The Journal of Heredity 2002:93(4)
© 2002 The American Genetic Association 93:293-300
Brief Communication |
A Case of Chloroplast Heteroplasmy in Kiwifruit (Actinidia deliciosa) That Is Not Transmitted During Sexual Reproduction
From Unité de Recherches sur les Espèces Fruitières et la Vigne, INRA, B. P. 81, F-33883 Villenave d'Ornon Cedex, France (Chat, Decroocq, and Decroocq); and Laboratoire de Génétique et d'Amélioration des Arbres Forestiers, INRA, B. P. 45, F-33611 Gazinet Cedex, France (Petit).
Address correspondence to J. Chat at the address above, or e-mail: chat{at}bordeaux.inra.fr.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
We report the first case of plastid chimera within the Actinidia genus, where plastid inheritance was believed to be paternal. The heterogeneity of chloroplast DNA observed in the hexaploid Actinidia deliciosa cultivar D uno involves the presence or absence of a particular MspI restriction site in the region between the psbC gene and the tRNA-Ser(UGA) gene. The heterogeneity was first observed using restriction fragment length polymorphism and then confirmed through cloning and sequencing. The analysis of the cloned fragments revealed the presence of two haplotypes: the most frequent type was found in 123 (88.5%) out of a total of 139 colonies screened. Partial sequences of the psbC-trnS fragment from both haplotypes revealed that the polymorphism occurs within the coding region of the psbC gene and consists of a synonymous transition. A contamination-free cross involving D uno as the male parent produced only plants characterized by the most frequent haplotype, indicating either selection bias against the rare type or more likely fixation of the frequent type in tissues leading to the formation of the male gametes. The MspI restriction profiles performed on various tissues suggest that the rarer type is absent from the histogenic layer LII and that D uno is a periclinal plastid chimera.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
In higher plants, each cell possesses many plastids and each plastid contains many copies of the circular plastid genome (Bendich 1987). Homoplasmy, defined as the homogeneity of organelle DNA within individual cells, is often observed and hence generally assumed. On the contrary, the occurrence of more than one type of organelle DNA in a given cell, called heteroplasmy, or in a given individual (called chimerism when sorting-out is complete) is infrequent. Homoplasmy is commonly explained by vegetative segregation and uniparental inheritance, occurring during vegetative and sexual reproduction, respectively (Birky 1983, 1995). Vegetative segregation, leading to sorting-out, has been compared with the classic concept of drift in population genetics, and applied to the population of organelle genomes in a cell lineage (Birky 1983). In the absence of mutation, homoplasmic cells produce only homoplasmic daughter cells after mitotic cell divisions. In contrast, when a heteroplasmic cell divides, the daughter cells may by chance receive only one type of organelle genome, leading to homoplasmic cells. After several mitotic divisions, the disappearance of heteroplasmic cells from the population is generally observed (Kirk and Tilney-Bassett 1978). The number of successive cell divisions leading to a complete sorting-out reaches theoretically 10N, where N is the number of organelle genomes in the mother cell (Michaelis 1955). Unless sorting-out is very slow, a fertilized heteroplasmic egg containing two kinds of plastids is thus expected to give rise to a mature plant consisting of clonal sectors with homoplasmic cells. Such chimeras are evident in variegated plants having sectors of green and white cells, such as in Pelargonium (Tilney-Bassett and Birky 1981). The prevalence of uniparental inheritance of organelles, which involves various molecular and cellular mechanisms (Birky 1995), also contributes to the higher frequency of homoplasmy.
Since then, heteroplasmy has been found in organisms belonging to various and distant groups. Mitochondrial heteroplasmy has been reported in many animals (Jenuth et al. 1996; Koehler et al. 1991; Solignac et al. 1987; Steel et al. 2000) including humans (reviewed by Chinnery et al. 2000). Plastid heteroplasmy has also been reported in a great number of plant taxa, for example, Gossypium (Lax et al. 1987), Medicago (Johnson and Palmer 1989; Lee et al. 1988), Oenothera (Chiu et al. 1988), Oryza (Moon et al. 1987), and Pelargonium (Tilney-Bassett and Birky 1981). Many of the cases of variegation known to date in cultivated plants are due to spontaneous mutations of the plastid genome (Kirk and Tilney-Bassett 1978). But heteroplasmy can also arise from biparental inheritance when each parent transmits its organelles to the zygote, as has been observed, for example, in Pelargonium (Tilney-Bassett and Birky 1981) or from uniparental inheritance when sorting-out in the parent is incomplete so that some heteroplasmic gametes are produced, as observed in Gossypium (Lax et al. 1987) and in Epilobium (Michaelis 1962). So far the extent of heteroplasmy has been difficult to estimate, as its detection was limited to the availability of phenotypic markers. As the number of molecular studies dedicated to the organelle genomes increases, additional cases of heteroplasmy should be discovered.
Previous studies in Actinidia, a genus comprising dioecious vines of various ploidy levels (2n = 2x to 6x) (Ferguson 1990), have established that plastids and mitochondria are inherited strictly paternally and strictly maternally, respectively. This unusual mode of inheritance among angiosperms was first demonstrated in interspecific crosses (Cipriani et al. 1995; Testolin and Cipriani 1997) before being confirmed at the intraspecific level in the kiwifruit (Actinidia deliciosa) (Chat et al. 1999). During our survey of plastid inheritance (Chat et al. 1999) we discovered that one region of the chloroplast (cp) DNA exhibits intraspecific variability within A. deliciosa. It is this polymorphism that allowed us to demonstrate the paternal cpDNA inheritance within controlled progeny of A. deliciosa. Later we realized that 1 of the 10 parents investigated could actually be heteroplasmic. No case of heteroplasmy had been reported so far in Actinidia.
In this study we demonstrate the presence of mixed populations of cpDNA molecules in the Actinidia cultivar D uno and we analyze the sequence polymorphism between the two types of cpDNA coexisting in that individual. We suggest different mechanisms to explain two apparently contradictory observations—the maintenance of cpDNA heterogeneity following asexual reproduction and the loss of cpDNA heterogeneity following sexual reproduction—by considering that organelle transmission is a quantitative trait rather than a qualitative one (Birky 1995), and by taking into account features of plant ontogenesis and reproduction.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
Plant Material
The plant material under study consists of the cultivar D uno, a male Italian rootstock selection (Bellini and Monastra 1986). This male was crossed with the female cultivar Hayward to produce the F1 population. Forty full sibs coming from the controlled cross performed by Chalak and Legave (1997) were planted in an orchard near Bordeaux and subsequently analyzed using molecular markers. The Corsica D uno parent has been propagated using node IBA-treated cuttings and one vegetative clone was kept to be grown in a container at INRA-Bordeaux. The D uno plant grown in Corsica and from which the pollen has been collected for the controlled cross is called the "original male parent" in the text, to distinguish it from its vegetatively propagated copy grown in Bordeaux, called the "vegetative clone." Both parents and all the progenies are hexaploids, as shown twice using flow cytometry (Chalak and Legave 1997). Two other genotypes have been used as controls in this study: M2, a putative source of pollen contamination for the F1 offspring, and Greensill.
Isolation of Genomic DNA
Total genomic DNA was isolated according to a modified CTAB procedure as detailed in Chat et al. (1999) or using the plant DNeasy kit (Qiagen). At least one DNA sample from each plant material was isolated from leaf tissue. Genomic DNA has been extracted from two leaf samples collected at random on the original male parent in 1995. Concerning the vegetative clone of D uno, several DNA samples were isolated from tissues collected in 2000: three DNA extracts isolated from separate leaf samples collected on distinct and consecutive developing buds, one DNA extract isolated from root samples collected at random on the vegetative clone, and one DNA extract isolated from stamens collected at random from flowers 1 or 2 days before anthesis. In 2001, additional DNA samples were isolated from three different leaf tissues of the vegetative clone of D uno, that is, the margin of the lamina, midrib, and petiole. All the DNA extracts were diluted at a final concentration of 5 ng/µl to be used as template.
Restriction Fragment Length Polymorphism (RFLP) of the cpDNA Region
The cpDNA region under study was located between the psbC and trnS genes, coding for the psII 44 kDa protein and the tRNA-Ser(UGA), respectively. Primers designed by Demesure et al. (1995) have been used to amplify this particular DNA region, called CS in the text. Conditions for polymerase chain reaction (PCR), digestion with the MspI restriction enzyme, and subsequent separation of the DNA fragments by electrophoresis have been described by Chat et al. (1999). Template used for PCR come either from total genomic DNA or from isolated bacterial colonies containing a plasmid with cloned CS fragments from the original male parent. The MspI restriction enzyme used in the present study is an isoschizomer of the HpaII used in Chat et al. (1999).
Cloning and Bacterial Transformation
Prior to cloning the CS region of the cpDNAs from D uno was amplified using the same conditions described above and purified through a QIAquick column. PCR products were then cloned using the pGEM-T Vector System kit following the manufacturer's instructions (Promega). Recombinant plasmids were introduced into Escherichia coli-competent cells (strain DH5
) using heat shock. Positive colonies were then selected on LB media containing 100-µg/ml ampicillin and the substrates 5-bromo-4-chloro-3-indol-ß-D-galactoside (X-gal) and isopropyl-1-thio-ß-D-galactoside (IPTG) (20 µg/ml each). Throughout, the term "colony" is used for the bacterial material, whereas the term "clone" is restricted to the plant material.
DNA Sequencing
Sequencing was carried out in both directions with M13 primers. The insert of at least two independent colonies has been sequenced for all haplotypes identified. For each type, sequences were aligned using Multalin version 5.4.1 (Corpet 1988) and a consensus sequence was obtained.
Amplification of Simple Sequence Repeat (SSR) from the Nuclear Genome
Six SSR primer pairs, UDK1996-001, -030, -406 (Huang et al. 1998) and 721, 722, 735 (Weising et al. 1996), were tested on the two parents, Hayward and D uno. As they all revealed polymorphism, they were subsequently used to determine the genotype of each of the 40 F1 offspring. PCR was performed using 10 ng of genomic DNA, 2 mM of MgCl2, 0.2 µM of each primer, 100 µM of dNTP, 1x PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), and 0.5 U Taq DNA polymerase in a total volume of 25 µl. Thermal cycling consisted of an initial denaturation step (94°C for 1 min) followed by 30 cycles each consisting of a 94°C denaturing step (50 s), a 55°C annealing step (50 s), and a 72°C elongation step (60 s). At the end of the reaction, a final extension period was included (72°C for 8 min). PCR products (1–1.2 µl/lane) were denatured at 94°C for 5 min in 60% formamide before being separated on a standard DNA sequencing gel containing 6% polyacrylamide (acrylamide:bis-acrylamide 19:1), 7.5 M urea, and 0.5x TBE at 80 W constant power for approximately 2 h.
Statistical Analyses
For SSR markers, we only selected alleles that were present in one parent and absent in the other. Given the difficulty to distinguish between one-dose versus multidose allele conditions within the F1 population, segregation analysis was performed based on the phenotype rather than the genotype (i.e., the segregation ratio corresponds to the presence [+] versus the absence [-] of the allele). Recent molecular data support the hypothesis of an autopolyploid origin of the hexaploid A. deliciosa (Testolin and Ferguson 1997; Testolin et al. 2001). In the gamete population originating from an autohexaploid with polysomic inheritance, ratios [+]:[-] of 1:1, 4:1, 19:1, and 1:0 are theoretically expected for alleles in simplex, duplex, triplex, and quadruplex conditions, respectively. In the present study, the maximum dosage observed for the maternal or paternal alleles was two (see Results section). The probability of the gamete being [+] corresponds to the random sampling without replacement of three alleles among the six parental ones (either paternal or maternal) and can be calculated according to the following formulas:
single dose allele:
 |
 |
The normal distribution, which approximates the binomial distribution in case of large samples, was used to derive the probability that a given male gamete of D uno (and subsequently a given F1 offspring) will inherit a particular ratio of cpDNA types (p) in the absence of bias. The mean ratio of each type of cpDNA considered for the normal distribution was obtained using the cloning strategy.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
Original Male Parent D uno
The D uno restriction pattern for the MspI digest of the CS fragment is shown in Figure 1. Two types of cpDNA, called type A and type B, can be distinguished within the D uno restriction pattern. The 400 bp restriction fragment observed in type B is cleaved into 300 bp and 100 bp restriction fragments in type A due to an extra MspI recognition site. Considering the relative intensities of the bands in the restriction pattern, it appears that D uno contains mostly type A cpDNA and only a few type B cpDNA. This pattern was repeated in different amplification reactions, where the 400 bp fragment always presented a reduced intensity after staining with ethidium bromide (data not shown). Moreover, as judged by visual inspection of the gels, we obtained similar relative band intensities of the restriction fragments when using different DNA extracts from the leaves of D uno as templates for PCR (Figure 1). Using DNA isolated from leaves collected in 1995 on the original male parent D uno, it was not possible to digest this 400 bp remaining fragment even when using supraoptimal restriction enzyme concentration (data not shown).
|
To test at which threshold a mixture of two populations of cpDNA template is detectable in our experimental conditions, we used a range of artificial mixtures of DNA extracts as templates for PCR amplification followed by digestion with MspI (data not shown). Twelve different mixtures ranging from 1% to 99% were obtained with DNA isolated from two apparently homoplasmic kiwifruit plants analyzed previously, that is, Greensill for type A cpDNA and Hayward for type B cpDNA (Chat et al. 1999). It appears that type B cpDNA is detectable in our experimental conditions when the corresponding ratio is at least 10% of the total. As judged visually, it seems that in the case of D uno leaves, the ratio of type B should be within the range of 10–30%.
To precisely evaluate the proportion of each population of cpDNA in D uno, but also to definitively confirm the occurrence of two cpDNA populations in this plant, we cloned the CS amplified fragments of D uno. Colonies containing a 1600 bp insert (size of the CS fragment) were analyzed by PCR-RFLP performed in the same way as for total genomic DNA (Figure 1). The relative ratio of each type of cpDNA was evaluated on 139 positive colonies. There were 16 type B colonies (11.5%), the 95% confidence interval being from 6.5% to 16.5%. This ratio is thus consistent with that established using artificial mixtures of DNA as templates in PCR.
To determine the molecular basis of the polymorphism, the CS insert fragments of two colonies per cpDNA type were sequenced. Both 5' and 3' terminal sequences of the CS fragments were obtained, corresponding to 540 bp and 600 bp long, respectively. No polymorphism was detected within the 3' terminal sequences. On the other hand, the 5' terminal sequences for the type A (EMBL: AJ459487) and type B (EMBL: AJ459488) cpDNA differ from each other by a single nucleotide at position 231 from the cloning site. It corresponds to the open reading frame of psbC at position 303. The substitution consists of a thymine (type B) being replaced by a cytosine (type A), or vice versa, resulting in a gain of restriction site for the MspI enzyme. The exact size of the MspI restriction fragments deduced from the sequences are as follows: 381 bp (type B) cleaved into 111 bp and 270 bp (type A), which correspond roughly to the sizes deduced from the acrylamide gels. The alignment performed between the psbC gene product of Arabidopsis thaliana (Swissprot: P56778) and the deduced amino acid sequence of A. deliciosa shows that the substitution is located at a silent third codon position of psbC.
Vegetative Clone of D uno
The vegetative clone of D uno was tested for cpDNA heterogeneity. For this purpose, leaves from three distinct and consecutive developing buds were collected in spring 2000. The PCR-RFLP results shown in Figure 1 revealed that all three DNA samples extracted contained both types of cpDNA in a proportion comparable to that of the original male parent. The type B cpDNA was also detected in the margin of the lamina, the midrib, and the petioles of the leaves, as well as in the stamens (Figure 2). On the contrary, no type B cpDNA was detected in the roots.
|
Sexual Offspring of D uno
We have shown previously that all 40 offspring originating from the cross Hayward x D uno had inherited the major paternal type A cpDNA (absent from the maternal plant). These results have been repeated for all 40 offspring in the present study and are illustrated for 15 of them in Figure 3. In order to confirm the parentage of the F1 plants, we used nuclear codominant markers. Six SSR primer pairs (Huang et al. 1998; Weising et al. 1996) that produced alleles present in one parent but absent in the other parent were selected. Genotypes of the parental clone have been determined unambiguously at 19 polymorphic loci. Assuming polysomic inheritance, chi-square tests indicated no segregation distortion in the F1 offspring for any of them. Moreover, the results obtained with 735 and 722 showed that SSR banding patterns of D uno and M2, a putative source of pollen contamination, differed from each other. As shown for 722 in Figure 4, half of the 40 F1 offspring exhibit the D uno-specific band (110 bp), but none of them exhibited that of M2 (106 bp). As a conclusion, the absence of significant deviation from the Mendelian expectation, as well as the absence of unexpected bands in the SSR banding patterns of the F1 population, excludes the possibility of pollen contamination.
|
|
From our experimental data, we know that type B cpDNA may not be detected under our experimental conditions if the corresponding ratio is lower than 10% of the total. No type B cpDNA has been detected from the visual inspection of the gel in any of the 40 F1 offspring. Given the proportion of type B in D Uno that was deduced from the cloning experiment (11.5%), the probability of not detecting type B by PCR in a given F1 offspring (computed using the binomial distribution) is relatively high (38.2%). On the other hand, if we consider the entire F1 population, the probability of not detecting type B in any of the offspring drops to (0.382)40 = 1.9 x 10-17.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
The variable cpDNA region of kiwifruit studied was initially identified in order to determine cpDNA inheritance in intraspecific crosses (Chat et al. 1999). During that study, polymorphic patterns for cpDNA were revealed among A. deliciosa clones using the CS-MspI combination. Among the 10 clones of A. deliciosa investigated in 1999, three clones—one male and two females—showed the extra MspI site that results in a 400 bp fragment being cleaved into two fragments. This extra site was shown here to result from a single silent substitution. In 1999 the male clone found to have the extra MspI site (D uno), but characterized also by the presence at lower frequency of the uncleaved fragment, had been misinterpreted as resulting from a partial digestion of the restriction enzyme site. Using a cloning strategy that provides a simple and efficient way to separate different PCR products, we have demonstrated here unambiguously that D uno possesses a mixed population of cpDNA. As judged visually from the restriction profiles, the two haplotypes appeared to be unequally represented in the leaf-sample DNA extract. Hypothesizing that no bias occurred during PCR or cloning, the proportion of the dominant type A cpDNA within D uno leaf tissue can be estimated at around 88%; such a proportion reflects only the cpDNA heterogeneity present in D uno leaf tissue, but may not hold for other organs.
How did that chloroplast heteroplasmy originate? It could be the result of plastid mutation at any stage of plant development (Kirk and Tilney-Bassett 1978). Frequently, however, plastid heteroplasmy is assumed to originate from biparental transmission of cpDNA. Biparental transmission of plastids was demonstrated as early as in 1909 in Pelargonium (Baur 1909). In the 1990s, documented analyses performed within the Actinidia genus revealed contrasted inheritance of its organelles, that is, paternal for the plastid and maternal for the mitochondria (Chat et al. 1999; Cipriani et al. 1995; Testolin and Cipriani 1997). No case of biparental cpDNA inheritance has been detected among the 262 Actinidia progenies analyzed so far. However, for a type II error of 1%, a rate of maternal transmission of cpDNA of up to 1.7% would be compatible with the available experimental data (Chat et al. 1999). Biparental inheritance could explain the origin of the cpDNA heterogeneity within D uno. This hypothesis is supported by the fact that both types of cpDNA, that is, type A and type B, already occur within A. deliciosa (Chat et al. 1999). Since D uno is a plant of unknown origin, unfortunately it was impossible to analyze the cpDNA of its parents. A de novo mutation seems an unlikely hypothesis to account for this case of heteroplasmy, since this scenario would require the occurrence of two independent mutation events within A. deliciosa leading to the same restriction profile. Our results indicate that the identification and characterization of within-individual cpDNA or mtDNA diversity, based on molecular techniques, may challenge our perception of the frequency of occurrence of biparental inheritance in plants.
A cross between the male cultivar D uno, containing both haplotypes (type A and type B), and the female cultivar Hayward, containing only the type B cpDNA, was analyzed to infer the transmission of the cpDNA heteroplasmic state across generations. Codominant nuclear molecular markers have here confirmed the parentage of the offspring. In our previous work, all the offspring have been found to inherit only the most frequent paternal cpDNA type (Chat et al. 1999). We have confirmed this finding using additional PCR-RFLP experiments with all the offspring. We can thus conclude that the rarer paternal type B is either absent from the offspring or below the detection threshold (i.e., below 10%). Given the high number of offspring analyzed, the results thus provide good evidence for the predominant transmission of the type A cpDNA molecule to the offspring, leading probably to the fixation of the major type A cpDNA within one generation.
To account for the maintenance of cpDNA heterogeneity after vegetative propagation and its loss after sexual reproduction, three hypotheses can be suggested: random drift consecutive to a bottleneck, selection bias in favor of the most frequent paternal type, or complete sorting-out resulting in a chimeric state, with the fixation of the major type A cpDNA in the tissue producing the male gametes.
Random Drift
Even if rare, the possibility of germ cells being heteroplasmic cannot be completely excluded. In plants, a plastid heteroplasmy was found to persist after several generations of sexual propagation in Gossypium (Lax et al. 1987) and in Senecio (Frey 1999). Hypothesizing heteroplasmy of the germ line cells of D uno, one possible explanation for the fixation of one cpDNA within a single sexual generation consists of drastic drift following a "germ-line bottleneck" (Chinnery et al. 2000). A plastid genetic bottleneck will occur when only a small proportion of the available cpDNA genomes repopulate the next generation. However, the probability given by the normal distribution not to detect any type B cpDNA among the 40 F1 offspring is very low (p = 1.9 x 10-17), suggesting that the absence of type B cpDNA in the offspring cannot be attributed to a genetic bottleneck only.
Selection Bias
Another explanation for the production of homoplasmic F1 offspring could be a selection bias. A selective advantage for a mutant or a wild-type cytoplasmic genome may operate during vegetative growth, but also during transmission from one generation to the next (Chinnery et al. 2000). Although the substitution differentiating the two haplotypes A and B is silent, cpDNA consists of a single linkage group, so that nonsilent substitutions or deleterious insertions or deletions may be linked with our marker. However, the fact that both cpDNA types were found fixed in different A. deliciosa individuals does not support the hypothesis of a strong detrimental effect of type B on the growth or the reproduction of the plant.
Chimera
A chimera is a stable genetic mosaic where at least two genetically distinct cell lineages coexist within the meristems (Szymkowiak and Sussex 1996). Of particular importance for the development of chimera are the developmental processes leading to the formation of the shoot apical meristem. The shoot apical meristem of most angiosperm species includes three histogenic layers: LI, LII, and LIII (reviewed in Szymkowiak and Sussex 1996; Tilney-Bassett 1986). As a consequence of vegetative segregation, two cpDNA molecules present in a cell are usually quickly sorted out into separate daughter cells (Birky 1983). Considering a heteroplasmic fertilized egg, each layer in a zygote could inherit either type of cpDNA. Vegetative segregation, associated with plant ontogenesis, would then produce stable plastid chimeras (Chesser 1998). The seemingly constant proportion of the two cpDNA types observed among the various leaf DNA samples of D uno suggests that sorting-out is complete.
In Actinidia, neither the number of meristem layers nor their contribution to the vegetative and reproductive organs have been investigated. Nevertheless, a hypothesis can be proposed, which is in agreement with most ontogenetic studies of dicotyledons and with our experimental data. In dicotyledons, epidermal tissues derive from LI, whereas the internal tissues of the stems, leaves, and flowers derive from both LII and LIII. Given the stable cpDNA mixture detected in the leaf tissues of D uno, it may be deduced that at least one of the three layers present in the shoot meristem contains the minor type B cpDNA. As the three consecutive buds of the vegetative clone exhibit the same restriction profile, the resultant chimera is more likely a periclinal chimera or a mesochimera. Germ cells that give rise to the gametes usually originate from LII (Marcotrigiano and Bernatzky 1995). Given that type B is not transmitted to the offspring, LII would be fixed for type A. Data from the three distinct leaf tissues provide information on the plastid genotypes of both LI and LIII. In general, all three layers contribute to the mature leaf. Near the midrib, and apart from the epidermis, LII usually gives rise to the palisade and lower mesophyll and LIII to the middle mesophyll. At the margins of the lamina, all the internal cells are generally derived from LII (Szymkowiak and Sussex 1996). For D uno, the margin of the lamina shows the same restriction profile as the midrib and the petiole, suggesting that the type B cpDNA is more likely present in LI than in LIII. This is in agreement with the restriction profile of the roots. Adventitious roots which form on a stem originate from the subepidermal tissues of the stem (Dolan et al. 1994; McPheeters and Skirvin 1983). Because adventitious roots from the vegetative clone of D uno exhibit only type A, it seems more likely that the layer containing type B is LI.
|
Conclusion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
In this study we have identified cpDNA heterogeneity within an Actinidia plant using restriction analysis, cloning, and sequencing. We have demonstrated that the polymorphism observed results from a silent substitution within the coding region of the plastid gene psbC. We believe that the observed cpDNA heterogeneity of D uno is the result of biparental inheritance of cpDNA, leading to the development of a heteroplasmic egg prior to the formation of a plastid periclinal chimera. The absence of transmission of the minor type from D uno to the offspring provides conclusive evidence that it is absent from the LII. Results of amplification using template DNA isolated from different organs, as well as ontogenetic patterns from most dicotyledons, indicate that the rarer cpDNA type is probably present in LI rather than in LIII. As a conclusion, it is likely that D uno is a periclinal chimera with the following structure: BAA, the first letter referring to LI.
|
Acknowledgments |
|---|
This work was supported by a grant from the European Union (INCO-DC IC18-CT97-0183). The authors thank Armand Mouras for helpful discussions.
|
Footnotes |
|---|
Corresponding Editor: William F. Tracy
Received December 17, 2001
Accepted June 10, 2002
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
|---|
-
Baur E, 1909. Das wesen und die Erblichkeitsverhältnisse der "Varietates albomarginatae hort." von Pelargonium zonale. Z Vererbungsl 1:330–351.
Bellini E and Monastra F, 1986. Propagazione, problemi vivaistici, scelta varietale e miglioramento genetico dell'Actinidia. In: l'Actinidia in Italia, Roma, Italy, (Attalla L BE, Costa G, Eynard I, Fraccaroli S, Giulivo C, Gorini F, Lalatta F, Monastra F, Monzini A, Xiloyannis C, Youssef J, Ugolini A, eds). Rome: Ministero dell'Agricoltura e delle Foreste; 27–52.
Bendich AJ, 1987. Why do chloroplasts and mitochondria contain so many copies of their genome. Bioessays 6:279–282.[CrossRef][ISI][Medline]
Birky CW Jr, 1983. Relaxed cellular controls and organelle heredity. Science 222:468–75.
Birky CW, 1995. Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution. Proc Natl Acad Sci USA 92:11331–11338.
Chalak L and Legave JM, 1997. Effects of pollination by irradiated pollen in Hayward kiwifruit and spontaneous doubling of induced parthenogenetic trihaploids. Sci Hort Amsterdam 68:83–93.
Chat J, Chalak L, and Petit RJ, 1999. Strict paternal inheritance of chloroplast DNA and maternal inheritance of mitochondrial DNA in intraspecific crosses of kiwifruit. Theor Appl Genet 99:314–322.[CrossRef]
Chesser RK, 1998. Heteroplasmy and organelle gene dynamics. Genetics 150:1309–1327.
Chinnery PF, Thorburn DR, Samuels DC, White SL, Dahl HHM, Turnbull DM, Lightowlers RN, and Howell N, 2000. The inheritance of mitochondrial DNA heteroplasmy: random drift, selection or both. Trends Genet 16:500–505.[CrossRef][ISI][Medline]
Chiu W-L, Stubbe W, and Sears BB, 1988. Plastid inheritance in Oenothera: organelle genome modifies the extent of biparental plastid transmission. Curr Genet 13:181–189.[CrossRef]
Cipriani G, Testolin R, and Morgante M, 1995. Paternal inheritance of plastids in interspecific hybrids of the genus Actinidia revealed by PCR-amplification of chloroplast DNA fragments. Mol Gen Genet 247:693–697.[CrossRef][ISI][Medline]
Corpet F, 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890.
Demesure B, Sodzi N, and Petit RJ, 1995. A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Mol Ecol 4:129–131.[Medline]
Dolan L, Linstead P, Poethig RS, and Roberts K. 1994. The contribution of chimaeras to the understanding of root meristem organisation. In: Shape and form in plants and fungi (Ingram DS and Hudson A, eds). London: Academic Press; 195–208.
Ferguson AR. 1990. The genus Actinidia. In: Kiwifruit: science and management (Warrington IJ and Weston GC, eds). Auckland, NZ: Ray Richards; 15–35.
Frey JE, 1999. Genetic flexibility of plant chloroplasts. Nature (Lond) 398:115–116.[CrossRef][Medline]
Huang WG, Cipriani G, Morgante M, and Testolin R, 1998. Microsatellite DNA in Actinidia chinensis: isolation, characterisation, and homology in related species. Theor Appl Genet 97:1269–1278.[CrossRef]
Jenuth JP, Peterson AC, Fu K, and Shoubridge EA, 1996. Random genetic drift in the female germline explains the rapid segregation of mammalian mitochondrial DNA. Nat Genet 14:146–151.[CrossRef][ISI][Medline]
Johnson LB and Palmer JD, 1989. Heteroplasmy of chloroplast DNA in Medicago. Plant Mol Biol 12:3–11.
Kirk JTO and Tilney-Bassett RAE, 1978. The plastids. Their chemistry, structure, growth and inheritance. Amsterdam: Elsevier.
Koehler CM, Lindberg GL, Brown DR, Beitz DC, Freeman AE, Mayfield JE, and Myers AM, 1991. Replacement of bovine mitochondrial DNA by a sequence variant within one generation. Genetics 129:247–255.[Abstract]
Lax AR, Vaughn KC, Duke SO, and Endrizzi JE, 1987. Structural and physiological studies of a plastome cotton mutant with slow sorting out. J Hered 78:147–152.
Lee DJ, Blake TK, and Smith SE, 1988. Biparental inheritance of chloroplast DNA and the existence of heteroplasmic cells in alfalfa. Theor Appl Genet 76:545–549.
Marcotrigiano M and Bernatzky R, 1995. Arrangement of cell layers in the shoot apical meristems of periclinal chimaeras influences cell fate. Plant J 7:193–202.
McPheeters K and Skirvin RM, 1983. Histogenic layer manipulation in chimaeral "Thornless Evergreen" trailing blackberry. Euphytica 32:351–360.[CrossRef]
Michaelis P, 1955. Uber Gesetzmässigkeiten der Plasmon-Umkombination und über eine Methode zur Trennung einer Plastiden-, Chondriosomen-, resp. Sphaerosomen-, (Mikrosomen)- und einer Zytoplasmavererbung. Cytologia 20:315–338.
Michaelis P, 1962. Uber gehäufte Plastidenabänderungen I. Biologisches Zentralblatt 81:91–128.
Moon E, Kao TH, and Wu R, 1987. Rice chloroplast DNA molecules are heterogeneous as revealed by DNA sequences of a cluster of genes. Nucleic Acids Res 15:611–630.
Solignac M, Génermont J, Monnerot M, and Mounolou JC, 1987. Drosophila mitochondrial genetics: evolution of heteroplasmy through germ line cell divisions. Genetics 117:687–696.
Steel DJ, Trewick SA, and Wallis GP, 2000. Heteroplasmy of mitochondrial DNA in the ophiuroid Astrobrachion constrictum. J Hered 91:146–149.
Szymkowiak EJ and Sussex IM, 1996. What chimaeras can tell us about plant development. Annu Rev Plant Physiol Plant Mol Biol 47:351–376.[CrossRef][ISI]
Testolin R and Cipriani G, 1997. Paternal inheritance of chloroplast DNA and maternal inheritance of mitochondrial DNA in the genus Actinidia. Theor Appl Genet 94:897–903.[CrossRef]
Testolin R and Ferguson AR, 1997. Isozyme polymorphism in the genus Actinidia and the origin of the kiwifruit genome. Syst Bot 22:685–700.[CrossRef]
Testolin R, Huang WG, Lain O, Messina R, Vecchione A, and Cipriani G, 2001. A kiwifruit (Actinidia spp.) linkage map based on microsatellites and integrated with AFLP markers. Theor Appl Genet 103:30–36.[CrossRef]
Tilney-Bassett RAE, 1986. Plant chimaeras. London: Edward Arnold.
Tilney-Bassett RAE and Birky CW Jr, 1981. The mechanism of the mixed inheritance of chloroplast genes in Pelargonium. Evidence from gene frequency distributions among the progeny of crosses. Theor Appl Genet 60:43–53.[CrossRef]
Weising K, Fung RWM, Keeling DJ, Atkinson RG and Gardner RC, 1996. Characterisation of microsatellites from Actinidia chinensis. Mol Breed 2:117–131.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||