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BMC Proceedings

Open Access

Identification of microsatellite loci in Pinus tecunumanii

  • Valderês Aparecida de Sousa1,
  • Camila C Mantello2,
  • Ananda Virginia de Aguiar1Email author,
  • Daiane Rigoni Kresting1,
  • Anete Pereira de Souza2 and
  • Laila Toniol Cardin3
BMC Proceedings20115(Suppl 7):P1

Published: 13 September 2011


Microsatellite LocusPositive CloneGermplasm CollectionCommercial ExploitationRepetitive Region


Pinus tecunumanii has displayed good performance in tropical regions of Brazil and showed high potential for commercial exploitation. Embrapa Forestry and its partners own many of the species seed production areas. In spite of its importance, the majority of P. tecunumanii germplasm collections remain still genetically uncharacterized. Thus identifying genetic markers is an important tool to genetically characterize these collections.We describe the initial steps to develop microsatellites for Pinus tecunumanii by enriched library construction with the ultimate goal of characterizing accessions of the germplasm collections of EMBRAPA.


The genomic-enriched library was constructed following the protocol described by [1]. The genomic DNA of P. tecunumanii was digested with AFAI and enriched in (CT)8 and (GT)8 repeats. Enriched fragments were amplified by polymerase chain reaction (PCR), connected to a pGEM T-easy vector and transformed into competent XL1- blue Escherichia coli cells. The positive clones were selected using the B-galactosidase gene and then grown overnight in an HM/F medium with ampicillin. After PCR 95 positive clones were sequenced in both directions using the T7 and SP6 primers as well as the Big Dye terminator Kit. The sequences were assembled and edited in Seqman (DNAStar), the repetitive regions were found using the Simple Sequence Repeat Identification Tool [2]. Primer select (DNAStar) and Primers Plus were used to design primer pairs flanking the microsatellite regions.

Results and conclusion

Of the ninety five sequences cloned only eleven contained microsatellite sequences and five showed repeats and adequate flanking regions for primer design. The observed proportion of dinucleotide was 5.26% (5), while trinucleotide and tetranucleotide proportions were 1.05% (1) and 5.26% (5), respectively. Ninety one percent of nucleotides were simply perfect and 9% were compost perfect. The explanation for this low yield (11.6%) can be attributed to the genomic-enriched procedure. To overcome this problem this procedure will be repeated. The obtained sequences will be used for validation of P. tecunumanii microsatellite primers and used to estimate the genetic diversity from the germplasm collection located in various regions of Brazil.



The authors would like to thank Selma Buzzetti de Moraes and Mario Luiz Teixeira de Moraes for their assistance with DNA extraction procedures and the Valor Florestal Company for collecting material used in this research.

Authors’ Affiliations

Embrapa Forestry, Brazilian Agricultural Research Corporation, Colombo, Brazil
UNICAMP, Campinas, São Paulo, Brazil
UNESP, Ilha Solteira, São Pauloa, Brazil


  1. Billote N, Risterucci AM, Baurens FC: Microsatellite enriched libraries: applied methodology for the development of SSR markers in tropical crops. Fruits. 1999, 54: 277-288.Google Scholar
  2. Temnykh S, DeClerck G, Lukashova A, Lipovich L, Cartinhour S, McCouch S: Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential. Genome Research. 2011, 11: 1441-1452.View ArticleGoogle Scholar


© Aparecida de Sousa et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.