TECHNICAL NOTE
Isolation and characterization of microsatellite loci
in the Brazilian orchid Epidendrum fulgens
F. Pinheiro Æ M. O. Santos Æ F. Barros Æ D. Meyer Æ
A. Salatino Æ A. P. Souza Æ S. Cozzolino
Received: 7 January 2008 / Accepted: 8 January 2008

Springer Science+Business Media B.V. 2008
Abstract Epidendrum fulgens has a patchy distribution
along the Atlantic Rainforest in the Brazilian coast, due to
the destruction of its native habitat. Here, we report on both
the development of nine new microsatellite markers iso-
lated from this species and the characterization of their
allele variability in two distant and unrelated populations.
The number of alleles observed for each locus ranged from
2 to 17 with an average of 6.4 alleles per locus. These
microsatellites should be valuable tools for studying the
effect of habitat fragmentation on the genetic structure of
E. fulgens populations.
Keywords Epidendrum
Orchidaceae
Microsatellites

Atlantic Rainforest
Cross-amplification
Epidendrum fulgens is an endemic orchid of coastal Bra-
zilian Atlantic Rainforest. In the past, this orchid had a
wide geographic distribution between the Brazilian States
of Rio de Janeiro and Rio Grande do Sul but, nowadays, its
populations are small and fragmented due to habitat loss.
The knowledge of patterns of genetic diversity and gene
flow is essential to guide conservation management deci-
sions and for understanding the genetic consequences of
habitat loss in fragmented populations. Therefore, the aim
of this study was to develop a set of polymorphic micro-
satellite (simple sequence repeat—SSR) markers for
E. fulgens for describing the population genetic structure of
this threatened species.
Total DNA was extracted from silica gel exsiccated
leaves of E. fulgens following the protocol of Doyle and
Doyle (1990). Marker isolation involved the construction
of a genomic library partially enriched for (CT)n and (GT)n
repeats by using biotinylated oligonucleotide sequences
bound to Streptavidin-coated magnetic particles as descri-
bed by Kijas et al. (1994) with modifications by Billote
et al. (1999). Microsatellite-enriched DNA fragments were
ligated into pGEM-T Easy vector (Promega) as described
by supplier and used to transform XL1Blue competent
E. coli cells (Stratagene). A total of 96 recombinant colo-
nies were obtained and sequenced using the BigDye v3.1
terminator kit on the ABI PRISM 3130 Sequence Analyser
(Applied Biosystems). For 25 clones, containing SSR
motifs, forward and reverse sequences were aligned in
SeqMan (DNASTAR package), and primers were designed
for 16 loci using the PRIMER3 software (http://frodo.
wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).
For each SSR, the forward primers were synthesized
with a 19 bp long M13 tail (50-CACGACGTTGTAA
AACGAC-30) following the amplification method of
Schuelke (2000), which involved three primers: a forward
F. Pinheiro (&)
A. Salatino
Departamento de Botaˆnica, IB/USP, 05508-900 Sao Paulo,
SP, Brazil
e-mail: biopinheiro@yahoo.com.br
F. Pinheiro
S. Cozzolino
Dipartimento delle Scienze Biologiche, Universita` degli Studi
Federico II, 80139 Naples, Italy
M. O. Santos
A. P. Souza
Departamento de Gene´tica e Evoluc¸a˜o, IB/UNICAMP,
13083-970 Campinas, SP, Brazil
M. O. Santos
A. P. Souza
Centro de Biologia Molecular e Engenharia Gene´tica (CBMEG),
UNICAMP, 13083-970 Campinas, SP, Brazil
F. Barros
Instituto de Botaˆnica, Sec¸a˜o de Curadoria do Herba´rio,
04301-012 Sao Paulo, SP, Brazil
D. Meyer
Departamento de Gene´tica e Biologia Evolutiva, IB/USP,
05508-900 Sao Paulo, SP, Brazil
123
Conserv Genet
DOI 10.1007/s10592-008-9514-4

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Conserv Genet
123

SSR-specific primer with the M13 tail at its 50 end, a
reverse locus-specific primer, and a universal M13 primer
labelled with a fluorescent dyes, 6-FAM or JOE (Applied
Biosystems) respectively. All PCR amplifications were
performed in a Applied Biosystems 2700 thermocycler in
10 ll reactions containing: 10 ng DNA template, 19 PCR
buffer, 2 mM MgCl2, 100 lM dNTPs, 1 pmol forward
primer, 4 pmol reverse primer, 0.4 pmol universal M13
primer and 0.5 U Taq polymerase (Amersham Pharmacia
Biotech). A ‘touchdown’ cycling program was used: 95C
for 3 min, then 10 cycles of 94C for 30 s, 58C decreasing
to 48C at 1C per cycle for 30 s, 72C for 30 s followed
by 40 cycles of 94C for 30 s, 48C for 30 s, 72C for 30 s,
followed by a final extension of 20 min at 72C. Micro-
satellite alleles were resolved on a 3130 DNA Sequence
Analyser and were sized with LIZ (500) standard by using
GENEMAPPER v3.7 software (Applied Biosystems).
A total of 40 individuals from two Brazilian populations
of E. fulgens (Imbituba and Guaratuba) were analyzed to
evaluate SSR polymorphism. ARLEQUIN 3.11 (Excoffier
et al. 2005) software was used to calculate observed (HO)
and expected (HE) heterozygosity, to test for departure
from Hardy–Weinberg equilibrium (HWE) and for linkage
disequilibrium between all pairs of loci. MICRO-CHECKER
(Van Oosterhout et al. 2004) software was used to quantify
genotyping errors. Nine SSRs were polymorphic, with
number of alleles per locus ranging from 2 to 17 with an
average of 6.4 alleles per locus. The observed and expected
heterozygosities (HO and HE) ranged from 0.10 to 0.90 and
0.05 to 0.91, with averages of 0.494 and 0.556, respectively
(Table 1). Four loci showed a significant departure from
Hardy–Weinberg equilibrium (P \ 0.001), three due to
heterozygote deficiency (EFF29, EFF58 and EFF70) in
Imbituba population, and one due to heterozygote excess
(EFF51) in the Guaratuba population. The small size of the
Imbituba population and fragmentation history of the
region may be the factors promoting the local observed low
levels of heterozygosity. No linkage disequilibrium was
detected among any pair of loci, and no genotyping errors
due to presence of null alleles, short allele dominance or
scoring of stutter peaks was detected.
Cross-species and cross-genera amplification were per-
formed on Epidendrum species and allied genera, on single
individuals by using the same amplification conditions
used for E. fulgens. Several positive amplifications occur-
red across all tested species (Table 2).
The primers proved to be useful in revealing levels of
diversity in E. fulgens and thus can be used to explore the
genetic structure of scattered populations across its actual
geographical range.
Acknowledgements We thank C. Palma-Silva and R. P. Romanini
for technical support and helpful discussions on this manuscript. This
work was supported by grants from FAPESP (06/54189-3) and CNPq
(471929/2006-9) to F. Pinheiro and F. Barros, and by awards from
CAPES to F. Pinheiro and CNPQ to F. Pinheiro, F. Barros, A. Sal-
atino and A. P. Souza.
References
Billote N, Risterucci A-M, Baurens F-C (1999) Microsatellite
enriched libraries: applied methodology for the development of
SSR markers in tropical crops. Fruits 54:277–288
Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue.
Focus 12:13–15
Excoffier L, Laval LG, Schneider S (2005) Arlequin ver. 3.0 An
integrated software package for population genetics data anal-
ysis. Evol Bioinform Online 1:47–50
Kijas JM, Fowler JC, Garbett CA, Thomas MR (1994) Enrichment of
microsatellites from the citrus genome using biotinylated
oligonucleotide sequences bound to streptavidin-coated mag-
netic particles. Biotechniques 16:656–660
Schuelke M (2000) An economic method for the fluorescent labelling
of PCR fragments. Nat Biotechnol 18:233–234
Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004)
MICRO-CHECKER: software for identifying and correcting
genotyping errors in microsatellite data. Mol Ecol Notes 4:535–
538
Table 2 Cross-species and genera amplification of nine microsatellite primers from Epidendrum fulgens within the subtribe Laeliinae
Species EFF6 EFF26 EFF29 EFF43 EFF45 EFF51 EFF58 EFF61 EFF70
Epidendrum xanthinum - + W + + - + W +
Epidendrum secundum - + W + + + + + ++
Pseudolaelia cipoensis - + W + - - W W -
Cattleya eldorado - - W + + - W + -
Prosthechea vespa - + W + - - W + -
Successful amplifications with a single band visualized (+), successful amplifications with more than one band visualized (++), weak ampli-
fications (W) and failed amplifications (-) are indicated
Conserv Genet
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