Multiple gene control of plastome-genome incompatibility and plastid DNA inheritance in interspecific hybrids of Zantedeschia

From interspecific hybridisation in the genus Zantedeschia, we have previously produced albino, variegated and virescent hybrids. The inhibition of chloroplast development in these hybrids is due to plastome-genome incompatibility. The albino hybrids did not develop prolamellar bodies in their etioplasts in darkness and formed only distended membranes, but no grana in light. This indicates that the block to chloroplast development occurs before or during the development of etioplasts. In albino leaf sectors of variegated hybrids, chlorophyll and carotenoid contents were only 2–4% of those in green plants. The mRNA levels for five plastid-development-related genes were also severely reduced, but they were still readily detectable using Northern hybridisation techniques. In the green sectors of variegated hybrids, chlorophyll content and mRNA levels of the fives genes were slightly reduced. We have earlier shown that F₁ hybrids between Z. aethiopica and Z. odorata were albino when Z. odorata plastids were present but virescent when Z. aethiopica plastids were present. When the F₁ hybrids with Z. aethiopica plastids were backcrossed to Z. odorata, the progeny were albino, virescent or green. Z. odorata plastids were inherited only from maternal parents in the F₁ progenies but inherited from either maternal or paternal parents in the backcross. The increased compatibility with, and inheritance of Z. odorata plastids in the backcross suggests that multiple genes play a major role in the plastome-genome incompatibility. An increase in the number of genes from the parent contributing the plastids improves chloroplast development in the hybrids of the genus Zantedeschia. Received: 29 November 1999 / Accepted: 2 December 1999     

The model plant Medicago truncatula exhibits biparental plastid inheritance

The plastid, which originated from the endosymbiosis of a cyanobacterium, contains its own plastid DNA (ptDNA) that exhibits a unique mode of inheritance. Approximately 80% of angiosperms show maternal inheritance, whereas the remainder exhibit biparental inheritance of ptDNA. Here we studied ptDNA inheritance in the model legume, Medicago truncatula. Cytological analysis of mature pollen with DNA-specific fluorescent dyes suggested that M. truncatula is one of the few model plants potentially showing biparental inheritance of ptDNA. We further examined pollen by electron microscopy and revealed that the generative cell (a mother of sperm cells) indeed has many DNA-containing plastids. To confirm biparental inheritance genetically, we crossed two ecotypes (Jemalong A17 and A20), and the transmission mode of ptDNA was investigated by a PCR-assisted polymorphism. Consistent with the cytological observations, the majority of F(1) plants possessed ptDNAs from both parents. Interestingly, cotyledons of F(1) plants tended to retain a biparental ptDNA population, while later emergent leaves tended to be uniparental with either one of the parental plastid genotypes. Biparental transmission was obvious in the F(2) population, in which all plants showed homoplasmy with either a paternal or a maternal plastid genotype. Collectively, these data demonstrated that M. truncatula is biparental for ptDNA transmission and thus can be an excellent model to study plastid genetics in angiosperms.     

Fungi in ectomycorrhizal associations of silver fir (Abies alba Miller) in Central Italy

 Naturally occurring ectomycorrhizas of silver fir (Abies alba Miller) were studied in two stands, one natural and one artificial, situated in Central Italy. A total of 25 mycorrhizal types was classified, for eight of which the mycobiont was identified at the species level. Analysis of macroscopic and microscopic features and matching of field-collected carpophores with associated mycorrhizas led to the tentative identification of several other types encountered during this study, at least at the genus level. No significant differences were noticed between natural and artificial stands in the relative richness of mycorrhizal types found on A. alba, indicating the maturity of the artificial stand with regard to succession of ectomycorrhizal fungi. Confocal laser scanning microscopy was used for visualization of mycorrhizal structures formed by Lactarius spp., without the need for specific staining with a fluorochrome, thanks to latex autofluorescence. This technique allowed observation of several structures in greater detail than with conventional light microscopy. Accepted: 13 February 1998     

Trisomy associated with loss of maturation capacity in a long-term embryogenic culture of Abies alba

 Karyological studies were made on a 6-year-old embryogenic cell line of Abies alba. Embryogenic cells were obtained from a mature zygotic embryo cultivated on modified MCM-medium and subcultured every 3 weeks. Three years after induction, part of the cell line was transferred to media supplemented with 500 or 1000 mg l^-1 caseine hydrolysate and 500 mg l^-1 L-glutamine. Approximately 3 years after addition of the organic nitrogen to the medium, morphological changes such as malformation of the suspensor cells and a loss of maturation capacity occurred. Chromosome counts revealed that all cells cultivated on medium with organic nitrogen were trisomic. Fluorescent-banding methods and comparison with an euploid cell line showed that the additional chromosome belonged to the group of long, metacentric chromosomes of Abies alba without secondary constriction. Those cells cultured on medium not supplemented with caseine hydrolysate and L-glutamine retained a stable chromosome number of 2n=24. Both normal and deformed suspensor cells were observed. The maturation frequency was very low. The emergence of aneuploidy within one cell line could be the consequence of high selection pressure caused by the different culture conditions. Received: 27 February 1997 / Accepted: 7 March 1997     

Cytological evidence for preservation of mitochondrial and plastid DNA in the mature generative cells of Chlorophytum spp. (Liliaceae)

Summary.  Following 4′,6-diamidino-2-phenylindole staining of mature pollen grains of Chlorophytum comosum, fluorescence microscopy confirmed that cytoplasmic nucleoids (DNA aggregates) were present in the generative cells, which indicated the possibility of biparental cytoplasmic inheritance. Electron and immuno-electron microscopy showed that both plastids and mitochondria were present in the generative cells, and both organelles contained DNA. These results indicate that mitochondria and plastids of C. comosum have the potential for biparental inheritance. Similar results were obtained with mature pollen grains of C. chinense. Therefore, we conclude the coincident biparental inheritance for mitochondria and plastids in the members of the genus Chlorophytum. Received June 28, 2002; accepted September 26, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: College of Life Science, Peking University, Bejing 100871, People's Republic of China.     

Potential for biparental cytoplasmic inheritance in Jasminum officinale and Jasminum nudiflorum

 Mature Jasminum officinale and J. nudiflorum pollen grains were stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined by epifluorescence microscopy. The pollen grains were found to be trinucleate, and the sperm cells in both species contained a large number of epifluorescent spots that corresponded to cytoplasmic DNA aggregates (nucleoids). The nucleoids of J. nudiflorum were observed to be dimorphic under the epifluorescence microscope, indicating that the sperm cells might contain both plastid and mitochondrial DNA. The nucleoids of J. officinale presented a similar appearance when stained with DAPI, but electron microscopic examination of the sperm cells revealed that they contained both plastids and mitochondria. When analyzed by DNA immunogold electron microscopy, gold particles were detected on both plastids and mitochondria. These findings demonstrated the preservation of plastid and mitochondrial DNA in mature sperm cells and thus the potential for biparental cytoplasmic inheritance in J. officinale and J. nudiflorum. Received: 8 August 1997 / Revision accepted: 25 February 1998     

Complementary genes control biparental plastid inheritance in Pelargonium

Summary Zonal pelargoniums exhibit biparental plastid inheritance. After G x W plastid crosses the progeny are a mixture of green, variegated and white embryos corresponding to a maternal, biparental or paternal inheritance of plastids, respectively. There are two patterns of segregation: type-I females have families in which the majority of embryos are green, variegated are of intermediate frequency and white are the least frequent. Type-II females have families in which green and white embryos are present at about the same frequency and variegated are the least common. The results of many selfs and crosses made within and between 8 type-I and 8 type-II plants led us to conclude that the type of female was determined by its genotype with respect to a pair of complementary genes. Plants giving rise to the type-II pattern contained one or two copies of the dominant alleles of both genes, whereas in the absence of either one or both dominant alleles the plants were type I. The genes were called Pr1/pr1 and Pr2/pr2, an adaptation of symbolism used previously. All 8 type IIs were double heterozygotes Pr1pr1, Pr2pr2, whereas we found 3 genotypes among the type Is, Pr1Pr1, pr2pr2; pr1pr1, Pr2Pr2 and pr1pr1, Pr2pr2. In unrelated experiments we found type IIs of which some were again double heterozygotes and others single heterozygotes Pr1pr1, Pr2Pr2 or Pr1Pr1, Pr2pr2. The model displaces an earlier model based on the proposed operation of a gametophytic lethal or incompatibility system.     

RFLP marker analysis supports tetrasonic inheritance in Lotus corniculatus L.

Lotus corniculatus is a tetraploid (2n=4x=24) perennial forage legume and has been reported to have tetrasomic inheritance for several traits, although it has also been reported to show disomic inheritance. Molecular markers were used to clarify whether tetrasomic inheritance, disomic inheritance, or a combination of both, was found within an F₂ population arising from a cross between two diverse L. corniculatus accessions. The inheritance of ”tetra-allelic” RFLP markers (markers with four segregating bands) indicated that disomic inheritance could not account for the phenotypic F₂ classes observed, and that only tetrasomic inheritance would explain the observed results. Goodness of fit tests for ”tetra-allelic” and ”tri-allelic” (three segregating bands) RFLP marker data suggested support for chromosomal-type tetrasomic inheritance. RFLP genotypes interpreted from autoradiographic signal intensity provided additional support for tetrasomic inheritance and the occurrence of preferential pairing between parental chromosomes. Bivalent pairing was predominant in the two parental lines and their F₁ hybrid in cytological analyses. L. corniculatus has been classified as both an autotetraploid and an allotetraploid species. RFLP evidence of tetrasomic inheritance gives support for L. corniculatus being classified as an autotetraploid species. Even though bivalent pairing occurs, as seen in other autotetraploid species, pairing between any of the four homologous chromosomes is possible. Preferential pairing in the F₁ hybrid suggests that genome differentiation appears to be minimal between homologs within an accession, while genome differentiation is greater between homologs from different accessions of this genetically diverse species. Received: 16 November 1999 / Accepted: 14 July 2000     

Quantitative cytology of the egg and central cell of Plumbago zeylanica and its impact on cytoplasmic inheritance patterns

Summary The egg and central cells of Plumbago zeylanica have an average volume of 543,000 m³ and 2,560,000 m³ respectively, with surface areas of 38,600 m² and 154,000 m². The egg contains an average of 39,900 mitochondria and 730 plastids. The majority of the plastids are perinuclear (> 60%) with less than 40% in lateral areas or near the filiform apparatus. After fertilization, the number of maternal organelles exceeds paternal organelles by a ratio of 11,000 for mitochondria and 154 for plastids. The central cell contains an average of 178,700 mitochondria and 1,840 plastids. After fertilization, these organelles far exceed the number of sperm organelles transmitted, by a ratio of approx. 14,000 for plastids and 1820 for mitochondria. Biparental inheritance of plastids in the embryo is possible, but not favored; the only comparable data in Oenothera and Impatiens reveals that biparental inheritance is possible in up to 124 ratios. Plants lacking biparental plastid inheritance do not contain plastids in the sperm, and thus the presence of even few sperm plastids may result in expression. The number of paternal mitochondria transmitted into the central cell is greater than that transmitted into the egg as the result of preferential fertilization with the mitochondrion-rich dimorphic sperm cell, although the ratio of paternal to maternal mitochondria is 11,000 in the egg and 1820 in the central cell. The similarity in these ratios suggests that there is a critical dosage of mitochondria that is permissible within the zygotic and endospermatic lineages. This may represent either: (1) a maximum permissible value to prevent expression of paternal mitochondrial genome, (2) a minimum ratio required in order to permit recombination of maternal and paternal mitochondrial genomes, or (3) a cytoplasmic genome balance number.Abbreviations mtDNA mitochondrial DNA - S_ua sperm cell unassociated with the vegetative nucleus - S_vn sperm cell physically associated with the vegetative nucleus     

Western white pine (Pinus monticola Dougl.) reproduction: II. Fertilisation and cytoplasmic inheritance

Fertilisation and proembryo development are described from transmission electron micrographs emphasising the origin and fate of the maternal and paternal mitochondria and plastids. During central cell and egg development mitochondria migrate toward the nuclei, forming a perinuclear zone consisting predominantly of maternal mitochondria and polysomes. At the same time, maternal plastids transformed and at fertilisation are excluded from the neocytoplasm. The pollen tube releases two sperm nuclei into the egg with cytoplasm from the generative cell and the tube cell. The leading sperm nucleus fuses with the egg nucleus and a small number of paternal mitochondria and plastids are taken into the perinuclear zone. The second sperm nucleus degenerates. As the zygote nucleus undergoes mitosis followed by free nuclear division and nuclear migration to the chalazal end of the archegonium, maternal and paternal organelles intermingle within the neocytoplasm. The result is paternal inheritance of plastids and biparental, but predominantly maternal, inheritance of mitochondria. This pattern is consistent within the Pinaceae but differs from some other conifer families. Received: 9 December 1999 / Revision accepted: 30 April 2000     

PCR-RFLP analysis of cpDNA in the genus Abies

 We used PCR-RFLP analysis of the chloroplast DNA of the genus Abies (family Pinaceae), to determine if the method could be employed to detect inter-specific variation in this genus and to study how the variation was distributed in different regions of the genome. Ten different chloroplast DNA regions, consisting of coding and non-coding DNA sequences, were amplified with specific primers in ten different Abies taxa. The amplification products were digested with several restriction enzymes. The results showed that the chloroplast genome is highly variable in most of the investigated taxa and contains multiple variable regions that appear to be distributed throughout the whole genome. Species-diagnostic markers were found for four of the ten investigated species. Unexpectedly, intra-specific variation was also detected in four species. It is likely that further studies, including larger sample sizes and/or more powerful methods for the detection of chloroplast DNA variation, will reveal additional variation for this genus. Received: 2 September 1998 / Accepted: 17 September 1998     

Quantitative,three-dimensional analysis of alfalfa egg cells in two genotypes: Implications for biparental plastid inheritance

In alfalfa (Medicago sativa L.), plastids are inherited biparentally. Patterns of plastid transmission vary according to the genotypes involved, but there is a strong bias in favor of male plastid transmission. Previous cytological studies on the male gametophyte of this species have not provided an adequate explanation for the differences in plastid transmission frequencies among genotypes. In the present study, we compared egg cells from genotypes classified as strong or weak plastid transmitters to determine whether plastid transmission strength is correlated with egg cell structure before fertilization. We found that plastids in the mature egg cells of the strong female (genotype 6–4) are significantly larger than in mature eggs of the weak female (genotype CUF-B), and that significantly more plastids are positioned in the apical portion of the mature egg cell of genotype 6–4 than in CUF-B. Immature eggs in the two genotypes show the same pattern as mature eggs with regard to plastid number and polarization. Since only the apical portion of the egg cell/zygote gives rise to the functional embryo, these results indicate that the potential input of female plastids, in terms of plastid size and number, may be an important factor in determining the inheritance patterns of these organelles in alfalfa.Support for this work by the United States Department of Agriculture under grant 88-37234-3876, the National Science Foundation under grant DCB-9103658, the Organized Research Fund of Northern Arizona University, and the Arizona Agricultural Experiment Station is gratefully acknowledged. We are indebted to Dr. Craig Caldwell, Northern Arizona University Computer Visualization Laboratory, for his expert help with the computer graphics.     

Spatiotemporal distribution of fruitbodies of ectomycorrhizal fungi in an Abies firma forest

 Spatial associations between ectomycorrhizal (ECM) fungi and their presumed host trees, and spatiotemporal associations among ECM fungi were surveyed for 3 years in an Abies firma-dominated forest in central Japan. A total of 39 species in 13 genera of ECM fungi were recorded, with more species in the Russulaceae than any other family. Russula ochroleuca, Russula sp.1 and Strobilomyces confusus tended to produce their fruitbodies on the forest floor directly under the crown of A. firma, whereas those of Inocybe cincinnata, Gomphus floccosus and G. fujisanensis were aggregated in limited areas outside the A. firma crown. Interspecific spatial associations were analysed for Russula sp.1, which was the most dominant species, and three other frequent species, I. cincinnata, S. confusus and R. ochroleuca. Pairwise, Russula sp.1 with I. cincinnata, with S. confusus or with R. ochroleuca showed an association which was exclusive, overlapping or independent, respectively. Fruiting phenologies differed in that S. confusus showed a peak density in the summer, whereas the other three species peaked in the autumn. These results suggest that the formation of ECM fruitbodies can be partitioned among the species both spatially and temporally. Accepted: 7 July 1998     

Genetic variation at chloroplast microsatellites (cpSSRs) in Abies nebrodensis (Lojac.) Mattei and three neighboring Abies species

Abies nebrodensis (Lojac.) Mattei (Sicilian fir) is an endangered species represented by only one population of 29 adult individuals occurring in a limited area of the Madonie Range in northern Sicily (Italy). Taxonomic boundaries between this taxon and the neighboring Abies species are not clear. In this study, we used six chloroplast simple-sequence repeats (cpSSRs) to investigate the population genetic structure and the distribution of chloroplast haplotypic variation in A. nebrodensis and three of the neighboring Abies species: Abies alba (Mill.), Abies numidica (De Lann) and Abies cepha-lonica (Loud.). Our aims were to quantify the level of cpDNA differentiation within the Abies populations and to shed light on the history of A. nebrodensis. Diversity levels based on the haplotype frequency at six cpSSRs were high, especially in A. alba and A. cephalonica. In all, we found 122 haplotypes among the 169 individuals analyzed, and the four species were distinguished from each other by their haplotype composition. The majority of the haplotypes (76%) were detected only once, but in A. nebrodensis seven individuals (41% of the sample population) shared the same haplotype. Moreover, the seven A. nebrodensis individuals with an identical haplotype showed a tendency to be geographically grouped within the population"s limited range. The analysis of molecular variance (AMOVA) showed a significant difference in the level of apportionment of gene diversity between the species A. alba and A. cephalonica (F_ST=0.191 and 0.012, respectively). AMOVA analysis conducted over all populations from the four species showed that 19% of the total cpSSR variation was attributable to differences among species, 6% was due to differences among populations within species, and 74% to differences within populations. The high percentage of unique haplotypes identified confirms the power of cpSSR haplotype analysis for identifying individual trees in individual Abies populations. Our results indicate that A. nebrodensis differs from the other three Abies species investigated and support its classification as an independent taxon. The results also showed a decreased level of variation in A. nebrodensis and suggested that the species has experienced a genetic bottleneck during the last two centuries. Received: 10 April 2000 / Accepted: 13 July 2000     

Differences in the initiation of the zygotic and parthenogenetic pathway in the Salmon lines of wheat: ultrastructural studies

 The ultrastructure of the egg apparatus of the sexual (aestivum)-Salmon line (aS) and the isogenic but alloplasmic (kotschyi)-Salmon line (kS) of the Salmon system of wheat was studied by transmission electron microscopy 3 days before and during anthesis. Additionally, the zygotic stage of aS, 17 h after pollination, was included. Metabolic activity of egg cells from the sexual line aS was low 3 days before anthesis and increased dramatically after pollination and fertilization. This timing of increased activity was evident because of changes occurring in the egg cell nucleus and nucleolus, polysomes, endoplasmic reticulum and Golgi apparatus, and the completion of the cell wall around the zygote. In contrast to the sexual line, the egg cell of the parthenogenetic line showed high activity 3 days before anthesis. The metabolic and ultrastructural characters observed in the nucleus and cytoplasm of the kS line 3 days before and during anthesis corresponded with those of the isogenic sexual line aS during anthesis and 17 h after pollination, respectively. High metabolic activity observed in the persistent synergid of kS may be connected with the occurrence of additional embryos in seeds (twins) of this line. Received: 25 November 1997 / Revision accepted: 16 April 1998     

Chloroplast DNA variation and evolution in the genus Lens Mill

 Chloroplast DNA (cpDNA) restriction site diversity was assessed by 21 enzyme/probe combinations in 30 accessions of six Lens species, including the recently recognized L. lamottei and L. tomentosus. A total of 118 fragments were scored and 26 restriction site mutations were identified. The cpDNA restriction pattern supports circumscribing L. lamottei and L. tomentosus as independent species. The value of the data for reconstructing phylogeny in the genus is discussed. The cpDNA of all 13 accessions of the lentil’s wild progenitor, L. culinaris subsp. orientalis, differed from that of the single lentil cultivars used in this study. This diversity indicates that other populations of this subspecies from Turkey and Syria examined by Mayer and Soltis (1994) are potentially the founder members of lentil. Examination of L. lamottei×L. nigricans hybrids between accessions having different restriction patterns showed paternal plastid inheritance in L. nigricans. Received: 2 July 1996 / Accepted: 19 July 1996     

The zygote and proembryo of alfalfa: quantitative,three-dimensional analysis and implications for biparental plastid inheritance

Summary Genetic studies have demonstrated biparental inheritance of plastids in alfalfa. The ratio of paternal to maternal plastids in the progeny varies according to the genotypes of the parents, which can be classified as strong or weak transmitters of plastids. Previous cytological investigations of generative cells and male gametes have provided no consistent explanation for plastid inheritance patterns among genotypes. However, plastids in the mature egg cells of a strong female genotype (6–4) were found to be more numerous and larger than in mature eggs of a weak female genotype (CUF-B), and the plastids in 6–4 eggs are positioned equally around the nucleus. In CUF-B, the majority of plastids are positioned below (toward the micropyle) the mid level of the nucleus, which is the future division plane of the zygote. Since only the apical portion of the zygote produces the embryo proper, plastids in the basal portion were predicted to become included in the suspensor cells and not be inherited. In the present study, we examined zygotes and a two-celled proembryo from a cross between CUF-B and a strong male genotype (301), a cross that results in over 90% of the progeny possessing paternal plastids only. Our results indicate that the distribution of plastids observed in the CUF-B egg cell is maintained through the first division of the zygote. Further, paternal plastids are similarly distributed; however, within the apical portion of the zygote and in the apical cell of the two-celled proembryo, the number of paternal plastids is typically much greater than the number of maternal plastids. These findings suggest that maternal and paternal plastid distribution within the zygote is a significant factor determining the inheritance of maternal and paternal plastids in alfalfa.     

Identifying different types of de-differentiated microspores from indica-japonica F1 hybrids with subspecies-differentiating RFLP probes in rice

The indica, japonica and intermediary types of de-differentiated microspores from indica-japonica hybrids were identified with 11 subspecies-differentiating RELP probes in rice (Oryza sativa L.). The results showed that the distribution of indica, japonica and intermediary types of de-differentiated microspores could be easily detected in a simple and quick way using the RFLP method. Moreover, the microspores from the same hybrid but inoculated onto different media, or microspores from different hybrids when inoculated onto the same medium, often displayed distinctive distribution curves of de-differentiated microspores types, indicating that the media employed in this experiment had high selectivity for the de-differentiation of certain types of microspores. The application of the RELP method to de-differentiated microspore identification is of great theoretical and practical significance in rice doubled-haploid breeding. Received: 27 February 1996 / Accepted: 14 June 1996     

Plastid DNA inheritance and plastome-genome incompatibility in interspecific hybrids of Zantedeschia (Araceae)

Plastid DNA (ptDNA) probes were used in RFLP analysis to determine ptDNA inheritance in interspecific hybrids in Zantedeschia. Biparental and maternal ptDNA inheritance was found in albino hybrids between the evergreen species Z. aethiopica and several winter-dormant species. From two albino hybrids, different types of ptDNA were detected in shoots derived from different parts of an embryo. This result indicates that plastids were sorted out during embryo development. Only maternal ptDNA was detected in the hybrids of Z. aethiopica × Z. odorata (a summer-dormant species) but paternal, biparental, and maternal ptDNA were found in the hybrids of the reciprocal cross. Z. odorata × Z. aethiopica. By correlating these ptDNA inheritance patterns with the leaf colour (albino, pale-green, and green) of the hybrids, it is suggested that the Z. odorata plastome is incompatible with the Z. aethiopica genome. The Z. aethiopica plastome is partially compatible with the Z. odorata genome but the development of Z. aethiopica plastids appears to be blocked by the presence of the Z. odorata plastids.