivji

c3t

DNA

isolation from macroalgae

199

sample was then extracted with an equal volume of

chloroform-isoamyl alcohol (24

1, v: v) by mixing

thoroughly enough to form a complete emulsion. The

mixture was centrifuged at 11 000

in a microfuge

for 30 to 60

to separate the 2 phases. The upper phase

(containing the DNA), was carefully transferred to a

new 1.5 m1 sterilized microfuge tube. One-fifth volume

of a

CTAB solution (5

CTAB, w/v, 0.7M NaCl),

pre-heated to 65 "C, was added and the sample mixed

thoroughly. The sample was then re-extracted with

an equal volume of chloroform: isoamyl alcohol, cen-

trifuged at 11 000

for 30

and the upper phase

transferred to a new 1.5 m1 sterilized microfuge tube.

Between 25 and 50 pg of yeast tRNA were added to the

sample as a carrier to aid in precipitation of the nucleic

acids. Between 1 and 1.5 volumes of CTAB precipita-

tion buffer

CTAB, w/v, 50 mM Tris-HCl (pH 8.0),

10 mM EDTA] was added very slowly (drop by drop)

and the tube contents mixed very gently by swirling.

The tube was placed on dry ice for 5 to 10 min until the

sample became viscous or frozen, and then centrifuged

(11 000

for 3 to 5 min. The supernatant was

removed and the pellet resuspended in 50 to 100 p1 of

warm (65 "C), high-salt TE buffer (10 mM Tns-HC1,

mM EDTA, 1 M NaCI, pH 8.0). Incubating the sample

at 65 "C for 2 to 10 min sometimes facilitated dissolving

the pellet. After the pellet was completely dissolved,

2 volumes of cold 95

ethanol were added and the

sample placed in dry ice for 10 to 15 min or at -20 'C

overnight. The sample was then centrifuged (1 1000

for 10 min, the pellet washed in 70

ethanol, re-

centr~fuged for 1 min, and dried under

vacuum for

to 30 mm. The dried pellet was re-suspended in

300 p1 TE (10 mM Tris-HC1, 1 mM EDTA, pH 8.0) and

prec~pitated for a second time by the addition of half

volume 7.5 M ammonium acetate and 2 volumes cold

'KD

ethanol. The sample was centrifuged

1000

for

min, washed in cold 70

ethanol,

and dried

under vacuum. The final, dry pellet was resuspended

in 20 to 200 ,u1 of TE buffer, depending on its size.

The ilveraye size and concentration of DNA ex-

tracted from the various algal species was estimated by

comparing the migration and fluorescence intensity of

undiyested algal DNA with standardized amounts of

undigested bacteriophage lambda DNA on agarose

gels (Maniatis et al. 1982).

Molecular methods. Restriction endonucleases and

T4-Liyase (Bethesda Research Laboratories, Gaithers-

burg, MD, USA) were used according to the supplier's

specifications. RNase A and RNase T1 were obtained

from Siyma Chemical Co. (St. Louis,

MO,

USA). The

probe used for Southern blot hybridizations was the

plasmid pBD4, which contains the yeast Saccharomyces

cerevisiae 5S, 18S, 5.8s and 25s ribosomal RNA genes

(Bell et al. 1977). The probe was labelled with 32P dCTP

(New England Nuclear, Boston, MA, USA) using the

random primer method of Feinberg

Vogelstein (1983).

Gels were blotted onto Nytran membranes (Schleicher

and Schuell, Keene, NH, USA), according to the manu-

facturer's instructions. DNA blots were hybridized

with the probe at 55

SSC (0.3M sodium chlo-

ride/0.03M sodium citrate), 1

SDS (sodium dodecyl

sulfate), 1M sodium chloride, for 16 to 24 h. After

hybridization, the blots were washed twice in

SSC at

room temperature, followed by two 30 min washes in 2X

SSC,

SDS at 55 "C, and two 30 min washes in

0.1X SSC at room temperature. Autoradiography using

intensifying screens (DuPont Company, Boston, MA)

was carried out at -70 "C for 1 to 5 d.

To determine if the extracted DNA was of sufficient

purity for cloning, DNA from the kelp Alaria marginata

was digested with EcoRI and ligated into the plasmid

vector pIC-7 (Marsh et al. 1984) using the shotgun

method outlined by Maniatis et al. (1982). Twenty-six

white, recombinant Escherichia col1 colonies were

randomly selected from LB-amplcillin-Xgal plates and

screened for cloned algal DNA inserts. The

col1

plasmids were isolated using the boiling lysis method

of Maniatis et al. (1982), digested with EcoRI to liberate

the cloned A. marginata DNA fragments, and sub-

jected to electrophoresis on a 0.8

agarose gel.

The primer designed for PCR amplification consisted

of the randomly chosen sequence GCATCACTGG.

Amplifications were performed in 50 p1 reactions with

1 ng of template DNA, 1 pM primer, 1.25 units of DNA

polymerase (Taq polymerase, Perkin-ElmerKetus),

and 0.2 mM of each dNTP in reaction buffer [50 mM

KC1, 10 mM Tris (pH

8.3), 1.5 mM MgC12, 0.01

BSA]. The reaction mix was overlaid with mineral oil,

denatured for 3 min at 93 "C, and amplified through

cycles in a Biocycles (Bios Corporation) thermal cycler

using the follotving temperature profile: 25 s at

"C,

primer annealing at 40 "C, and

min extension at

72 "C. A final extension for 2 min at 72 "C was per-

formed after completion of the 25 cycles.

RESULTS

DNA

isolation

and

yields

Using the method described, DNA was obtained

from all the species examined. DNA yields were

variable, ranging from approximately 10 to 70 ng per

mg of frozen algal tissue, and depended on the species

as well as the age and overall condition of the tissue.

Older and thicker tissues generally gave lower yields

than younger tissues, although ths relationship seemed

to be reversed in the case of the kelp Nereocystis

luetkeana.

200 Mar Ecol. Prog. Ser 84 197-203, 1992

The DNAs obtained from most of the algae were approximately the same posit~on as undigested

substantially of htgh molecular weight, migrating in lambda DNA [48 kb (kilobases)] in agarose gels

(Fig.

1).

The only exceptions were the green alga

UIva

sp., and the red articulated coralline alga

Bossiella

sp., which consistently yielded degraded DNA. Lyo-

philization of the algal tissue before

DNA

extract~on

also seemed to increase DNA degradation, at least in

the few species tested (Fig. 1). This observation is

consistent with our findings using higher plants and

fungi (data not shown).

Flg.

1 Agarose gel of undigested total DNA isolated from

varlous marine macroalgae and the eelgrass Zostera marina.

molecular size standards [undigested bacteriophage

lambda DNA and 1 kb ladder marker

(BRL)];

lyophilized

tissue; astc,risk: repeated attempts to isolate DNA from these

algae. Ldnes. 1, Irjdaea cordata

(It);

Gracilana sp. (lt),

3, Gastroclonium coulteri (It);

Sal-gassum mutrcum

(It),

Nereocystis luetkeana

(It);

Iridaea col-data, 7, Gracilana

sp.;

'8,

Bossiella sp

9, Gastroclon~um coulteri; 10, Nereocystis

luetkeana, 1.2, Petalonia debilis; '13, Ulva sp.; '14, Ulva sp.,

15, Cladophoropsls membranaceae, 16, Caulerpa van-

bosseae; 17, Acetabulana crenulata, '18, BossieUa sp

;

19, Der-

besia sp., 20, Sphacelaria sp., 21, Smithora naiadum,

22, Zostera manna (eelgrass)

Fig

2 Agarose gel of

restriction

endo-

nuclease d~gested red algal DNAs DNAs In

Lanes 2 to

and 9 to 13 were d~gested with

EcoRI, and

Lanes 7

and 14 to

with

BamHI Lanes 10 and 17 contaln non-

sto~chiomrtr~c amounts of ceslum chloride

gradrcnt pur~fird nuclear, chloroplast and

rnitochondr~al DNAs from Porphyra yezoensis

(see Sh~vji 1991 for methods) Lanes 1, mole-

cul~~r cve~ght markers

Gnffithsia pacifica

and 8, Smlthora naiadum, 4, Rhodymenia

Utility of

DNA

for molecular biological studies

Susceptibility of the various algal DNA samples

to digestion by 4 commonly used restriction endo-

nucleases are shown in Figs.

and Table 1. With

few exceptions (indicated in Table

l),

the DNAs are

sutticiently pure tor restriction endonuclease digestion

and Southern blot hybridizations. The DNA isolated

from the eelgrass

Zostera marina

had a dark brown

pigmentation that did not seem to interfere with diges-

iiur~

ii~e

erlciuiiuciedse

Edlllkii.

uii~e~ erlciu-

nuclease enzymes were tested on this species however.

The yeast nbosomal DNA (rDNA) probe detected

homologous DNA sequences in all the plants tested,

except the green alga

Acetabularia crenulata

(Figs. 4

5).

Ribosomal DNA restriction fragment length poly-

morphisms (RFLPs) were readily detected among

species of the red algal genus

Porphyra

(Fig. 4). Use of

the rDNA probe also revealed RFLPs among individual

plants obtained from different

Nereocyst~s luetkeana

populations separated by short geographic distances.

The north Seattle population differs in its hybridiza-

tion patterns from the more southern Vashon Island

and Tacoma Narrows populations, when using DNAs

Branchioglossum sp

6, Indaea cor-

data. 7, CXraclldna sp

9 and 18, Porphyra

torta conchoc~l~s, 10 and 17, Porphyra

yrloensls cnnchocells 11 and 16, Porphyra

thuretli,

and 15, Porphjra nereocystls,

and 14, Porphyra m~nlata

Shiqi et a1

DNA isolati

Ion from macroalgae 201

Fig 3. Agarose gel of restriction endonuclease digested DNAs

from brown and green algae and the eelgrass Zostera marina

All DNAs digested with EcoRI, except for Z. marina (BamHI)

Lanes. 1, molecular weight markers; 2, Alaria marginata;

3. Petalonia deb~lls; 4, Sphacelaria sp.;

Lam~nana sac-

charina;

Macrocyst~s integrifolla,

Costaria costata;

Nereocystis luetkeana blade;

Nereocystis luetkeana

stipe (lyophilized); 10. Fucus sp.; 11, Caulerpa vanbosseae;

12. Cladophoropsis membranaceae; 13, Derbesia sp.; 14, Aceta-

bularia crenulata;

15,

Zostera marina

Fig 4. Autoradiograph showing hybridization of Saccharo-

myces cerevisiae ribosomal DNA gene probe to red algal

DNAs. All DNA5 digested with EcoRI, except for Lanes

and

(BamHI). Lanes: 1, Gdffithsia pac~f~ca, 2, Smithora naladum,

3, Rhodymenia sp.,

Branchioglossum sp.;

Iridaea cordata;

Gracilarja sp.;

S. naiadurn;

Porphyra torta;

Porphyra

yezoensis; 10, Porphyra thuretii; 11, Porphyra nereocystis;

12,

Poiphyra mniata. Arrowheads in Lane 11 indicate posi-

tions of hybridizing bands evident upon longer exposures

Fig.

Autoradiograph showing hybridization of Saccharo-

myces cerevisiae ribosomal DNA gene probe to DNAs from

brown and green algae and the eelgrass Zostera marina.

Arrowheads indicate the 3 hybridization bands evident with

Nereocystis luetkeana stipe tissue, but absent with blade

tissue. DNA in Lanes 1 to 3 and

to 13 digested w~th EcoRI.

DNA in Lanes 4 to

and 14 digested with BamHI. Lanes:

1, Alaria marginata; 2, Petalonia debilis;

Sphacelana sp.;

N. luetkeana (Vashon Island population);

luetkeana

(Tacoma population),

N luetkeana (N. Seattle population);

luetkeana (N. Seattle population, blade tissue);

N luetkeana (N. Seattle population, stipe tissue);

Fucus

;

10, Caulerpa vanbosseae, 11, Cladophoropsis mem-

branaceae, 12, Derhesia sp., 13, Acetabulana crenulata,

14, Zostera marina

digested with the enzymes BamHl (Fig. 5) and EcoRl

(not shown). Interestingly, rDNA polymorphisms that

may be tissue-specific were also detected in blade and

stipe tissue from this kelp (Fig. 5).

Shotgun cloning of Alaria marginata DNA using the

pIC-7 plasmid vector resulted in the successful cloning

of numerous EcoRI DNA fragments, ranging in size

from approximately 1.5 to

kb (data not shown), indi-

cating that inhibitors of DNA ligase were not present in

the DNA preparation.

Results of PCR amplifications using the arbitrary

sequence primer and DNAs from

species are shown

in Fig.

The results indicate no inhibition of the

amplification reactions by components of the DNA

preparation.

DISCUSSION

The procedure outlined here allows extraction of

high molecular weight DNA from a wide diversity of

marine macroalgae. The method is rapid and eco-

nomical, utilizing only a few microfuge tubes per algal

202

Mar. Ecol. Prog. Ser.

Fig.

Fingerprinting algal

genomes using

PCR

and an

arbitary sequence primer. Lanes:

Porphyra

torfa;

Petalonia

dehzlis;

Nereocyrtic lr~etk~ana;

molecular weight markers

sampie irom beginning to end oi the procedure. Tne

DNA yields obtained appear generally higher than

those obtained with ultracentrifugation methods (e.g.

1 ng mg-l: Fain et al. 1988;

ng mg-': Roe11

Morse

1991).

Despite the wide diversity of potentially enzyme-

inhibiting, secondary compounds found in red, brown

and green seaweeds, the method appears to have

general applicability, yielding DNA of sufficient purity

for enzymatic manipulations used most commonly in

molecular biological studies. Our inability to extract

undegraded DNA from Ulva sp., and the articulated

coralline alga Bossiella sp., even after repeated at-

tempts with both fresh and frozen tissue, may reflect

high nuclease activities in these algae. High levels of

nuclease activity have also been found in leaves of

wheat and maize (Jones

Boffey 1984). DNA degra-

dation may also have occurred in Bossiella sp., due to

the extensive grinding required to break open the

calcified cells. Alternative methods of tissue grinding,

coupled with the addition of higher concentrations of

EDTA and/or extra organic-phase extractions, might

result in isolation of higher quality DNA from such

algae.

The ability to rapidly isolate restrictable and clonable

DNA from macroalgae should facilitate studies on the

genetics, population biology, systematics and evolution

of seaweeds. The utility of the DNAs isolated here for

detecting genetic differen.ces among algal populations

is illustrated by the discovery of RFLPs among Nereo-

cystis luetkeana populations separated by relatively

short geographic distances (i.e. the north Seattle popu-

lation is about

and 64 km north of the Vashon Island

and Tacoma Narrows populations, respectively).

The difference in rDNA hybridization patterns be-

tween blade and stipe tissues of Nereocystis luetkeana

was unexpected, and warrants some comment. These

differences might result from the presence of endo-

phytic algae that occur preferentially on the stipe.

Alternatively, we speculate that such differences could

also occur as a result of underrepresentation, or loss of

some rRNA genes in the blade tissues. Such an occur-

rence has been described in several higher plants

(Grisvard

Tuffet-Anghileri 1980, Cullis 1986, Rogers

Bendich 1987a, b).

Our study also demonstrates the utility of using

yeast ribosomal RNA genes as probes for detecting

RFLPs in all

macroalgal divisions. Plants contain

multiple copies of ribosomal RNA genes, usually

arranged as tandemly repeated units separated by

regions (intergenic spacers) of variable length and

DNA sequence (Rogers

Bendich 1987a). The rapid

evolution of intergenic spacer regions

indicated by

changes in DNA sequence and restriction enzyme

recognition sites, thus providing a readily detectable

source of genetic variation ~poiymorphismsj between

species, populations, and in some cases individual

plants (Appels

Dvorak 1982, Rogers

Bendich

1987a). Because of the highly conserved nature of

eukaryotic ribosomal RNA genes, such genes from

other organisms can be used as probes to detect

RFLPs in the macroalgae. Bhattacharya

Druehl

(1989) and Bhattacharya et al. (1990) have demon-

strated the utilty of a nematode ribosomal DNA

probe to detect genetic differences among popula-

tions of the kelp Costaria costata. Species differences

are readily detectable within the genus Porphyra

when yeast ribosomal genes are used as the probe

(Fig.

4).

Such genetic polymorphisms have been

found to be useful for resolving taxonomic problems

in the phenotypically plastic macroalgae (Goff

Coleman 1988).

The utility of the isolated algal DNAs for use in PCR

studies is demonstrated by the successful amplification

of DNA segments using a primer of arbitrary sequence.

Amplification using short, arbitrary sequence primers

has been shown to be useful for detecting genetic

varlation among higher plant cultivars (Gustavo et al.

1991). This technique may also prove useful for

detecting strain and population differences in the

macroalgae.

In conclusion, the DNA

isolation

method described

yields DNA of sufficient purity for use in a variety of

molecular biological studies, and is of general applica-

bility for isolation of DNA from diverse red, brown, and

green macroalgae. The method also has the advan-

tages of being simple, rapid, inexpensive, and only

requiring a small amount of algal tissue.

Shivji et al.: DNA isolation from macroalgae

203

Acknowledgements. We thank

Geselbracht for assistance

in field collection of the seaweeds,

Duffield for the labora-

tory cultures, and

Stiller for performing the PCR amplifica-

tion~. This work was supported in part by the AK Foundation,

NSERC (Canada), Tetra Tech., Inc., Washington Sea Grant

Program, and the Egtvedt Food Research Fund.

LITERATURE CITED

Appels, R., Dvorak,

(1982). The wheat ribosomal DNA

spacer region: its structure and variation in populations

and among species. Theor. appl. Genet. 63: 337-348

Bell, G.

I.,

DeGennaro,

J.,

Gelfand, D.

H.,

Bishop, R.

J.,

Valenzuela, P., Rutter, W.

(1977). Ribosomal RNA genes

of Saccharomyces cerevisiae.

biol. Chem. 252(22):

8118-8125

Bhattacharya, D., Baillie, D. L., Druehl,

D. (1990). Popula-

tion analysis of the kelp Costana costata (Phaeophyta)

using a polymorphic ribosomal DNA probe. P1. Syst. Evol.

170: 177-191

Bhattacharya. D., Druehl.

D. (1989). Morphological and

DNA sequence variation in the kelp Costaria costata

(Phaeophyta). Mar. Biol. 102: 15-23

Cullis, C. A. (1986). Plant DNA variation and stress. In:

Gustafson, J. P,, Stebbins,

L.. Ayala. F. J. (eds.)

Genetics, development and evolution. Plenum Publishing,

New York,

143-155

Fain,

R., Druehl,

D.,

Baillie, D. L. (1988). Repeat and single

copy sequences are differentially conserved in the evo-

lution of kelp chloroplast DNA.

Phycol. 24: 292-302

Feinberg, A. P., Vogelstein, B. (1983). A technique for radio-

labelling DNA restriction endonuclease fragments to high

specific activity. Analyt. Biochem. 132: 6-13

Goff,

J.,

Coleman. A. W. (1988). The use of plastid DNA

restriction endonuclease patterns in delineating red algal

species and populations.

Phycol. 24: 357-368

Grisvard,

J.,

Tuffet-Anghileri, A. (1980). Variations in the

satellite DNA content of Cucumis

me10

In relation to

dedifferentiation and hormone concentration. Nucleic

Acids Res. 8: 2843-2858

This article was submitted to the editor

Gustavo, C-A., Bassam,

J.,

Gresshoff, P.

(1991). DNA

amplification fingerprinting using very short arbitrary

oligonucleotide primers. Biotechnology 9: 553-557

Jones. M. C., Boffey,

A. (1984). Deoxyribonuclease activl-

ties of wheat seedlings. FEBS Lett. 174: 215-218

Maniatis, T., Fritsch,

F.,

Sambrook, J. (1982). Molecular

cloning: a laboratory manual. Cold Spring Harbor Labora-

tory Press, New York

Marsh,

L.,

Erfle,

M.,

Wykes, E. (1984) The plc plasmid and

phage vectors with versatile cloning sites for recombinant

selection by insertional inactivation. Gene 32: 481-485

McCandless,

(1981). Polysaccharides of the seaweeds.

In: Lobban, C. S., Wynne. M. J. (eds.) The biology of

seaweeds. Blackwell Scientific Publications, Oxford, p.

559-588

Parsons, T

J.,

Maggs,

A., Douglas,

E. (1990). Plastid

DNA restriction analysis links the heteromorphic phases

of an apomictic red algal life history. J. Phycol. 26:

495-500

Ragan,

A. (1981). Chemical constituents of seaweeds. In:

Lobban, C.

S.,

Wynne, M. J. (eds.) The biology of

seaweeds. Blackwell Scientific Publications, Oxford, p.

589-626

Roell, M.

K.,

Morse, D.

(1991). Fractionation of nuclear,

chloroplast, and mitochondrial DNA from Polysiphonia

boldii (Rhodophyte) using a rapid and simple method for

the simultaneous isolation of RNA and DNA. J. Phycol. 27.

299-305

Rogers, S.

O.,

Bendich, A. J. (1985). Extraction of DNA from

milligram amounts of fresh, herbarium and mummified

plant tissues. Plant Mol. Biol. 5. 69-76

Rogers,

O.,

Bendich, A.

(1987a). Ribosomal DNA in

plants: variability in copy number and in the intergenic

spacer. Plant Mol. Biol. 9: 509-520

Rogers.

0..

Bendich, A.

(1987b). Heritability and vari-

ability in ribosomal RNA genes of Vicia faba. Genetics

117: 285-295

Shivji, M.

(1991). Organization of the chloroplast genome in

the red alga Porphyra yezoensis. Curr. Gen. 19: 49-54

Su,

X.,

Gibor, A. (1988). A method for RNA isolation from

marine macroalgae. Analyt. Biochem. 174: 650-657

Manuscript

first received: February

17,

1992

Revised version accepted: June

1992