Journal of Heredity 2004:95(4):353-355
© 2004 The American Genetic Association
Brief Communication |
Maternal Inheritance of Mitochondria in Eucalyptus globulus
From the School of Plant Science and Cooperative Research Centre for Sustainable Production Forestry, University of Tasmania, Private Bag 55, Hobart, Tasmania 7001, Australia.
Address correspondence to René E. Vaillancourt at the address above, or e-mail: R.Vaillancourt{at}utas.edu.au.
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Abstract |
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It is important to verify mitochondrial inheritance in plant species in which mitochondrial DNA (mtDNA) will be used as a source of molecular markers. We used a polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) approach to amplify mitochondrial introns from subunits 1, 4, 5, and 7 of NADH dehydrogenase (nad) and cytochrome oxidase subunit II (cox2) in Eucalyptus globulus. PCR fragments were then either sequenced or cut with restriction enzymes to reveal polymorphism. Sequencing cox2 showed that eucalypts lack the intron between exons 1 and 2. One polymorphism was found in intron 2–3 of nad7 following restriction digests with HphI. Fifty-four F1 progeny from seven families with parents distinguishable in their mitochondrial nad7 were screened to show that mitochondria were maternally inherited in E. globulus. These results constitute the first report of mitochondrial inheritance in the family Myrtaceae.
It is important to determine the mode of inheritance of mitochondria in plants because this may impact on plant fitness through involvement in interspecific cross incompatibility, cytoplasmic male sterility, and disease resistance (Quenzar et al. 2001). Mitochondria are also commonly used as a source of genetic markers in studies of gene flow and phylogeography (e.g., Carreel et al. 2002; Dumolin-Lapègue et al. 1997a; Jøhnk and Siegismund 1997) and interpretation of such studies relies on knowledge of their mode of inheritance. Furthermore, the assumption of maternal inheritance, and thus dispersal by seed only, is the reason the mitochondrion is now viewed as a prime target for gene insertions. For example, Farre and Araya (2001) reported that the transformation of purified wheat mitochondria resulted in gene incorporation with faithful transient expression. Strict maternal inheritance should, in theory, imply that transgenes placed in organelles will be easier to contain than those located in the nucleus, because nuclear genes are transmitted by both seeds and pollen, and seed dispersal is lower than that of pollen in many plant species.
Mitochondrial inheritance in angiosperms is still relatively poorly studied, while in gymnosperms, a large fraction of the species, genera, and families have been studied (Reboud and Zeyll 1994). Mitochondria are generally believed to be maternally inherited in angiosperms, however, some exceptions have been found (e.g., Birky 1995; Havey 1997; Laser et al. 1997; Reboud and Zeyll 1994; Tsukamoto et al. 2000). In Chlamydomonas, strict paternal transmission of mitochondria is enforced by degradation of maternal copies of the mitochondria in the zygotes (Nakamura et al. 2003). In seed plants the mechanism(s) behind non-Mendelian inheritance of mitochondria is little understood, except in Hordeum vulgare, in which it appears to be due to degradation or exclusion of mitochondria during pollen development and occurs before fertilization (Sodmergen et al. 2002). Given that many studies used a relatively small sample size, it is possible that exceptions to strictly maternal mitochondrial inheritance in angiosperms are not rare. In several cases, nonmaternal inheritance has been found in interspecific hybrids or in some intraspecific cases only with some parents (Dulieu et al. 1990; Rajora and Mahon 1994). Chloroplast inheritance has been better studied. Among seed plants, chloroplasts may be inherited either biparentally or predominantly from the maternal or paternal parent (Birky 1995; Harris and Ingram 1991; Mogensen 1996). Maternal chloroplast inheritance has been reported for Eucalyptus (Byrne et al. 1993; McKinnon et al. 2001).
We used a polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) technique to analyze mitochondrial inheritance in the progeny from seven full-sib families of Eucalyptus globulus Labill. This species is native to Tasmania and southeastern Australia. While E. globulus is widely planted in temperate regions of the world and occupies at least 1.7 million hectares of plantation worldwide (Tibbits et al. 1997), its native trees are also exploited for wood/pulp production. This species has been the focus of evolutionary and population genetic studies (Freeman et al. 2001; McKinnon et al. 2004; Nesbitt et al. 1995). Demonstrating maternal inheritance for the mitochondrion will validate its use in E. globulus as a marker for seed dispersal.
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Materials and Methods |
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Mitochondrial inheritance in E. globulus was determined using trees from an incomplete diallel among eight parent trees (samples 529, 536, 655, 656, 657, 658, 659, 660) possessing widely different chloroplast haplotypes. By screening parents that were different in their cpDNA we hoped to maximize the chance of finding differences in their mtDNA. Progeny and parental DNA were extracted by McKinnon et al. (2001). We used the mitochondrial primers cox2/1-2r, nad4/2-3r, nad5/1-2r, nad7/2-3r, and nad7/3-4r from Dumolin-Lapègue et al. (1997b), and also nad1/b-c and nad4/1-2r from Demesure et al. (1995). PCR amplifications were performed as detailed in Dumolin-Lapègue et al. (1997b). PCRs of 20 µl contained primers at 0.2 µM, dNTPs at 100 µM, MgCl2 at 2 mM, 1x Taq buffer, 2 µg of bovine serum albumin (BSA), 1 U of Taq polymerase, and 20 ng of DNA template. All amplifications were carried out using 1 cycle of 4 min at 94°C, followed by 30 cycles of 45 s at 92°C, 45 s at 46°C or 56°C, 2 min at 72°C, and a final cycle of 10 min at 72°C. PCR optimization using a temperature gradient revealed an optimum annealing temperature of 46°C for cox2/1-2r and 56°C for all other primer pairs. PCR fragment size in E. globulus was compared to that of the following outgroups: Eucalyptus tenuiramis, Corymbia ficifolia, and Angophora floribunda (DNA from Steane et al. 2002).
DNA sequencing was undertaken on cox2/1-2r for the eight E. globulus parents and the outgroups. Sequencing was carried out using a Beckman Coulter Dye Terminator Cycle Sequencing kit, employing half reactions and these were electrophoresed on a Beckman Coulter CEQ 2000 automated sequencer. We looked for polymorphism in the other mitochondrial fragments using restriction digests. Restriction digest reactions (30 µl) contained 20 µl of PCR product, 1x buffer, 100 µg/ml of BSA, and 5.7 U of enzyme. TaqI digestions were incubated for 2 h at 65°C, while AseI, BamHI, BanI, BanII, BciI, BglII, BspEI, ClaI, EcoRI, HgaI, HincII, HphI, KpnI, MspI, NciI, NcoI, PflMI, PstI, SspI, XhoI, and XmnI digests were incubated at 37°C for 2 h. The number of restriction enzymes used differed between fragments (Table 1). Restriction digest products were concentrated in a vacuum centrifuge prior to electrophoresis. All PCR/digest products were visualized by ultraviolet (UV) fluorescence after electrophoresis on 2% agarose gels in 1x TAE containing ethidium bromide (0.8 µg/ml).
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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PCR product sizes are presented in Table 1. No polymorphism in uncut PCR fragment size was found among the E. globulus samples and outgroups. Nad4/2-3r and nad7/3-4r failed to amplify over the 40°C–60°C temperature range. Every DNA template tested was identical in cox2 sequence. Sequence comparisons with Oenothera villaricae (GenBank X00212) confirmed the PCR fragment identity as that of cox2. There was 97.2% sequence homology between the two cox2 sequences. We report that the cox2 intron between exons 1 and 2 was absent in E. globulus, Eucalyptus tenuiramis, Corymbia ficifolia, and Angophora floribunda. Only one of the mitochondrial fragment/restriction enzyme combinations tested (see Table 1) yielded polymorphism within E. globulus. This relatively low frequency of polymorphism in mitochondrial introns, especially because samples exhibiting plastid polymorphism were chosen for screening in order to improve the chances of finding polymorphism, is in accordance with other studies that show low rates of nucleotide substitutions in the plant mitochondrial genome (Palmer et al. 2000).
A polymorphism was discovered in nad7/2-3r whereby E. globulus 658 lacked a restriction site for the enzyme HphI (fragments = 850 and 450 bp), while all other samples (E. globulus and outgroups) possessed this restriction site (fragments = 520, 330, and 450 bp). All progenies available from crosses involving parent 658 were screened using this polymorphism. Our testing of mitochondrial inheritance in E. globulus across 7 different families and 54 samples failed to find any evidence of pollen parent transmission (Table 2). While the results cannot exclude low levels of mitochondria transmission through pollen, since only 54 samples were screened, they indicate that maternal inheritance is the norm for E. globulus, as it is for most plant species (Birky 1995; Reboud and Zeyll 1994). Mitochondrial markers can be used in E. globulus to study seed dispersal and phylogeography, although judging from the difficulty in finding polymorphism, using chloroplast markers would seem easier. This is the first report of mitochondrial inheritance in the family Myrtaceae.
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Acknowledgments |
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We would like to thank the Australian Research Council for funding this study.
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Footnotes |
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Corresponding Editor: James Hamrick
Received September 15, 2003
Accepted April 1, 2004
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References |
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