Microsatellite markers for the giant kelp Macrocystis pyrifera

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Feb 20, 2009 - Filipe Alberto Æ Allison Whitmer Æ Nélson C. Coelho Æ ... Ó Springer Science+Business Media B.V. 2009 ... 2007). Forests of this brown alga.
Conserv Genet (2009) 10:1915–1917 DOI 10.1007/s10592-009-9853-9

TECHNICAL NOTE

Microsatellite markers for the giant kelp Macrocystis pyrifera Filipe Alberto Æ Allison Whitmer Æ Ne´lson C. Coelho Æ Mackenzie Zippay Æ Elena Varela-Alvarez Æ Peter T. Raimondi Æ Daniel C. Reed Æ Ester A. Serra˜o

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 ecosystems on the planet, forming a complex food-web system F. Alberto (&)  N. C. Coelho  E. Varela-Alvarez  E. A. Serra˜o CCMAR, CIMAR-Laborato´rio Associado, University of Algarve, Campus de Gambelas, Faro, Portugal e-mail: [email protected] 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

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 characterization of hyper-variable microsatellite markers that should prove extremely useful for addressing this knowledge gap. Genomic DNA was isolated using an initial nuclei isolation (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 streptavidincoated magnetic particles and biotinylated probes (Magnesphere, 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, 37C), 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

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R-TGCTACAAAAAGAGGACACAC

F-CAAAACAAGAGGGGAACAC

R-GGCAGGTCTCGTCTTCTG

F-GGAAATGCGGCACTAAAG

R-AAAAGGGTGTGGCATCTT

F-ACTCGCTCAAGGTAAGCC

R-ACCGTGTAGCATGAGTCTATG

F-GTTCCAGCTTGGTATTCAAA

R-GCAATGGTGGGTGAAAGA

F-ACGGGTTTTACGAGGAGTG

R-TTCGGTTCATCTACATACTCG

F-CAACAACTAGCGTACCTTGAG

(CT)14

(GA)15..(GA)7

(CT)32

(GA)10…(GA)3…(GA)8

(GA)27

(CT)19

198

201

258

227

257

128

154

170

158

176

156

301

151

217

226

192

Clone size (bp)

60–55

60–55

62–60

65–60

60–55

60–55

60–55

65

65

65

55–50

55–50

55–50

55–50

65

55–50

PCR annealing (C)





nA



nA

nA

nA







nA

nA

nA

nA





Microchecker

17

27

27

9

22

9

14

7

3

4

34

28

24

4

13

10

A

186–231

180–265

160–264

224–237

230–290

116–132

141–195

164–172

156–164

173–181

125–204

281–337

96–168

216–222

222–247

169–193

Size range (bp)

0.906

0.877

0.918

0.775

0.909

0.650

0.786

0.690

0.625

0.531

0.930

0.929

0.922

0.4690

0.809

0.667

He

0.036

0.057

0.133***

0.025

0.097***

0.132*

0.111***

0.016

-0.112

-0.173

0.284***

0.204***

0.345***

0.499***

0.064*

0.101*

FIS

*P \ 0.05, **P \ 0.01, ***P \ 0.001

Locus name and GenBank accession number, primer sequence, motif repetition, clone size, PCR annealing temperature, presence of null alleles estimated with Microchecker, number of alleles found, fragment size range in base pairs, gene diversity, and inbreeding coefficient, were estimated for the Carpentaria bed

Mpy-19 (FJ624224)

Mpy-17 (FJ624223)

Mpy-14 (FJ624222)

Mpy-11 (FJ624221)

Mpy-9 (FJ624220)

Mpy-8 (FJ624219)

(GA)16

R-CTGCGTCCATTTGAGCCAC F-CGCATTCATTTTTCGCAC

Mpy-7 (FJ624218)

R-CAGGCTTGGTGTTGTTGC

(AG)11

(CT)10

(CT)9

(CT)27

(GA)21

(GA)31

(CT)9CC(CT)4

F-CGGAAGGAGAGAGGGCAAG

R-CGGAGAACAGGGAGAGCAG

F-TGACGCGTTCATCGTGTTG

R-GACCAGATGCAGAGATGACAG

F-TTGCTCCTCCTGCTGCTAC

R-GGGACATAAAGCACAGA

F-ATAGTAGCAAACCTCACAAG

R-CTCTCACTCTCACACTCCT

F-GGTAAACACGCCTCC

R-ACCCTTTTAGTGCCG

F-GGAGACAAAGCCAAGA

R-CCGAACCACACTCAAC

F-TCTTTCCTTTTCTCTTCTCT

Mp-BC-25 (FJ624217)

Mp-BC-19 (FJ624216)

Mp-BC-18 (FJ624215)

Mp-BC-16 (FJ624214)

Mp-BC-13 (FJ624213)

Mp-BC-9 (FJ624212)

Mp-BC-8 (FJ624211)

(CT)11

R-GTCTCTGTTGCCGTCGTTG F-AACCCACTCCACTCCT

Mp-BC-4 (FJ624210)

R-CTTCATAGTGCCCTTGTAT

(CT)22

F-AAACGTTGGTCACTCGCAC

Mp-BC-3N (FJ624209)

Repeat motif

Primer sequences

Locus name (Genebank no.)

Table 1 Characterization of sixteen microsatellite loci for the giant kelp Macrocystis pyrifera

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Conserv Genet (2009) 10:1915–1917

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, California 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 94C, followed by 24 cycles of ‘‘touchdown’’ PCR consisting of 30 s at 94C, 30 s at 64C (reduced by 0.5C each subsequent cycle), and 30 s at 72C, 10 additional cycles consisting of 30 s at 94C, 30 s at 52C and 40 s at 72C, and a final elongation step at 72C for 10 min. All PCR reactions were performed on a GeneAmp 9700 thermocycler (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 manually 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 heterozygote 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

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then what was previously obtained with less variable ITS markers (Coyer et al. 2001). Our isolation and characterization 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 Fundac¸a˜o para a Cieˆncia e Tecnologia pos-doctoral grant [SFRH/BPD/14945/2004 to F.A.], and grant MEGIKELP [PTDC/MAR/65461/2006].

References Alberto F (2009) MsatAllele_1.0: an R package to visualize the binning of microsatellite alleles. J Hered. doi:10.1093/jhered/ esn110 Billote N, Lagoda PJL, Risterucci A et al (1999) Microsatellite enriched libraries: applied methodology for the development of ISSR markers in tropical crops. Fruits 54:277–288 Coyer JA, Smith GJ, Andersen RA (2001) Evolution of Macrocystis spp. (Phaeophyceae) as determined by ITS1 and ITS2 sequences. J Phycol 37:574–585. doi:10.1046/j.1529-8817.2001.037001574.x Graham MH (2004) Effects of local deforestation on the diversity and structure of Southern California giant kelp forest food webs. Ecosystems (NY, Print) 7:341–357. doi:10.1007/s10021-0030245-6 Graham MH, Va´squez JA, Buschmann AH (2007) Global ecology of the giant kelp Macrocystis: from ecotypes to ecossystems. Oceanogr Mar Biol Annu Rev 45:39–88 Koblizkova A, Dolezel J, Macas J (1998) Subtraction with 30 modified oligonucleotides eliminates amplification artifacts in DNA libraries enriched for microsatellites. Biotechniques 25:32 Raymond M, Rousset F (1995) Genepop (version-1.2)—populationgenetics software for exact tests and ecumenicism. J Hered 86:248–249 Rozen S, Skaletsky HJ (2000) Primer 3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386 Van Oosterhout C, Hutchinson WF, Wills DPM et al (2004) MICROCHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4:535–538. doi: 10.1111/j.1471-8286.2004.00684.x Varela-Alvarez E, Andreakis N, Lago-Leston A et al (2006) Genomic DNA isolation from green and brown algae (Caulerpales and Fucales) for microsatellite library construction. J Phycol 42:741– 745. doi:10.1111/j.1529-8817.2006.00218.x

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