Primers for 52 polymorphic regions in the Quercus rubra chloroplast, 47 of which amplify across 11 tracheophyte clades
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Postglacial migration studies in Quercus rubra L. (northern red oak) are hampered by low levels of population differentiation in the widely used universal chloroplast primers. We sequenced the large single copy (LSC) regions of the Q. rubra and Quercus ellipsoidalis chloroplasts to enable us to query additional regions for future studies on migration and speciation. Using 454 sequencing of long-range PCR amplicons and Sanger sequencing for gap closure, we report 65 coding sequences from Q. rubra and 59 from Q. ellipsoidalis. Comparison of our de novo assembly of the LSC region sequence for Q. rubra to Q. rubra chloroplast sequence (NCBI Reference Sequence: NC_020152.1) from a different tree revealed 106 polymorphisms, all within intergenic regions, that can serve as tools for postglacial migration studies and taxonomic studies within the Lobatae. Sequence alignment for the 59 complete coding regions in common for theQ. rubrachloroplast reference sequence, our Q. rubra sequence and our Q. ellipsoidalis sequence revealed no sequence polymorphisms and no indels. We also report the 52 primer pairs we used for gap closure, including 53 new primer pairs not previously reported. We tested these 52 primer pairs against 11 species representing the Tracheophyta and detected 47 that produced amplicons in all 11 species. The new universal primers we have identified provide additional tools for resolving the taxonomic relationships among the congeneric taxa of forest trees in the temperate and subtropical forests of the Northern Hemisphere.
KeywordsQuercus rubra Chloroplast genome Universal primers Long-range PCR
We thank Meg Staton (Clemson University Genomics Institute) for bioinformatics assistance; John Tan, Brent Harker, and Rory Carmichael (Notre Dame Genomics and Bioinformatics Core Facilities) for DNA sequencing and genome assembly; and Amy Fernow for assistance with PCR reactions. Funding for Daniel Borkowski was provided in part by the National Science Foundation (NSF IOS-1025974). The University of Notre Dame Core Genomics Facility provided funding for the 454 sequencing.
Data archiving statement
We have submitted chloroplast genome contigs to GenBank at NCBI, and the accession numbers will be provided as soon as they are available.
- Aziz R, Bartels D, Best A, DeJongh M, Disz T, Edwards R, Formsma K, Gerdes S, Glass E, Kubal M, Meyer F, Olsen G, Olson R, Osterman A, Overbeek R, McNeil L, Paarmann D, Paczian T, Parrello B, Pusch G, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST server: rapid annotations using subsystems technology. BMC Genomics 9(1):75PubMedCentralPubMedCrossRefGoogle Scholar
- Jansen R, Kaittanis C, Saski C, Lee S-B, Tomkins J, Alverson A, Daniell H (2006) Phylogenetic analyses of Vitis (Vitaceae) based on complete chloroplast genome sequences: effects of taxon sampling and phylogenetic methods on resolving relationships among rosids. BMC Evol Biol 6(1):32PubMedCentralPubMedCrossRefGoogle Scholar
- Jansen RK, Saski C, Lee S-B, Hansen AK, Daniell H (2011) Complete plastid genome sequences of three rosids (Castanea, Prunus, Theobroma): evidence for at least two independent transfers of rpl22 to the nucleus. Mol Biol Evol 28(1):835–847. doi: 10.1093/molbev/msq261 PubMedCentralPubMedCrossRefGoogle Scholar
- LeCorre V, Kremer A (1998) Cumulative effects of founding events during colonisation on genetic diversity and differentiation in an island and stepping-stone model. J Evol Biol 11(4):195–512Google Scholar
- Little ELJ (1979) Atlas of United States trees. J For 77(3):188Google Scholar
- McCleary TS, Robichaud RL, Nuanes S, Anagnostakis SL, Schlarbaum SE, Romero-Severson J (2009) Four cleaved amplified polymorphic sequence (CAPS) markers for the detection of the Juglans ailantifolia chloroplast in putatively native J. cinerea populations. Mol Ecol Resour 9(2):525–527. doi: 10.1111/j.1755-0998.2008.02465.x PubMedCrossRefGoogle Scholar
- Petit RJ, Aguinagalde I, de Beaulieu JL, Bittkau C, Brewer S, Cheddadi R, Ennos R, Fineschi S, Grivet D, Lascoux M, Mohanty A, Muller-Starck GM, Demesure-Musch B, Palme A, Martin JP, Rendell S, Vendramin GG (2003) Glacial refugia: hotspots but not melting pots of genetic diversity. Science 300(5625):1563–1565PubMedCrossRefGoogle Scholar
- Rozen S, Skaletsky HJ (2000) Primer3 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 365–386Google Scholar
- 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(1):142–166. doi: 10.3732/ajb.92.1.142 PubMedCrossRefGoogle Scholar