Methods for sampling, DNA extraction and AFLP analyses
In 2008, we mapped, and collected leaf or cambium samples of, all V. michelii saplings (d.b.h. < 10 cm, minimum height = 130 cm) in these two plots. Diameter at breast height (d.b.h.) was measured for each sapling. For genetic analysis, leaf or cambium samples were kept in 2 mL tubes with silica gel until DNA extraction.A total of 627 juveniles (374 in the logged plot, 253 in the control plot; Fig. 1) and 12 adult trees were collected and genotyped.
For all samples, total genomic DNA was extracted from cambium or leaf tissues according to the hexadecetyltrimethyl ammonium bromide (CTAB) procedure (Doyle and Doyle 1987). DNA quality was assessed by spectrophotometry or by agarose gel electrophoresis.Amplified fragment-length polymorphism (AFLP) reactions were performed following Voset al(1995). DNA samples were digested with the restriction enzymes EcoRI and MseI, and the digested fragments were ligated with EcoRI and MseI adapters. Pre-amplification was conducted using primers combinations with one additional selective nucleotide: EcoRI+A+ MseI+A. Selective amplifications were performed with the following primer combinations: MseI+AAA / EcoRI+AAG, MseI+AAA / EcoRI+ACC, and MseI+AAA / EcoRI+ATG. The EcoRIprimers for the selective amplifications were labeled fluorescently. Both amplification steps were performed using an ABI 9700 thermocycler (Applied Biosystems, USA). AFLP fragments were separated on an ABI 3130xl capillary sequencer (Applied Biosystems, USA) and scored using GeneMapper® v3.7 in terms of the presence or absence of identified AFLP fragments. Peaks were visually inspected and only those showing a clear presence/absence pattern, and whose peaks were higher than 20 units, were retained for subsequent analyses. AFLP markers with extremely low frequencies (pN/3, with N the number of genotyped individuals) were removed following Lynch and Milligan (1994) After the exclusion of extreme-frequency fragments, to exclude low-quality samples, individual samples were ranked according to the total number of peaks produced with all primer combinations, and the 5% lower tail was excluded from subsequent analyses.
After removal of AFLP bands with extreme frequencies, individuals with poor banding profiles, and markers involved in too many genotypic disequilibria, a dataset including genotypes for 11 mature trees and 524 juveniles (312 in the logged plot, of which 139 in the eastern subplot (P9E) and 173 in the western subplot (P9W); 212 in the control plot, of which 121 in the eastern subplot (P11E) and 91 in the western subplot (P11W)), characterised at 687 AFLP markers, was obtained.
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