The Polystichum Homepage
Welcome to the website for the hollyferns, members of the fern genus Polystichum and allies.
Barrington Laboratory Protocols
The AFLP protocol was adapted from Vos. et al. (1995) with additional modifications as suggested by the Wolf Lab at Utah University (Wolf 2000), the Gastony Lab at Indiana University (Nakazato et al. 2006), previous studies in the Barrington Lab (Sigel and Barrington 2008), as well as modifications specific to this study.
Restriction of the genomic DNA was performed using the restriction nucleases EcoRI and MseI (New England Biolabs, Ipswich, Massachusetts, USA). Ligation of the sticky end adaptors (obtained from Invitrogen, Carlsbad, California, USA) to the restriction sites was also achieved in this initial reaction following the Wolf (2000) protocol. The following adjustments were made: 20µl of the enzyme master mix was made to avoid pipetting volumes under 0.4µl. The master mix had the following components: 0.02µl of MseI, 0.05µl of EcoRI, 0.025µl of T4 Ligase, and 0.655µl ddH2O were used per 1µl of total master mix product. Each reaction volume totaled 11µl, each containing 100ng of DNA suspended in 5.5µl ddH2O. Samples were incubated for two hours at 37°C in a thermal cycler. In preparation for pre-selective amplification 3µl of the restriction/ligation product was diluted with 24.5µl TE 0.1 buffer.
In the first round of PCR, preselective amplification was achieved with primers that complement the EcoRI and MseI adaptors plus one additional nucleotide i.e. MseI+C (5’ GAT GAG TCC TGA GTA AC 3’) and EcoRI+A (5’ GAC TGC GTA CCA ATT CA 3’). Each preselective reaction totaled 25µl, comprising the following: 3µl diluted restriction/ligation product, 2.5µl ExTaq Buffer (TaKaRa), 0.1µl Ex Taq DNA polymerase (TaKaRa), 0.75µl of 50mM MgCl2, 1.2µl of 2.5mM dNTP's, 16.45µl ddH2O, and 0.5µl of 10µM of each of the primers. Reaction samples were initially denatured for two minutes at 72°C, followed by 30 cycles of: 94°C for 30 seconds, 56°C for 30 seconds and 72°C for two minutes, ending with 30 minutes at 6°C. 15.5µl of the preselective amplification product was diluted with 100µl of TE 0.1 buffer. The quality of the undiluted preselective and restriction/ligation products were visualized on a 1.5% TBE-agarose gel.
In the second round of PCR, selective amplification was achieved using the MseI+CAG and EcoRI+AAC (NED, yellow) primers. Each selective primer has an identical sequence to its corresponding preselective primer with an additional two bases, the EcoRI primer is fluorescently tagged for visualization on the ABI 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, California, USA). Selective reactions were set up in 18µl aliquots comprising the following: 3.6µl of the diluted preselective DNA product, 1.8µl Ex Taq Buffer, 1.8µl Ex Taq DNA polymerase, 0.5µl MgCl2, 0.864µl dNTPs, 1.44µl of the 0.4mM MseI selective primer (Invitrogen), 3.66µl of the 0.08mM EcoRI selective primer (Invitrogen), 0.144µl BSA, and 5.872µl ddH2O. Reactions were initially denatured at 94°C for two minutes, followed by 13 cycles of 94°C for 30 seconds, 65°C for 30 seconds (reduced by 0.7°C per cycle), 72°C for two minutes, and 24 cycles of 94°C for 30 minutes, 56°C for 30 seconds, 72°C for two minutes, with a final hold at 72°C for 30 minutes.
Bonin, A., Ehrich, D., and Manel, S. 2007. Statistical analysis of amplified fragment length polymorphism data: a toolbox for molecular ecologists and evolutionists. Molecular Ecology. 16: 3737-3758
Nakazato, T., Jung, M. K., Housworth, E. A., Rieseberg, L. H., and Gastony, G. J. 2006. Genetic map-based analysis of genome structure in the homosporous fern Ceraopteris richardii. Genetics. 173: 1585-1597.
Sigel, E. S. and Barrington, D. S. 2008. Polyphyly of polyploidy species: an inquiry into the origin of Drypteris campyloptera and Dryopteris dilatata. M.Sc. thesis, Department of Botany, The University of Vermont, Burlington, VT.
Whitlock, R., Hipperson, H., Mannarelli, M., Butlin, K., and Burke, T. 2008. An objective, rapid and reproducible method for scoring AFLP peak-height data that minimizes genotyping error. Molecular Ecology Resources 8: 725-735.
Wolf, P. G. 2000. AFLP Protocol [online]. Available from http://bioweb.usu.edu/wolf/aflp_protocol.htm [cited 15 July 2008].
Vos, P., Hogers, R., Bleeker, M., Reijans, M., Lee, T. Y. D., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kulper, M. and Zabeau, M. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23: 4407-4414.
Please be in touch with me with your questions about and insights into the holly ferns - Dave Barrington, Proprietor