Comparison of levels of genetic diversity detected with AFLP and microsatellite markers within and among mixed Q. petraea (Matt.) Liebl. and Q. robur L. stands
Envoyé à Silvae Genetica le 30/05/01
Stéphanie Mariette1*, Joan Cottrell2, Ulrike M. Csaikl3, Pablo Goikoechea4, Armin König5, Andrew J. Lowe6, Barbara C. Van Dam7, Teresa Barreneche1, Catherine Bodénès1, Réjane Streiff1, Kornel Burg3, Katrin Groppe5, Robert C. Munro6, Helen Tabbener2 and Antoine Kremer1
1INRA, Equipe de Génétique et Amélioration des Arbres Forestiers, BP45, F-33610 Cestas, France.
2Forest Research, Northern Research Station, Roslin, Midlothian, Scotland, EH25 9SY, United Kingdom.
3Österreichisches Forschungszentrum, Seibersdorf Ges. m. b. H., A-2444 Seiberdorf, Austria.
4NEIKER, Dpto Producción y Protección Vegetal, Granja Modelo-Arkaute, Apdo 46, 01080 Vitoria Gasteiz, Spain.
5BFH, Institut für Forstgenetik und Forstpflanzenzuechtung, Sieker Landstrasse 2, 22927 Grosshandorf, Germany.
6Centre for Ecology and Hydrology, CEH, Edinburgh Bush Estate, Penicuik, Midlothian, EH26 0QB, Scotland, United Kingdom.
7Alterra Green World Research, POB 47, Droevendaalsesteeg 3, 6700 AA Wageningen, The Netherlands.
*Corresponding author :
INRA, Laboratoire de Génétique et Amélioration des Arbres Forestiers, BP45, 33610 Cestas, France.
Running title: AFLP markers, microsatellites and Quercus spp.
In this study, we compare the genetic diversity within and among Quercus spp. populations assessed with two contrasting types of molecular markers: a limited number of highly polymorphic microsatellite markers and numerous less informative AFLP markers. Seven mixed stands of Quercus petraea and Quercus robur were analysed with six microsatellite markers and 155 AFLP loci. Genetic differentiation and genetic diversity within each population was assessed. The intra- and inter-locus variances were calculated and the results were used to compare the genetic diversity between populations. The rankings of populations provided by the two types of markers were compared. The results obtained with the two types of markers revealed the same general trend. The genetic diversity within population and the genetic differentiation among populations were greater in Q. petraea than in Q. robur. The genetic differentiation was generally higher when AFLP markers were used as compared to microsatellites, and it was also the case when only polymorphic AFLP fragments were used. For AFLP, the inter-locus variance was always much higher than the intra-locus variance, and explains why it was not possible to distinguish populations based on the level of diversity for this marker system. Finally, no significant positive correlation was found between the level of within-population assessed with the two markers.
Keywords: microsatellite, AFLP, genetic diversity, genetic differentiation, Quercus robur, Quercus petraea.
The assessment of genetic diversity with molecular markers in natural populations follows a two-stage sampling: (i) sampling of populations and individuals and (ii) sampling of loci within the genome. The associated components of sampling variance have been termed “intra-locus variance” and “inter-locus variance” respectively and in theory the inter-locus variance should be much higher than the intra-locus component (Nei 1987). On the basis of allozymic data, Nei concluded that “a large number of loci should be examined even if the number of individuals per locus is small”. The larger inter-locus variance is likely to be the result of large differences in the mutation rates across the loci within the genome. There have been major advances in molecular techniques in recent years and as a result there is currently a wide range of markers available (Karp et al. 1997). Markers such as microsatellites are based on sequence data and this makes their development expensive. However, they have the advantage that they are codominant markers. In contrast markers such as Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) are cheap to develop as no knowledge of DNA sequence is required for their development. They provide information on many loci which are randomly distributed throughout the genome; however they are usually dominant markers (Breyne et al. 1997). Consequently, for a given investment of time and money they provide information on a wider range of loci than microsatellites, but the information at a given locus is less specific. As a result, for a given amount of resources, two contrasting sampling strategies can be adopted to assess genetic diversity using molecular markers: (i) selection of highly informative markers at a few loci (microsatellites), (ii) sampling of numerous less informative markers randomly distributed within the genome (RAPD or AFLP). It is not yet clear that these two extreme strategies will produce similar results when used to measure within- and among-populations diversity. Most of studies that compare different types of markers focused on allozymes and RAPD markers: Baruffi et al. (1995), Cagigas et al. (1999), Isabel et al. (1995), Lannér-Herrera et al. (1996) or Le Corre et al. (1997) all gave allozyme and RAPD diversity data sets that do not provide congruent results. Still few comparative studies involve AFLP though they provide a high number of markers. We report here on the comparison of the levels of genetic diversity within and among populations of two closely related white oak species Q. petraea (Matt.) Liebl. and Q. robur L. using these two contrasted types of nuclear molecular markers: microsatellite and AFLP.
The two species are sympatric and generally occupy different but proximal ecological niches. However, leaf and fruit interspecific differences are clearly recognized (Dupouey & Badeau 1993). Genetic variation in Q. petraea and Q. robur populations has previously been analysed in several studies using allozymes (Müller-Starck & Ziehe 1991, Kremer et al. 1991, Müller-Starck et al. 1993; Kremer & Petit 1993). Nevertheless, due to different population sampling strategies, results were not congruent and could not be directly compared. Zanetto et al. (1994) showed that the level of diversity within Q. petraea populations is slightly higher than within Q. robur and that the proportion of variation partitioned among populations within species is low for both species. In addition, Q. petraea populations are more differentiated than those of Q. robur. Studies with allozymes also showed that both species share the same alleles and exhibit only small differences in allele frequencies, the two species exhibit extremely low genetic intraspecific differentiation. Analysis of total proteins confirmed results found with allozymes that is a low level of genetic differentiation between Q. petraea and Q. robur (Barreneche et al. 1996). Bodénès et al. (1997b) investigated the geographic variation of the species differentiation throughout their natural range. After screening 2800 PCR amplification products using random primers, they found only two per cent of the amplified fragments that exhibited significant frequency differences between the two species and none of them was species specific. Finally, Bacilieri et al. (1994) and Streiff et al. (1998) studied the spatial genetic structure of the two species in the same oak mixed stand (“La Petite Charnie”) respectively with allozymes and microsatellites: only slight differences in the levels of genetic diversity were found between the two species.
Within the framework of a research project supported by the European Union, seven mixed Q. petraea and Q. robur stands were selected in six different countries. Within each stand, every tree was analysed with six microsatellite markers by each laboratory within the project. In addition, approximately 45 samples of each species from each stand were screened with 155 AFLP loci by one of the laboratory (INRA). These data sets were used to compare the levels of diversity within and between the two species. The objectives of this study were twofold: (i) to compare contrasting marker systems for the assessment of gene diversity in oaks. The main purpose was to verify whether these markers would provide the same ranking when populations (within each species) were ordered by their level of diversity and (ii) to compare the level of diversity among populations (within each species) by considering the whole genome. A method was developed here to assess the inter-locus sampling variance and the intra-locus sampling variance of gene diversity and was used to compare the level of diversity between populations.