Báo cáo khoa học: "Ribosomal DNA and chloroplast DNA polymorphisms in a mixed stand of Quercus robur and Q petraea"

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article Original Ribosomal DNA and chloroplast DNA polymorphisms in a mixed stand of Quercus robur and Q petraea RJ Petit DB A Kremer 2 Wagner 1 INRA, laboratoire de génétique et d’amélioration des arbres forestiers, BP 45, 33611Gazinet Cedex, France; 2 Department of Forestry, University of Kentucky, Lexington, KY 40546-0073, USA More than 70 trees belonging to the morphologically distinguishable species Quercus Summary — robur L and Quercus petraea (Matt) Liebl were sampled in a mixed stand located in western France. The ribosomal DNA repeat was characterized by a high level of length polymorphism; while chloro- plast DNA in our sample was nearly fixed at 2 previously identified polymorphic regions. Overall, very little differentiation was found between species using both markers. The implications for our un- derstanding of this complex of species are discussed. Quercus petraea / Quercus robur / gene flow/ diversity/ sympatry Résumé — Polymorphisme de l’unité ribosomique et de l’ADN chloroplastique dans une fu- taie mixte de chênes pédonculé et sessile. Plus de 70 chênes des 2 espèces Quercus robur L et Q petraea (Matt) Liebl, ont été échantillonnés dans une parcelle de régénération située dans l’ouest de la France. Nous avons étudié le polymorphisme de longueur de l’unité ribosomique ainsi que le polymorphisme de l’ADN chloroplastique. La région codant pour les gènes ribosomiques est très variable. Au contraire, les 2 régions de l’ADN chloroplastique étudiées sont pratiquement mono- morphes. La différenciation interspécifique pour ces deux marqueurs est négligeable. Les implica- tions de ces résultats pour notre compréhension de ce complexe d’espèces sont discutées. Quercus petraea / Quercus robur/ flux de gènes / diversité / sympatrique
INTRODUCTION tracted from young leaves taken from flushing buds on branches collected in winter and forced in the greenhouse later in the spring. DNA was also Molecular markers have already provided extracted from leaves of seedlings germinated in the greenhouse. After digestion of the DNA by en- biologists with an impressive amount of tax- donucleases, repetitive DNA fragments were re- onomic data. However, recent studies of vealed by ethidium bromide staining of 0.9% aga- chloroplast DNA (cpDNA) variation in plants rose gels after 36-48 h of migration at 1 V/cm. indicate that some species may share iden- Negatives of the gels were taken under UV illumi- tical cpDNA genotypes (Rieseberg and Sol- nation at 254 nm. Two chloroplast DNA polymor- tis, 1991). In the genus Quercus, we have phisms were studied using the restriction endonu- cleases HindIII and Cfol. Polymorphic fragments shown that some European white oaks were verified as chloroplastic by comparison with share their cpDNA genotypes and that the Southern (1975) blots using cpDNA probes (frag- pattern of cpDNA variation is primarily geo- ments of the cpDNA of Petunia hybrida digested graphic, regardless of the species sampled by PstI (Palmer et al, 1983)). Similarly, we found (Kremer et al, 1991).Wittemore and Schaal that HindIII-digested rDNA fragments could also (1991) found similar results in American be detected directly by ethidium bromide staining. Two non-overlapping gel zones, named rRNA1 white oaks. They also showed that riboso- and rRNA2 (fig 1) had fragments which hybridized mal DNA polymorphisms could be used to with the complete rDNA repeat of wheat (pTA 71, identify some oak species. In these 2 stud- cf Gerlach and Bedbrook, 1979). We present here ies, sample sizes per population were low. the results of the polymorphism observed in the We have therefore sampled more than 70 10 kb region (rRNA1). trees in a mixed oak stand and analyzed both molecular markers, in order to study intrapopulation and interspecific diversity. Measurement and scoring of the rDNA repeat polymorphisms MATERIALS AND METHODS Negatives were scanned using a laser densitom- By comparison with a commercially availa- eter. ble size marker (1 kb ladder, Bethesda Research Laboratories), the sizes of several monomorphic Sampling chloroplast fragments were estimated using the procedure described by Schaffer and Sederoff A full description of the stand is given in the chap- (1981).These fragments were then used as natu- ter by Bacilieri et al. This 4-ha stand is located in ral internal markers in each lane to estimate the the Petite Charnie Forest near Le Mans in west- sizes of polymorphic rDNA fragments. Indeed, ern France. In order to regenerate the stand be- the presence of size markers within a lane ena- fore the final harvest, 426 trees of both species bles a more accurate estimate of fragments in had been left by the foresters. Individual trees that lane than in other lanes, since there is often were sampled for our study in the pure Quercus at least a slight shift among lanes, caused, for ex- petraea zone, in the pure Q robur zone and in the ample by unequal amounts of DNA present in mixed Q robur/Q petraea zone. Species identifi- each lane or by smiling effects. cation was based on several morphological mark- ers as explained by Bacilieri et al (1992). RESULTS DNA extraction and analysis cpDNA The method of total DNA extraction and analysis Seventy-two individuals (adults or seed- of cpDNA variation has been described previous- lings from different mother trees) ly (Kremer et al, 1991).Adult-tree DNA was ex- were
48 of Quercus petraea and 24 of bands and a single seedling (1.4%) with 3 analyzed: Quercus robur. Seventy-one had the same bands. Since we have preliminary results genotype (with HindIII: variant 5.8 kb; with from controlled crosses indicating that Cfol: variant 4.3-4.5 kb). A single Q pe- these length variants behave as alleles of traea individual had the HindIII variant 2.6- a single gene locus (Petit, unpublished 3.2 kb and the Cfol variant 4.3-4.5 kb. data) and in order to estimate the length frequencies, length variants variant present in single-banded individuals were given a weight of 2 (ie, these individuals rDNA were considered homozygous). Frequency distributions are given in figure 2 for Quer- cus robur and in figure 3 for Q petraea. A Twenty-one adult trees and 8 seedlings (from seeds collected from 8 different G-test of comparison of the species with 7 mother trees) of Quercus robur and 29 variant classes (by pooling the smaller and adult trees and 12 seedlings (also from larger, less frequent, variants) was non- seeds collected on different trees) of Quer- significant at the 5% probability level (df = 6, P = 0.19). We therefore pooled the 70 cus petraea were analyzed. The sizes of individuals of both species to calculate the the variants ranged from 10.24 to 9.46 kb. In order to compare the species, we unbiased gene diversity (0.829) and its pooled the results from the adult trees and standard deviation (0.016) using Nei and seedlings. There were 27 individuals Roychoudhury’s (1974), equations 2 and (38.6%) with 1 band, 42 (60%) with 2 12.
DISCUSSION for nuclear genes.) Note that, despite the small number of chloroplast polymor- phisms studied, the high level of differenti- high level of total diversity, but a low lev- A ation found in our first study is representa- el of intrapopulation diversity (only 8.8% of tive of any cpDNA polymorphism if the total) was found for cpDNA polymor- recombination does not occur in the chlo- phisms in a previous study (Kremer et al, roplast genome. In some situations, how- 1991).This result was obtained with a ever, an important local mixing of two large number of populations but a small cpDNA genotypes was observed, and the sample size per population. Therefore, the analysis of cpDNA genetic structure of results obtained in the present study, such populations would be of great inter- which confirm that some populations can est. The absence of cytoplasmic differenti- indeed be almost fixed for a single cyto- ation between species found in the present type, support our initial sampling proce- study also reflects a more general trend dure for the study of cpDNA diversity and (Kremer et al, 1991). differentiation in oaks: a large number of The level of diversity of the rDNA re- populations with a limited number of indi- viduals rather than the reverse. (Clearly, peat, on the other hand, is extremely high (0.829). The average value of intrapopula- such a sampling scheme is not appropriate
tion diversity for isozymes in oaks is 0.134 volume) found large differences in allelic (Kremer and Petit, this volume). For Quer- frequencies between the species for most cus petraea it is 0.277 (Kremer et al, allozymes studied, especially in the adult 1991).Itis difficult to compare our esti- stage. Even though our sample size is mate with other published measurements smaller, it is clear that many allozymes are of rDNA diversity in natural plant popula- differentiated than the rRNA gene more re- tions, since sample sizes varied greatly. gion. Moreover, it is often not reported whether How should we interpret this discrepan- length polymorphism corresponds to 1 or cy among nuclear markers, and among more loci. As Learn and Schaal (1987) some nuclear markers and the cytoplasmic have shown, the amount of diversity can- markers? Spirito (1990) studied theoreti- not at present be predicted from character- cally the reduction of neutral gene flow istics such as life-history traits; this diversi- caused by a single selected gene in plants. ty ranges from no length variation at all to It is obvious from his results that, in alloga- extreme cases with up to 20 variants per mous plants, neutral genes unlinked to the plant in Vicia faba (Rogers and Bendich, selected gene are easily exchanged even 1987) and a great deal of within-population if the selection is high. Rieseberg and Sol- variation. In oaks, Bellarosa et al (1990) tis (1991) present empirical evidence indi- found that the variability of the rDNA units cating that cytoplasmic gene flow may be was low for Quercus suber and Q trojana. high even when nuclear gene flow is very Whittemore and Schaal (1991) state that low. This requires that the various cyto- for American white oaks, "appreciable types have similar selective values in the length variation was observed. All plants species nuclear backgrounds. Information examined contained repeat types between about selective pressures (ie, disruptive 9 and 10.5 kb in length, each individual selection) that preserve species integrity having from one to three repeat types in despite high gene flow are sorely needed this length range. Variation within this to improve our understanding of this com- range is high within populations, and these plex of species. length variants are not useful for compar- ing different species or localities."They did, however find a shorter repeat type dis- REFERENCES tinctive of a group of species. In contrast, a shorter repeat is also present in the oaks Bacilieri R, Roussel G, Ducousso A (1993) Hy- we studied (rRNA2 region) but it is not bridization and mating system in a mixed specific to one of them. stand of sessile and pedunculate oak. Ann Sci For 50 (suppl 1), 122s-127s The absence of significant rDNA differ- Bellarosa R, Delre V, Schirone B, Maggini F between the species in the mixed ences (1990) Ribosomal RNA genes in Quercus oak stand was unexpected, because tan- spp (Fagaceae). Plant Syst Evol 172, 127- demly repeated DNA sequences, such as 139 the rRNA gene unit, are very often consid- Dover G (1982) Molecular drive: a cohesive ered to be excellent species markers. Do- mode of species evolution. Nature 299, 111- ver (1983) stated that molecular drive in 117 repeated gene families may lead to a co- Kremer A, Petit RJ (1993) Gene diversity in nat- hesive mode of species evolution, ie, spe- ural populations of oak species. Ann Sci For cies may become differentiated more 50 (suppl 1),186s-202s quickly than by drift alone. Moreover, in Kremer A, Petit RJ, Zanetto A, Fougère V, Du- the same population, Bacilieri et al (this cousso A, Wagner DB, Chauvin C (1991) Nu-
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