Supplementary Methods - Word file (24 KB )

Supplementary Methods
Sampling and DNA extraction:
A total of 348 individuals of U. minor and 92 individuals of U. glabra were used for the
analysis of chloroplast diversity. Most of these samples come from ex situ collections
from France (Cemagref, Eric Collin), Italy (CNR, Alberto Santini) and Spain (DGB,
Salustiano Iglesias). Additional field samples were collected from France (provided by
E. Collin), Greece (Stephanos Diamandis), Italy (A. Santini), Spain (Miguel A.
Cogolludo) and Britain (Max Coleman), sampled trees were at least 50 m apart, in order
to minimise relatedness and clonal origin.
Twenty-three of those U. minor samples (from Spain, England and Central Italy) were
genotyped using nuclear microsatellites and AFLPs.
Total DNA was isolated from leaves as described1.
Chloroplast DNA analysis:
Chloroplast DNA polymorphism was studied by PCR-RFLP procedure. A set of five
primer pairs (DT, SfM, HK, K1K2 and VL) were amplified as described2,3. PCR
products were digested with restriction enzymes TaqI (fragments DT, K1K2), MvaI
(SfM), HaeIII (HK) and HinfI (VL) following manufacturer’s (Boehringer Mannheim)
instructions. The digested fragments were separated on 8% polyacrylamide, 1xTBE
gels, and visualised using silver staining. For each polymorphic fragment in the
digestion profiles, length variants were numbered by decreasing molecular weight.
Haplotypes were defined according to combination of different variants observed in
fourteen polymorphic digestion fragments, considering the five amplification fragments
together. Arlequin 2.000 software4 was used to calculate the Minimum Spanning Tree
(MST) of the haplotypes.
Nuclear microsatellites:
Seven nuclear microsatellites (Ulmi1-11 (AY520826), Ulmi1-21 (AY520827), Ulmi198 (AY520829), Ulmi1-165 (AY520830), Ulmi1-177 (AY520832), Ulmi2-16
(AY520835), Ulmi2-20 (AY520837))5 were analysed. PCR amplification, fragment
visualisation and score were carried out as described5.
AFLP analysis:
AFLP analysis was performed as described6 with slight modifications7. Preamplification
was carried out using EcoRI + A / MseI + C primers. Two primer combinations were
used for selective amplification: EcoRI + ACT / MseI + CAG and EcoRI + ACT / MseI
+ CCA. EcoRI + ACT primer was labelled in its 5’ end with IRDye800 (MWG Biotech
AG, Germany). Amplified fragments up to 500 bp were resolved in 8% acrylamide, 7M
urea, 0.8xTBE gels on a 4200 Li-Cor automated DNA sequencer (Li-Cor Biosciences,
Lincoln, NE, USA). Only polymorphic scorable bands were considered in this analysis.
The presence / absence of these bands were visually scored by two persons
independently and binary coded (as 1 or 0). TREECON software8 was used to calculate
the distance matrix among samples (Nei and Li genetic distance option), which was
subjected to cluster analysis by UPGMA.
1. Dellaporta, S. L., Wood, J. & Hicks, J. B. A plant DNA minipreparation: Version II.
Plant Mol. Biol. Report 1, 19-21 (1983).
2. Demesure, B., Sodzi, N. & Petit, R. J. A set of universal primers for amplification of
polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Mol.
Ecol. 4, 129-131 (1995).
3. Dumolin-Lapègue, S., Pemonge, M. H., Petit, R. J. An enlarged set of consensus
primers for the study of organelle DNA in plants. Mol. Ecol. 6, 393-397 (1997).
4. Schneider, S., Roessli, D. & Excoffier, L. ARLEQUIN Ver. 2.000: a Software for
Population Genetics Data Analysis (Department of Anthropology, University of
Geneva, Switzeland, 1999).
5. Collada, C., Fuentes-Utrilla, P., Gil, L & Cervera, M. T. Characterization of
microsatellite loci in Ulmus minor Miller and cross-amplification in U. glabra Hudson
and U. laevis Pall. Mol. Ecol. Notes (in press).
6. Vos, P. et al. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23,
4407-4414 (1995).
7. Cervera, M. T. et al. Improved AFLP analysis of tree species. Can. J. For. Res.
30(10), 1608-1616 (2000).
8. Van de Peer, Y. & De Wachter, R. TREECON for Windows: a software package for
the construction and drawing of evolutionary trees for the Microsoft Windows
environment. Comput. Applic. Biosci. 10, 569-570 (1994).
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