Abstract
One of the most significant challenges confronting orchid researchers is the lack of specific molecular markers, mainly for species in the Neotropics. Here we report the first set of specific chloroplast microsatellite primers (cpSSR) developed for Neotropical orchids. In total, nine polymorphic cpSSR loci were isolated and characterized in four species occurring in the Brazilian Atlantic Rainforest: Epidendrum cinnabarinum, E. denticulatum, E. fulgens and E. puniceoluteum. Levels of intraspecific polymorphism were characterized using two populations for each species, with 13–20 individuals each. Allele numbers varied from two to three per locus, while the number of haplotypes ranged from three to six per species. Extensive differentiation among the taxa was detected. All markers were successfully cross-amplified in eight other different genera. These cpSSRs markers will enable novel insights into the evolution of this important Neotropical genus.
References
Cozzolino S, Noce ME, Musacchio A, Widmer A (2003) Variation at a chloroplast minisatellite locus reveals the signature of habitat fragmentation and genetic bottlenecks in the rare orchid Anacamptis palustris (Orchidaceae). Am J Bot 90:1681–1687
Demesure B, Sodzi N, Petit RJ (1995) A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Mol Ecol 4:129–131
Ebert D, Peakall R (2009) Chloroplast simple sequence repeats (cpSSRs): technical resources and recommendations for expanding cpSSR discovery and applications to a wide array of plant species. Mol Ecol Resour 9:673–690
Ebert D, Hayes C, Peakall R (2009) Chloroplast simple sequence repeat markers for evolutionary studies in the sexually deceptive orchid genus Chiloglottis. Mol Ecol Resour 9:784–789
Excoffier L, Laval LG, Schneider S (2005) ARLEQUIN version 3.0: an integrated software package for population genetics data analysis. Evol Bioinform Online 1:47–50
Fay M, Cowan RS (2001) Plastid microsatellites in Cypripedium calceolus (Orchidaceae): genetic fingerprints from herbarium specimens. Lindleyana 16:151–156
Hágsater E, Soto Arenas MA (2005) Epidendrum L. In: Pridgeon AM, Cribb PJ, Chase MW, Rasmussen FN (eds) Genera Orchidacearum 4. Oxford University Press, Oxford, pp 236–251
Hedrén M, Nordström S, Ståhlberg D (2008) Polyploid evolution and plastid DNA variation in the Dactylorhiza incarnata/maculata complex (Orchidaceae) in Scandinavia. Mol Ecol 17:5075–5091
Lexer C, Fay MF, Joseph JA, Nica MS, Heinze B (2005) Barrier to gene flow between two ecologically divergent Populus species, P. alba (white poplar) and P. tremula (European aspen): the role of ecology and life history in gene introgression. Mol Ecol 14:1045–1057
Nei M (1978) Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583–590
Peakall R (2007) Speciation in the Orchidaceae: confronting the challenges. Mol Ecol 16:2834–2837
Pinheiro F, Santos MO, Barros F, Meyer D, Salatino A, Souza AP, Cozzolino S (2008) Isolation and characterization of microsatellite loci in the Brazilian orchid Epidendrum fulgens. Conserv Genet 9:1661–1663
Provan J, Powell W, Hollingsworth PM (2001) Chloroplast microsatellites: new tools for studies in plant ecology and evolution. Trends Ecol Evol 16:142–147
Sang T, Crawford DJ, Stuessy TF (1997) Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). Am J Bot 84:1120–1136
Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18:233–234
Shaw J, Lickey EB, Beck JT, Farmer SB, Liu W, Miller J, Siripun KC, Winder CT, Schilling EE, Small RL (2005) The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. Am J Bot 92:142–166
Shaw J, Lickey EB, Schilling EE, Small RL (2007) Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III. Am J Bot 94:275–288
Small RL, Ryburn JA, Cronn RC, Seelanan T, Wendel JF (1998) The tortoise and the hare: choosing between noncoding plastome and nuclear Adh sequences for phylogenetic reconstruction in a recently diverged plant group. Am J Bot 85:1301–1315
Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol 17:1105–1109
Tate JA, Simpson BB (2003) Paraphyly of Tarasa (Malvaceae) and diverse origins of the polyploid species. Syst Bot 28:723–737
Acknowledgments
We thank Felipe N. M. de Assis for help during field work, Robyn S. Cowan, Dion Devey and Edith Kapinos for helping in the laboratory. This research was funded by grants from the Prance Fellowship in Neotropical Botany under the Kew Latin American Research Fellowship Programme (KLARF) to FP, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq—471929/2006-9) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP—06/54189-3) to FB. FP received fellowship from CNPq.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Pinheiro, F., Palma-Silva, C., de Barros, F. et al. Chloroplast microsatellite markers for the Neotropical orchid genus Epidendrum, and cross-amplification in other Laeliinae species (Orchidaceae). Conservation Genet Resour 1, 505 (2009). https://doi.org/10.1007/s12686-009-9121-9
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s12686-009-9121-9