American Journal of Botany: e213–e215. 2012.

AJB PRIMER NOTES & PROTOCOLS IN THE PLANT SCIENCES

MICROSATELLITE DEVELOPMENT FOR THE RELICTUAL CONIFER

ARAUCARIA ARAUCANA (ARAUCARIACEAE) USING
NEXT-GENERATION SEQUENCING1

M. ANGELA MARTÍN2,6, CLAUDIA MATTIONI3, ILARIA LUSINI3, FERNANDO DRAKE4,

MARCELLO CHERUBINI3, M. ANGEL HERRERA5, FIORELLA VILLANI3, AND LUIS M. MARTÍN2
2Departamentode Genética, E.T.S.I.A.M., Universidad de Córdoba, Campus de Excelencia Internacional Agroalimentario
(ceiA3), ES-14071 Córdoba, Spain; 3Istituto di Biologia Agroambientale e Forestale (IBAF), Consiglio Nazionale delle Ricerche
(CNR), Viale Marconi, 2 05010 Porano (TR), Italy; 4Departamento de Manejo de Bosques y Medioambiente, Facultad de
Ciencias Forestales, Universidad de Concepción, Casilla 160-C, Concepción, Chile; and 5Departamento de Ingeniería
Forestal, E.T.S.I.A.M., Universidad de Córdoba, Campus de Excelencia Internacional Agroalimentario (ceiA3), ES-14071
Córdoba, Spain

• Premise of the study: In this study, the 454 GS-FLX genome sequence system was used for the identification and characteriza-
tion of microsatellites in Araucaria araucana, one of the most important and endangered species of Chilean and Argentinean
native forests.
• Methods and Results: A total of 35 876 reads were identified, 96% of which were within the size range selected for further
analysis. Of these, 1563 contained a microsatellite insert suitable for primer design. Ten simple sequence repeat (SSR) markers
provided easily interpretable patterns and were used to evaluate the genetic diversity in four populations of the species. The 10
microsatellites showed high polymorphism levels, with a total of 99 alleles and 32 private alleles. The observed heterozygosity
was high and ranged from 0.513 to 0.723.
• Conclusions: The primers presented in this study display high genetic diversity and may provide useful information for the
design of conservation strategies in Araucaria araucana.

Key words: Araucaria araucana; Araucariaceae; conservation; endangered species; microsatellite.

Araucaria araucana (Molina) K. Koch (Araucariaceae) is a
relict conifer in South America’s temperate forest and a repre-
sentative symbol of Chilean forest biodiversity due to its ende-
micity and longevity. In addition to being part of the impressive
landscape in native forest ecosystems, it presents high cultural
and social importance for the indigenous Pehuenche commu-
nity. The species occurs in the Andean region at the border of
Chile and Argentina and in the Coastal Cordillera of Chile.
However, this current distribution is a remnant of a more exten-
sive former distribution that has been severely diminished by its
intense exploitation in the past for timber, logging, and seeds,
as well as by natural disturbances (Veblen, 1982). This, along
with its slow growth and poor regeneration capacity, makes it
particularly susceptible to external pressures. For these reasons,
it is included in Appendix I of the Convention on International
Trade of Endangered Species of Wild Flora and Fauna (CITES)
1 Manuscript
received 31 October 2011; revision accepted 14 January 2012.
This research was partially support by grants from the Research Service
of Concepción University (Chile) (No. 207-141-018-1.0) and the Spanish
Agency for International Development Cooperation, Spanish Ministry for
Foreign Affairs and Cooperation (A/023099/09 and A/030789/10). The
first author is grateful to Agrifood Campus of International Excellence
(ceiA3) from the Spanish Ministry of Education and the Ministry of
Science and Innovation for financial support.
6 Author for correspondence: ge2macum@uco.es
doi:10.3732/ajb.1100519
and listed as Vulnerable on the Red Data list published by the
International Union for Conservation of Nature (IUCN, 1996).
Both the identification and development of microsatellite
markers represent significant challenges, especially in the case
of organisms for which there are no sequence data, as is the case
for A. araucana. There is only one study that reports the devel-
opment of six specific microsatellite loci in A. araucana (Marconi
et al., 2011). On the other hand, microsatellite cross-species
amplification has been reported among the two South American
Araucaria species, A. angustifolia (Bertol.) Kuntze and A.
araucana (Salgueiro et al., 2005; Moreno et al., 2011), although
the number of transferred loci with high amplification quality
was low. Recently, DNA enrichment strategies involving hybrid-
ization with probes containing simple sequence repeat (SSR)
sequences to genomic DNA fragments have been introduced.
The coupling of multiple, simultaneous enrichments of micro-
satellite motif–enriched libraries with high-throughput 454 py-
rosequencing provides an efficient procedure for efficient
microsatellite isolation. In this study, the 454 GS-FLX genome
sequence system was used for the identification of microsatel-
lites in A. araucana.
METHODS AND RESULTS
Samples of A. araucana leaves were collected from Chilean natural popula-
tions in 2007 (Drake et al., 2009) and 2011. Leaf material was dried in silica gel
for 24 h and vacuum packed until DNA extraction. Voucher specimens for every

American Journal of Botany: e213–e215, 2012; http://www.amjbot.org/ © 2012 Botanical Society of America
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TABLE 1. Characterization of 10 microsatellite markers developed in Araucaria araucana.

Locus Primer sequences (5′–3′) Repeat motif Size range (bp) Ta (°C) Fluorescent label GenBank accession no.

Ara2027 F: AGGAAGGCATTTTGGCTTGG (AC)22 110–184 56 NED JN896693

R: TGGTCATCTTAATGGTACTTTGATTG
Ara5179 F: GCTTATAGACTCGACTTGCCAC (CA)15 125–126 56 NED JN896694
R: CGGATCCACCATTTGTAACTTTG
Ara5182 F: TGATGTGAGCCAAAATCAAAATC (TG)15 159–240 56 6-FAM JN896695
R: AGGAGAGAGTCATGAAGCCG
Ara5595 F: AGTCCAAAATAGACATAGGCATCC (CA)12 106–120 56 NED JN896696
R: TGGGAAAATCAAACCCTCGC
Ara11382 F: GGAAAGTAGCAAGGCCTCAAC (AC)14 117–234 56 VIC JN896697
R: TGCCTAAAACATCCCTTGGAC
Ara11384 F: TGATTGATGTGATTGGCTACAAATTC (CA)16 113–145 56 VIC JN896698
R: TGTTTGCATGCTTGGAGTGG
Ara20681 F: AACTAAAAACCTTAAATGCTCATCG (GT)12 163–188 56 VIC JN896699
R: CAATCCTCAAATTAGCCCATGC
Ara23863 F: CCATGGAGGTAGGCCTTGTG (AC)13 195–197 56 6-FAM JN896700
R: GCATGCTTGTAGGTTCTACCC
Ara27058 F: ACTTGTACGAATTCAATGGATGG (GT)15 225–226 56 6-FAM JN896701
R: CAGACTTGGCAATGGTGGTC
Ara35269 F: CCAGCCACCTTTCATTTCCC (TGT)11 149–159 56 6-FAM JN896702
R: TGCATTGACTCTAACGACAGC
Note: Ta = annealing temperature.

sampled population are deposited at the Istituto di Biologia Agro-ambientale e
Forestale–Consiglio Nazionale delle Ricerche (IBAF-CNR) (Porano, Italy)
with duplicates at the Genetics Department of Cordoba University (Cordoba,
Spain) (Appendix 1). Genomic DNA was extracted from 30 mg of leaf tissue
using the QIAGEN DNeasy Plant mini Kit (QIAGEN, Valencia, California,
USA). Microsatellite sequences were isolated using the high-throughput ge-
nomic sequencing approach described by Abdelkrim et al. (2009). Two micro-
grams of genomic DNA were analyzed on a Roche 454-GS-FLX platform
(Roche, Zurich, Switzerland) using a 1/16th run and the GS-FLX titanium re-
agents. A total of 35 876 reads were identified, 96% of which were within the
size range selected for further analysis (more than 80 bp long). The average
length was 285 base pairs. Of these, 4263 reads had a microsatellite insert with
a tetra- or a trinucleotide of at least six repeat units or a dinucleotide of at least
10 repeat units, and 1563 contained a microsatellite insert suitable for primer
design. Twenty primer pairs were designed using the software Primer3 (Rozen
and Skaletsky, 2000). To test them, 12 samples were amplified and their ampli-
fication products run on agarose gel. Amplification was performed using 10 ng
of DNA, 10 mM Tris-HCl, 1.5 mM MgCl2 reaction buffer, 200 μM dNTP, 0.3 μM
primer (both), and 0.5 U of Hotstart Taq polymerase. Cycling parameters were:
15 min of denaturation at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at
56°C, and 30 s at 72°C, and a final extension step at 72°C for 30 min. Ten
primer pairs did not amplify in at least 80% of the samples and were excluded
from further analysis. The forward primers of the 10 remaining microsatellites
were labeled with a fluorochrome (6-FAM, VIC, NED; Applied Biosystems,
Foster City, California, USA). Based on the size of the products, three multi-
plex-PCR mixtures were designed: (A) Ara2027, Ara5182, Ara11382,
Ara35269; (B) Ara5179, Ara11384, Ara27058; (C) Ara5595, Ara20681,
Ara23863. Amplifications were performed following the Type-it Kit (QIA-
GEN) protocol, in a 12.5 μL reaction volume containing 20 ng of genomic
DNA. Cycling parameters were: 5 min at 95°C followed by 28 cycles of 30 s at
95°C, 90 s at 56°C, and 30 s at 72°C, and a final extension step at 60°C for 30
min. Amplified samples were mixed with GeneScan 500-bp internal-lane size
standard (Applied Biosystems) and separated using an ABI PRISM 3100 DNA
sequencer. Data were analyzed using GeneScan and Genotyper software pro-
grams (Applied Biosystems).
The details of the 10 microsatellite loci are shown in Table 1. All primers
reliably amplified microsatellite regions in the assessed material. Polymor-
phism was evaluated in 84 individuals from four A. araucana populations
(Table 2). Genetic parameters as number of alleles per locus (Na), observed
heterozygosity (Ho), expected heterozygosity (He), inbreeding coefficient (FIS),
and deviation from Hardy–Weinberg equilibrium were calculated using Arle-
quin 3.1 software (Schneider et al., 2000). A total of 99 alleles were detected,
with a mean of 9.9 alleles per locus and 32 private alleles (12 in the Nahuelbuta
population, eight in the Icalma and Interlagos populations, and four in the Vi-
llarrica population). Polymorphic information content (PIC) ranged from
0.045 to 0.901, with a mean of 0.577. Ho and He ranged from 0.513–0.723 and

TABLE 2. Characteristics of the 10 microsatellite loci evaluated in four populations of Araucaria araucana.

Icalma (N = 23; 38°50′S, Interlagos (N = 22; 38°39′S, Nahuelbuta (N = 20; 37°49′S, Villarrica (N = 19; 39°27′S,
71°22′W) 71°49′W) 73°01′W) 71°50′W)
Locus Na Ho He FIS Na Ho He FIS Na Ho He FIS Na Ho He FIS

Ara2027 14 0.739 0.889 0.169* 13 0.636 0.882 0.279* 11 0.750 0.791 0.052 9 0.842 0.828 −0.017
Ara5179 1 0.000 0.000 — 2 0.000 0.351 1.000* 2 0.000 0.180 1.000* 1 0.000 0.000 —
Ara5182 4 0.739 0.689 −0.073 2 1.000 0.500 −1.000 2 0.900 0.495 −0.818 4 0.316 0.704 0.551*
Ara5595 6 0.783 0.686 −0.140 6 0.136 0.388 0.649* 4 0.400 0.524 0.236* 4 0.316 0.514 0.385*
Ara11382 10 0.565 0.827 0.317* 10 0.773 0.733 −0.054 11 0.650 0.799 0.186* 9 0.684 0.820 0.166*
Ara11384 16 0.870 0.871 0.002 12 0.773 0.802 0.036 12 0.900 0.858 −0.050 12 0.737 0.873 0.156*
Ara20681 8 0.783 0.807 0.030 7 0.636 0.768 0.161 9 0.750 0.801 0.064 4 0.421 0.464 0.093
Ara23863 2 0.783 0.476 −0.643 2 0.727 0.463 −0.571 2 0.600 0.420 −0.429 2 0.684 0.450 −0.520
Ara27058 1 0.000 0.000 — 2 0.000 0.165 1.000* 1 0.000 0.000 — 1 0.000 0.000 —
Ara35269 5 0.522 0.579 0.100 5 0.500 0.525 0.047 5 0.800 0.735 −0.088 2 0.105 0.100 −0.056
Average 6.5 0.723 0.744 0.029 6.1 0.518 0.570 0.090 5.8 0.639 0.638 0.000 4.6 0.513 0.610 0.132
Note: FIS = inbreeding coefficient; He = expected heterozygosity; Ho = observed heterozygosity; Na = number of alleles per locus.
* P < 0.05.
May 2012] AJB PRIMER NOTES & PROTOCOLS—ARAUCARIA ARAUCANA MICROSATELLITES
0.570–0.744, with averages of 0.600 and 0.640, respectively (Table 2). The single
locus fixation index (FIS) showed negative values in two loci and positive and
significant deviation from zero in five loci. This deficit in heterozygotes could
be attributed to the presence of null alleles, and the five loci were tested with
Micro-Checker 2.2.3 (van Oosterhout et al., 2004). Significant evidence of null
alleles was detected in loci Ara2027, Ara11382, Ara5179, and Ara5595. How-
ever, at the population level, the inbreeding coefficient did not display any sig-
nificant deviation from zero, showing that all populations were in equilibrium
(Table 2). The linkage disequilibrium tested among 45 possible pairwise loci
considering 1000 permutations, and after sequential Bonferroni correction, in-
dicated that all loci were genetically independent.
CONCLUSIONS
Araucaria araucana forests are the focus of increasing con-
servation concern because their widespread overexploitation in
the past has resulted in a decline of the species’ forested area
and the erosion of its genetic diversity. In this respect, the 10
microsatellites reported in this study have shown high polymor-
phism levels and a reliable and easily interpretable pattern.
Given that the extent and distribution of genetic diversity are
key elements for the evolutionary potential of species, these
primers may provide a baseline for the establishment of conser-
vation strategies for A. araucana.
LITERATURE CITED
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MARTIN, AND L. M. MARTÍN. 2009. Networking sampling of Araucaria
APPENDIX 1. List of vouchers of Araucaria araucana populations used in this study. Vouchers are deposited at IBAF-CNR (Porano, Italy) and the Genetics
Department of Cordoba University (Cordoba, Spain).
Species Collection no. Accession no.
A. araucana CH1105 CH1105/01–CH1105/20
A. araucana CH1106 CH1106/01–CH1106/23
A. araucana CH1107 CH1107/01–CH1107/22
A. araucana CH1108 CH1108/01–CH1108/19
e215
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Locality
Nahuelbuta N.P., Angol district, Bio-Bio región,
Chile (37°49′S, 73°01′W)
Icalma city, Lonquimay district, Araucania región,
Chile (38°50′S, 71°22′W)
Interlagos Route, Mellipeuco district, Araucania región,
Chile (38°39′S, 71°49′W)
Villarrica city, Villarrica district, Araucania región,
Chile (39°27′S, 71°50′W)