The Journal of Heredity 2001:92(6)
© 2001 The American Genetic Association 92:507-509
Brief Communication |
Inheritance of the Chloroplast Genome in Sorbus aucuparia L. (Rosaceae)
From Unité d'Ecologie et de Biogéographie, Université Catholique de Louvain, Place Croix du Sud 4-5, B-1348 Louvain-la-Neuve, Belgium.
Address correspondence to O. Raspé at National Botanical Garden of Belgium, Department of Bryophytes and Thallophytes, Borein van Bouchout, B-1860 Meise, Belgium, or e-mail: raspe{at}br.fgov.be.
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Abstract |
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Chloroplast DNA (cpDNA) inheritance was investigated in Sorbus aucuparia using progenies obtained from six controlled crosses between individuals of known haplotype. Polymerase chain reaction (PCR) amplification, followed by restriction analysis, was used to characterize 248 offspring for either of two polymorphic cpDNA fragments. All offspring exhibited the maternal haplotype, which indicates maternal inheritance of chloroplasts in S. aucuparia. Power analysis of the test showed that the frequency of paternal transmission of chloroplasts, if any, should not exceed 1.84% (with 99% confidence).
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Introduction |
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The genus Sorbus L. (Rosaceae: Maloideae), mostly distributed in northern temperate regions, comprises about 100 species of trees and shrubs (Clapham et al. 1962). Several of these species are commonly used for decorative planting, and the enormous variation in the color of fruit and autumn foliage provides a tremendous horticultural potential (McAllister 1986, 1996). In particular, several cultivars of Sorbus aucuparia have been selected and clonally propagated: cv. Fructu Luteo (with golden fruits), cv. Asplenifolia (which has deeply cut foliage), and cv. Sheerwater Seedling (which has a particularly upright habit). The taxonomy of the genus is complicated by the common occurrence of hybridization, as in other genera of the Maloideae (Campbell and Dickinson 1990). The resulting hybrids often are apomictic and locally distributed (Clapham et al. 1962; Hull and Smart 1984; Májovsk
and Bernátová 1996). Chloroplast DNA (cpDNA) markers have been increasingly used to address plant evolutionary issues. This is because of the markedly different characteristics of cpDNA compared to the nuclear genome, as well as the recent development of efficient molecular tools. The most striking and general differences between chloroplast and nuclear genomes are that the former show uniparental inheritance in most plant species (Birky 1995; Reboud and Zeyl 1994), a clonal mode of evolution (i.e., no recombination), and a slow rate of evolutionary change (Wolfe et al. 1987). These properties have been exploited in studies dealing with a variety of aspects of plant evolutionary biology such as phylogeny (Gielly and Taberlet 1994; Kron et al. 1999; Palmer et al. 1988), interspecific gene flow and hybridization (Ferguson et al. 1999), and plant phylogeography (Petit et al. 1997; Taberlet et al. 1998; Tremblay and Schoen 1999), as well as to get insights into the relative contribution of pollen and seed movement in intraspecific gene flow (McCauley 1995). Although maternal inheritance is the most common mode of transmission of the chloroplast genome in angiosperms, there is evidence of at least partially paternal transmission in many species (e.g., kiwifruit [Chat et al. 1999]; Turnera ulmifolia [Shore and Triassi 1998]; also see Reboud and Zeyl [1994]). It is therefore advisable to check the chloroplast inheritance if such markers are to be used for analyses assuming strict maternal inheritance of this genome. For example, Birky et al. (1989) showed that equilibrium values of organelle diversity within and between populations are sensitive to the degree of paternal transmission.
This study is part of an ongoing research program that is investigating the genetic variation and population structure of S. aucuparia in relation to the life history of the species. The aim of the present research was to test the hypothesis of strict maternal inheritance in S. aucuparia cpDNA in order to use cpDNA markers in population genetic studies. I used the Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) approach (Demesure et al. 1995) because it allows for analysis of a large number of samples, which is necessary to detect rare events of biparental or paternal transmission that might occur. To the best of my knowledge, this is the first study in the Maloideae aiming to test the maternal inheritance of chloroplasts in progeny arrays. The Maloideae, a subfamily of the Rosaceae, comprise several genera of significant economic importance, such as Malus (apple), Pyrus (pear), Cotoneaster, and Pyracantha.
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Materials and Methods |
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Plant Material
I performed six intraspecific crosses in wild populations located on the Plateau des Tailles (Upper Ardenne, Belgium). I enclosed inflorescences with mesh bags to exclude pollinators. Emasculation was unnecessary since the species has been shown to be self-incompatible using both experimental pollinations (Raspé et al. 2000a) and molecular analysis of the gene encoding self-incompatibility RNases (O. Raspé and J. R. Kohn, unpublished data). It has also been shown that unpollinated bagged inflorescences do not produce any fruit (Raspé et al. 2000a). Therefore it can be assumed that the seeds analyzed in this study were the result of neither selfing nor apomixis. Eight different individuals of known chloroplast DNA haplotype (Raspé et al. 2000b) were used for the performed crosses. The number of offspring analyzed per cross is given in Table 1. Two mutations (indels, i.e., insertions or deletions) were used to track the transmission of the chloroplast genome. In the cross 8.25 x 8.2, the two parents differed for a mutation (an indel of 10 bp) located in a fragment between the tRNA-Cys and tRNA-Asp encoding genes (CD fragment; Raspé et al. 2000b). In all other crosses the parents differed for an indel of 18 bp located in a fragment between the tRNA-Asp and tRNA-Thr encoding genes (DT fragment; Raspé et al. 2000b). I characterized each progeny by restriction analysis of one of these two cpDNA fragments amplified by PCR using the universal primers designed by Demesure et al. (1995) (Table 1).
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DNA Extraction, Amplification, and Restriction Analysis
DNA extraction, amplification, and restriction procedures were as described in Raspé et al. (2000b) and Saumitou-Laprade et al. (1999) and are briefly outlined below. Total DNA was isolated from young seedlings (only the two cotyledons were present) with a rapid procedure modified from Edwards et al. (1991). I set up PCR reactions using AmpliTaq polymerase (Perkin-Elmer) in 1x buffer with 3.5 mM MgCl2, 200 µg/ml bovine serum albumin, 200 µM dNTPs, and 0.2 µM of each primer. Amplifications were carried out by a first denaturation step of 5 min at 95°C, 30 cycles of 45 s at 92°C, 45 s at 57.5°C (fragment CD) or 48°C (fragment DT) and 4 min (fragment CD) or 2 min (fragment DT) at 72°C, followed by a final elongation step of 10 min at 72°C (Raspé et al. 2000b). Since the PCR products were too large to detect small indels less than 20 bp long, digestion with restriction endonucleases was necessary prior to electrophoresis of the fragments. The restriction enzymes best suited for each PCR fragment have been determined by Raspé et al. (2000b). The CD fragment was codigested with HinfI and NdeII, and electrophoresed on 6.5% polyacrylamide gels. The DT fragment was digested with HinfI and electrophoresed on 1.8% agarose gels. After staining with ethidium bromide, gels were photographed under ultraviolet (UV) light with a digital camera.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Among the 248 progeny tested, none showed the paternal haplotype or both maternal and paternal alleles (heteroplasmic progeny) (Figure 1), which indicates that the chloroplast genome is maternally inherited in S. aucuparia. I assessed the power of my analysis using the binomial model presented by Milligan (1992). According to this model, the probability of paternal transmission P can be computed with the following equation:
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Published data on chloroplast inheritance in the Rosaceae are scarce and often incidental. Matsumoto et al. (1997) reported that an apple cultivar, Malus x robusta, which is known to be the result of a cross between M. baccata and M. prunifolia, exhibited the same RFLP profile as M. baccata, the female parent, whereas it was different from the M. prunifolia profile. Similarly Kaneko et al. (1986) showed that five individuals of Prunus yedoensis were characterized by an RFLP fragment present in the putative female progenitor (P. pendula) but not in the male parent (P. lannesiana). To my knowledge, the only study of chloroplast inheritance in the Rosaceae, testing the transmission of a chloroplast marker in the progeny from a controlled cross, is that of Brettin et al. (2000). In their study, all 19 progeny analyzed from a cross between P. cerasus cv. Rheinische Schattenmorelle and cv. Erdi Botermo had a restriction fragment characteristic of the former, that is, the female parent.
In a previous article, a surprisingly low level of cpDNA differentiation among S. aucuparia populations was reported (GSTc = 0.286; Raspé et al. 2000b), which is less than half the GSTc value usually observed in other temperate tree species (see Raspé et al. 2000b for a comparison). It was suggested that this result could be accounted for by the species' life history (bird-dispersed seeds and colonizing habit). However, the GSTc estimate was based on the assumption of strict maternal inheritance of the chloroplast genome, and one could argue that the low level of differentiation we observed might be the result of occasional biparental inheritance. Indeed, biparental inheritance would lead to increased gene flow for cpDNA because chloroplasts would migrate in both seeds and pollen. The present study rules out this possibility. Birky et al. (1989, Figure 1) reported that with a level of paternal transmission as low as 2%, GST was only slightly lower compared to the case where there is no paternal transmission. Therefore, even if paternal leakage of chloroplasts occurred with a frequency of 1.84% (i.e., the maximum allowed by the data), it could not be solely responsible for the low observed differentiation among populations.
In conclusion, I have shown that cpDNA markers can be used as maternally inherited markers in population genetics studies in S. aucuparia.
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Acknowledgments |
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I thank L. Dhondt for technical help as well as R. A. Wesselingh, A.-L. Jacquemart, and three anonymous referees for comments on the manuscript. This research was supported by the Région Wallonne, Service de la Conservation de la Nature, and the Belgian National Fund for Scientific Research, where I was a postdoctoral researcher.
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Footnotes |
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Corresponding Editor: David B. Wagner
Received August 18, 2000
Accepted June 19, 2001
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References |
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