TECHNICAL NOTE

Microsatellite markers for the giant kelp Macrocystis pyrifera

Filipe Alberto Æ Allison Whitmer Æ Nelson C. Coelho ÆMackenzie Zippay Æ Elena Varela-Alvarez ÆPeter T. Raimondi Æ Daniel C. Reed Æ Ester A. Serrao

Received: 15 January 2009 / Accepted: 4 February 2009 / Published online: 20 February 2009

� Springer Science+Business Media B.V. 2009

Abstract We report the isolation and characterization of

16 microsatellite loci to study the population genetics

of the giant kelp, Macrocystis pyrifera. Markers were

obtained by screening a genomic library enriched for

microsatellite motifs. Of the 37 primer pairs defined, 16

amplified clean polymorphic microsatellites and are

described. These loci identified a number of alleles ranging

from three to forty (mean = 16.5, and gene diversity

ranging from 0.469 to 0.930 (mean = 0.774). The isolation

and characterization of these highly polymorphic markers

will greatly benefit much needed studies on the molecular

ecology of this important macroalga.

Keywords Macrocystis pyrifera � Giant kelp �Microsatellites � Macroalga � Genetic diversity

The giant kelp Macrocystis pyrifera is the world’s largest

benthic organism, and the most widely distributed kelp in

the seas (Graham et al. 2007). Forests of this brown alga

sustain one of the most productive, and dynamic ecosys-

tems on the planet, forming a complex food-web system

that is dependent on giant kelp itself as source of energy

and shelter (Graham 2004). Although much is known about

the ecology and population biology of giant kelp, the

influence of its reproductive and dispersal biology on

genetic structure across multiple spatial scales is poorly

understood. Here we report on the isolation and charac-

terization of hyper-variable microsatellite markers that

should prove extremely useful for addressing this knowl-

edge gap.

Genomic DNA was isolated using an initial nuclei iso-

lation (Varela-Alvarez et al. 2006) followed by standard

cetyltrimethyl ammonium bromide (CTAB) extraction

procedures, and digested with AfaI (RsaI) (GE Healthcare

Europe). Total digested DNA was purified and ligated to

annealed AfaI adaptors (AdapF: 50-TCTTGCTTACGCGT

GGACTA-30 and AdapR: 50-TAGTCCACGCGTAAGCA

AGAGCACA-30). The enrichment procedure followed the

protocol from Billote et al. (1999) which used streptavidin-

coated magnetic particles and biotinylated probes (Magne-

sphere, Promega, Madison, WI). We used a 50-biotinylated

(CT)15 probe, with a 30-dideoxyC end, to avoid the probe to

work as a primer in the following PCR step (Koblizkova

et al. 1998). The enriched ssDNA was amplified by PCR

using the AdaptF as a primer to recover double strand DNA.

This was ligated into pGEM-T Easy vector (Promega,

Madison, WI) and transformed into DH5a competent cells.

A total of 768 positive clones were transferred to

microplates containing 150 ll of LB/Ampicilin solution,

incubated (4 h, 37�C), diluted 59 in ultrapure water

(Sigma), and heated (10 min) to provoke cell lysis. This

solution was used as DNA template for PCR with standard

SP6 and T7 primer amplification, and the products were

transferred to Hybond N? nylon membranes (Amersham)

and hybridized with a 32P radiolabeled (CT)15 probe. Insert

sizes were estimated by agarose gel electrophoresis of the

F. Alberto (&) � N. C. Coelho � E. Varela-Alvarez �E. A. Serrao

CCMAR, CIMAR-Laboratorio Associado, University

of Algarve, Campus de Gambelas, Faro, Portugal

e-mail: falberto@ualg.pt

A. Whitmer � M. Zippay � D. C. Reed

Marine Science Institute, University of California,

Santa Barbara, CA 93111, USA

P. T. Raimondi

Department of Ecology and Evolutionary Biology, Center

for Ocean Health, Long Marine Lab, University of California,

100 Shaffer Road, Santa Cruz, CA 95060, USA

123

Conserv Genet (2009) 10:1915–1917

DOI 10.1007/s10592-009-9853-9

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1916 Conserv Genet (2009) 10:1915–1917

123

PCR product. A total of 60 clones were selected to be

sequenced based on their size and hybridization result.

Sixteen primer pairs were drawn with Primer 3 (Rozen and

Skaletsky 2000) from the ones that showed sufficiently large

flanking regions and long microsatellite regions. We also

tested an additional group of 21 primer pairs, isolated from a

library produced by Genetic Identification Services (primers

drawn using Designer PCR version 1.03, research Genetics,

Inc.).

Blade tissue from 180 individuals was collected from a

300 m 9 15 m area in the kelp bed off Carpinteria, Cali-

fornia to test for loci polymorphism. PCR reactions in

15 ll contained &20 ng of DNA, 0.3–0.6 lM of each

primer (Table 1) 60 lM of DNTPs, 2.0 mM of MgCl2 (see

Table 1 for locus optimisations), 1 ll 109 PCR buffer

(200 mM Tris-HCL (pH 8.4), 500 mM KCl) and 0.5 U Taq

DNA polymerase (Invitrogen, Life Technologies). Cycling

conditions consisted of an initial denaturing step of 4 min

at 94�C, followed by 24 cycles of ‘‘touchdown’’ PCR

consisting of 30 s at 94�C, 30 s at 64�C (reduced by 0.5�C

each subsequent cycle), and 30 s at 72�C, 10 additional

cycles consisting of 30 s at 94�C, 30 s at 52�C and 40 s at

72�C, and a final elongation step at 72�C for 10 min. All

PCR reactions were performed on a GeneAmp 9700 ther-

mocycler (PE Applied Biosystems).

Fragment length was analysed on an ABI PRISM 3130

DNA analyser (Applied Biosystems) using the GeneScan-500

LIZ standard. Raw allele sizes were scored with STRAND

(http://www.vgl.ucdavis.edu/informatics/STRand/), binned

using the R package msatAllele (Alberto 2009), and manu-

ally reviewed for ambiguities. GENEPOP (Raymond and

Rousset 1995) was used to estimate linkage disequilibrium

and conformity to the Hardy–Weinberg equilibrium.

A total of 16 loci were retained after amplification and

polymorphism screening. The levels of genetic diversity

were high; the number of alleles ranged from 3 to 34, and

gene diversity from 0.469 to 0.930 with a mean value of

0.774 (Table 1). A total of 32 pairs of loci out of 120 tests

had significant linkage disequilibrium after Bonferoni

correction was applied. Ten loci showed significant het-

erozygote deficiency (Table 1), probably caused by null

allele presence: Using MICROCHECKER software (Van

Oosterhout et al. 2004) we estimated that 8 loci were

affected by the presence of null alleles. The levels of

genetic diversity revealed by these loci are much higher

then what was previously obtained with less variable ITS

markers (Coyer et al. 2001). Our isolation and character-

ization of these microsatellite loci will greatly facilitate

new studies designed to advance our understanding of the

population genetics, molecular ecology and conservation

biology of this ecologically and economically important

species.

Acknowledgments We thank E. Hoaglund and T. Crombie for

technical assistance. Financial support for this work was provided by

the US National Science Foundation grant numbers OCE96-14091,

OCE99-82105, and OCE06-20276 and by Fundacao para a Ciencia e

Tecnologia pos-doctoral grant [SFRH/BPD/14945/2004 to F.A.], and

grant MEGIKELP [PTDC/MAR/65461/2006].

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