The basis of natural and artificial postzygotic hybridization barriers in Arabidopsis species

The success or failure of interspecific crosses is vital to evolution and to agriculture, but much remains to be learned about the nature of hybridization barriers. Several mechanisms have been proposed to explain postzygotic barriers, including negative interactions between diverged sequences, global genome rearrangements, and widespread epigenetic reprogramming. Another explanation is imbalance of paternally and maternally imprinted genes in the endosperm. Interspecific crosses between diploid Arabidopsis thaliana as the seed parent and tetraploid Arabidopsis arenosa as the pollen parent produced seeds that aborted with the same paternal excess endosperm phenotype seen in crosses between diploid and hexaploid A. thaliana. Doubling maternal ploidy restored seed viability and normal endosperm morphology. However, substituting a hypomethylated tetraploid A. thaliana seed parent reestablished the hybridization barrier by causing seed abortion and a lethal paternal excess phenotype. We conclude from these findings that the dominant cause of seed abortion in the diploid A. thaliana x tetraploid A. arenosa cross is parental genomic imbalance. Our results also demonstrate that manipulation of DNA methylation can be sufficient to erect hybridization barriers, offering a potential mechanism for speciation and a means of controlling gene flow between species.     

Rice interspecies hybrids show precocious or delayed developmental transitions in the endosperm without change to the rate of syncytial nuclear division

In angiosperms, interspecific crosses often display hybrid incompatibilities that are manifested as under‐proliferation or over‐proliferation of endosperm. Recent analyses using crosses between Arabidopsis thaliana and its related species with different ploidy levels have shown that interspecific hybridization causes delayed developmental transition and increased mitotic activity in the endosperm. In this study, we investigated endosperm development in interspecific crosses between diploid Oryza species. In a cross between female O. sativa and male O. punctata, we found that the hybrid endosperm was reduced in size and this cross was associated with precocious developmental transition. By contrast, the cross between O. sativa and O. longistaminata generated enlarged hybrid endosperm at the mid‐point of seed development and this cross was associated with delayed developmental transition. Subsequently, the hybrid endosperm displayed a shriveled appearance at the seed maturation stage. We found that the accumulation of storage products and the expression patterns of several marker genes were also altered in the hybrid endosperm. By contrast, the rate of syncytial mitotic nuclear divisions was not significantly affected. The gene OsMADS87 showed a maternal origin‐specific expression pattern in rice endosperm, in contrast to its Arabidopsis homologue PHERES1, which shows paternal origin‐specific expression. OsMADS87 expression was decreased or increased depending on the type of developmental transition change in the hybrid rice endosperm. Our results indicate that one of the interspecies hybridization barriers in Oryza endosperm is mediated by precocious or delayed developmental alterations and de‐regulation of OsMADS87, without change to the rate of syncytial mitotic nuclear division in the hybrid endosperm.     

Basis of difference between reciprocal crosses involving Triticum boeoticum and T. urartu

Summary The boeoticum () X urartu () F₁ hybrids gave small, plump and viable seeds while the reciprocal crosses with T. urartu as the female parent had long, shrivelled and non-viable seeds. Reciprocal nuclear-substitution lines comprising the nucleus of one species into the cytoplasm of the other were developed through repeated backcrossing and were crossed as female parents with respective non-recurrent parents (the cytoplasm donors). The difference between the reciprocal crosses was presumably attributable to different boeoticum urartu genomic ratios in the triploid endosperm rather than to the cytoplasmic difference between the diploid wheats. The endosperm with two doses of the boeoticum and one of the urartu genome resulted in small, plump and viable seed while the endosperm of the reciprocal crosses with two doses of the urartu and one of the boeoticum genome led to large but shrivelled and non-viable seeds irrespective of the cytoplasmic type. One dose of the paternal genome in the triploid endosperm is probably not expressed in the presence of two doses of the maternal genome thereby leading to the difference between the reciprocal crosses. The results reported here indicate that difference between reciprocal crosses may not always be attributed to cytoplasmic difference between the parental species.     

Genetic control of seed proteins in wheat

Summary Electrophoretic profiles of crude protein extracts from seed of F₁ hybrids and reciprocal crosses among diploid, tetraploid and hexaploid wheats were compared with those of their respective parental species. The electrophoretic patterns within each of three pairs of reciprocal crosses, T.boeoticum X T.urartu, T.monococcun X T. urartu and T.dicoccum X T. araraticum, were different from one another but were identical with those of their respective maternal parents. Protein bands characteristic of the paternal parents were missing in F₁ hybrid seed suggesting that the major seed proteins in wheat were presumably regulated by genotype of the maternal parent rather than by the seed genotype. However, in another three pairs of reciprocal crosses, T.boeoticum X T. durum, T.dicoccum X T.aestivum and T. zhukovskyi x T. aestivum, protein bands attributable to the paternal parents were present in the F₁ hybrid seeds indicating that the seed proteins were not always exclusively regulated by the maternal genotype. The expression of paternal genomes is presumably determined by dosage and genetic affinity of the maternal and paternal genomes in the hybrid endosperm. The maternal regulation of seed protein content is probably accomplished through the maternal control over seed size. The seed protein quality may, however, depend upon the extent of expression of the paternal genome.     

Polycomb group gene function in sexual and asexual seed development in angiosperms

In sexually reproducing angiosperms, double fertilization initiates seed development, giving rise to two fertilization products, the embryo and the endosperm. In the endosperm, a terminal nutritive tissue that supports embryo growth, certain genes are expressed differentially depending on their parental origin, and this genomic imbalance is required for proper seed formation. This parent-of-origin effect on gene expression, called genomic imprinting, is controlled epigenetically through histone modifications and DNA methylation. In the sexual model plant Arabidopsis, the Polycomb group (PcG) genes of the plant Fertilization Independent Seed (FIS)-class control genomic imprinting by specifically silencing maternal or paternal target alleles through histone modifications. Mutations in FIS genes can lead to a bypass in the requirement of fertilization for the initiation of endosperm development and seed abortion. In this review, we discuss the role of the FIS complex in establishing and maintaining genomic imprinting, focusing on recent advances in elucidating the expression and function of FIS-related genes in maize, rice, and Hieracium, and particularly including apomictic Hieracium species that do not require paternal contribution and thus form seeds asexually. Surprisingly, not all FIS-mediated functions described in Arabidopsis are conserved. However, the function of some PcG components are required for viable seed formation in seeds formed via sexual and asexual processes (apomixis) in Hieracium, suggesting a conservation of the seed viability function in some eudicots.     

Imprinting of the MEDEA polycomb gene in the Arabidopsis endosperm.

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, which is a tissue that supports embryo development. MEDEA (MEA) encodes an Arabidopsis SET domain Polycomb protein. Inheritance of a maternal loss-of-function mea allele results in embryo abortion and prolonged endosperm production, irrespective of the genotype of the paternal allele. Thus, only the maternal wild-type MEA allele is required for proper embryo and endosperm development. To understand the molecular mechanism responsible for the parent-of-origin effects of mea mutations on seed development, we compared the expression of maternal and paternal MEA alleles in the progeny of crosses between two Arabidopsis ecotypes. Only the maternal MEA mRNA was detected in the endosperm from seeds at the torpedo stage and later. By contrast, expression of both maternal and paternal MEA alleles was observed in the embryo from seeds at the torpedo stage and later, in seedling, leaf, stem, and root. Thus, MEA is an imprinted gene that displays parent-of-origin-dependent monoallelic expression specifically in the endosperm. These results suggest that the embryo abortion observed in mutant mea seeds is due, at least in part, to a defect in endosperm function. Silencing of the paternal MEA allele in the endosperm and the phenotype of mutant mea seeds supports the parental conflict theory for the evolution of imprinting in plants and mammals.     

Genomic imprinting and endosperm development in flowering plants

Genomic imprinting, the parent-of-origin-specific expression of genes, plays an important role in the seed development of flowering plants. As different sets of genes are imprinted and hence silenced in maternal and paternal gametophyte genomes, the contributions of the parental genomes to the offspring are not equal. Imbalance between paternally and maternally imprinted genes, for instance as a result of interploidy crosses, or in seeds in which imprinting has been manipulated, results in aberrant seed development. It is predominantly the endosperm, and not or to a far lesser extent the embryo, that is affected by such imbalance. Deviation from the normal 2m:1p ratio in the endosperm genome has a severe effect on endosperm development, and often leads to seed abortion. Molecular expression data for imprinted genes suggest that genomic imprinting takes place only in the endosperm of the developing seed. Although far from complete, a picture of how imprinting operates in flowering plants has begun to emerge. Imprinted genes on either the maternal or paternal side are marked and silenced in a process involving DNA methylation and chromatin condensation. In addition, on the maternal side, imprinted genes are most probably under control of the polycomb FIS genes.     

Maternal and paternal effects on the germination time of non‐dormant seeds of a monocarpic perennial species,Aster kantoensis (Compositae)

In plant species producing non‐dormant seeds, the germination time (from the start of imbibition to radicle emergence) is the main factor determining the timing of seedling emergence. We investigated maternal and paternal genetic effects on the germination time of non‐dormant seeds of a monocarpic perennial, Aster kantoensis Kitamura (Compositae). Three sets of reciprocal diallel crosses among five plants were conducted to produce genetic variation in seeds, and the germination time of the progeny of each parent was determined. The effects of the maternal parent and the interaction of maternal and paternal parents on the germination time of progeny were significant in all sets, and the effect of the paternal parent was significant in two of the three sets. This result means that the germination time of the progeny of a maternal or paternal parent can vary with the genotype of its mating partners. Because variation in the emergence time of seedlings contributes to avoiding seedling loss owing to unpredictable environmental changes, genetic variation in the germination time among the progeny of each parent mating with multiple partners could contribute to the establishment of the parent's seedlings in species producing non‐dormant seeds in the field.     

Genome‐wide screen of genes imprinted in sorghum endosperm,and the roles of allelic differential cytosine methylation

Imprinting is an epigenetic phenomenon referring to allele‐biased expression of certain genes depending on their parent of origin. Accumulated evidence suggests that, while imprinting is a conserved mechanism across kingdoms, the identities of the imprinted genes are largely species‐specific. Using deep RNA sequencing of endosperm 14 days after pollination in sorghum, 5683 genes (29.27% of the total 19 418 expressed genes) were found to harbor diagnostic single nucleotide polymorphisms between two parental lines. The analysis of parent‐of‐origin expression patterns in the endosperm of a pair of reciprocal F₁ hybrids between the two sorghum lines led to identification of 101 genes with ≥ fivefold allelic expression difference in both hybrids, including 85 maternal expressed genes (MEGs) and 16 paternal expressed genes (PEGs). Thirty of these genes were previously identified as imprinted in endosperm of maize (Zea mays), rice (Oryza sativa) or Arabidopsis, while the remaining 71 genes are sorghum‐specific imprinted genes relative to these three plant species. Allele‐biased expression of virtually all of the 14 tested imprinted genes (nine MEGs and five PEGs) was validated by pyrosequencing using independent sources of RNA from various developmental stages and dissected parts of endosperm. Forty‐six imprinted genes (30 MEGs and 16 PEGs) were assayed by quantitative RT–PCR, and the majority of them showed endosperm‐specific or preferential expression relative to embryo and other tissues. DNA methylation analysis of the 5’ upstream region and gene body for seven imprinted genes indicated that, while three of the four PEGs were associated with hypomethylation of maternal alleles, no MEG was associated with allele‐differential methylation.     

A study of hybridisation involving the sweet potato and related species

Overwhelming evidence points to an American origin for the sweet potato Ipomoea batatas (L.) Lam. Attempts have been made to identify related diploid species from Mexico, and to use these in hybridisation experiments with I. batatas. The sweet potato is a poor seed setter but abundant bloom occurs in Jamaica very late in the year. Attempts at hybridisation between I. batatas (2n=90) and I. trichocarpa (Elliott) (2n=30) or I. gracilis (2n=30) has been tolerably successful. A very high degree of self-incompatibility was demonstrated in all three species investigated but successful crosses were made using different plants of I. trichocarpa. An investigation of pollen viability showed that in all cases pollen could germinate but pollen tube growth was abnormal in incompatible pollinations. I. trichocarpa hybridised readily with I. batatas when the former was used as female parent. Embryo development in such a cross proceeded slowly, and stopped before cotyledon formation. No viable seeds were obtained. A comparison of embryo development in hybrid and normal seeds brought to light anomalies in development and structure of endosperm and maternal tissue in the hybrid.     

Parent-of-origin effects on seed development in Arabidopsis thaliana require DNA methylation

Some genes in mammals and flowering plants are subject to parental imprinting, a process by which differential epigenetic marks are imposed on male and female gametes so that one set of alleles is silenced on chromosomes contributed by the mother while another is silenced on paternal chromosomes. Therefore, each genome contributes a different set of active alleles to the offspring, which develop abnormally if the parental genome balance is disturbed. In Arabidopsis, seeds inheriting extra maternal genomes show distinctive phenotypes such as low weight and inhibition of mitosis in the endosperm, while extra paternal genomes result in reciprocal phenotypes such as high weight and endosperm overproliferation. DNA methylation is known to be an essential component of the parental imprinting mechanism in mammals, but there is less evidence for this in plants. For the present study, seed development was examined in crosses using a transgenic Arabidopsis line with reduced DNA methylation. Crosses between hypomethylated and wild-type diploid plants produced similar seed phenotypes to crosses between plants with normal methylation but different ploidies. This is consistent with a model in which hypomethylation of one parental genome prevents silencing of alleles that would normally be active only when inherited from the other parent - thus phenocopying the effects of extra genomes. These results suggest an important role for methylation in parent-of-origin effects, and by inference parental imprinting, in plants. The phenotype of biparentally hypomethylated seeds is less extreme than the reciprocal phenotypes of uniparentally hypomethylated seeds. The observation that development is less severely affected if gametes of both sexes (rather than just one) are 'neutralized' with respect to parent-of-origin effects supports the hypothesis that parental imprinting is not necessary to regulate development.     

Paternal regulation of seed development in wheat hybrids

Summary Diallel crosses among Triticum boeoticum (4 lines from different geographical areas), T.urartu, Aegilops squarrosa and Ae. speltoides exhibited reciprocal differences in hybrid seed morphology, endosperm development, and embryo viability. T. urartu and Ae. squarrosa as females with T. boeotiaum and Ae. speltoides lead to shrivelled inviable seed. T.boeoticum accessions as female with Ae.speltoides also lead to shrivelled seeds. The reciprocal crosses produced plump seeds which either resembled the maternal parent or showed size differences. By altering the endospermic genome ratios, hybrid seeds with 1 (PF)/1 (PM) showed extreme shrivelling whereas those with 4 (PF)/1 (PM) were medium shrivelled to plump. Genetic experiments involving hybrids of T. boeoticum, T. urartu and T. monococcum showed that a factor is present in pollen or male gametes, which shows dosage effect and which, by interacting with the maternal genome, leads to endosperm abortion.     

Asymmetric reproductive interference: The consequences of cross‐pollination on reproductive success in sexual–apomictic populations of Potentilla puberula (Rosaceae)

Apomixis evolves from a sexual background and usually is linked to polyploidization. Pseudogamous gametophytic apomicts, which require a fertilization to initiate seed development, of various ploidy levels frequently co‐occur with their lower‐ploid sexual ancestors, but the stability of such mixed populations is affected by reproductive interferences mediated by cross‐pollination. Thereby, reproductive success of crosses depends on the difference in ploidy levels of mating partners, that is, on tolerance of deviation from the balanced ratio of maternal versus paternal genomes. Quality of pollen can further affect reproductive success in intercytotype pollinations. Cross‐fertilization, however, can be avoided by selfing which may be induced upon pollination with mixtures of self‐ and cross‐pollen (i.e., mentor effects). We tested for reproductive compatibility of naturally co‐occurring tetraploid sexuals and penta‐ to octoploid apomicts in the rosaceous species Potentilla puberula by means of controlled crosses. We estimated the role of selfing as a crossing barrier and effects of self‐ and cross‐pollen quality as well as maternal: paternal genomic ratios in the endosperm on reproductive success. Cross‐fertilization of sexuals by apomicts was not blocked by selfing, and seed set was reduced in hetero‐ compared to homoploid crosses. Thereby, seed set was negatively related to deviations from balanced parental genomic ratios in the endosperm. In contrast, seed set in the apomictic cytotypes was not reduced in hetero‐ compared to homoploid crosses. Thus, apomictic cytotypes either avoided intercytotype cross‐fertilization through selfing, tolerated intercytotype cross‐fertilizations without negative effects on reproductive success, or even benefitted from higher pollen quality in intercytotype pollinations. Our experiment provides evidence for asymmetric reproductive interference, in favor of the apomicts, with significantly reduced seed set of sexuals in cytologically mixed populations, whereas seed set in apomicts was not affected. Incompleteness of crossing barriers further indicated at least partial losses of a parental genomic endosperm balance requirement.     

EMBRYO GROWTH AND SEED SIZE IN RAPHANUS SATIVUS: MATERNAL AND PATERNAL EFFECTS IN VIVO AND IN VITRO

Theories on the evolution of the angiosperm seed disagree as to the effects of different plant tissues on embryo growth. To examine the relative contributions of maternal and paternal genes on embryo growth, we conducted controlled crosses in the greenhouse with wild radish plants (Raphanus sativus), looked for maternal, paternal, and interaction effects on embryo development, and compared the performance of embryos within fruits and in embryo culture. Maternal plant identity affected fruit set, seeds per fruit, embryo developmental stage, and mean seed weight. In embryo culture, maternal effects were found for cotyledon size and embryo weight. Paternal effects were fewer or smaller in magnitude than maternal effects. The identity of the pollen donor affected embryo developmental stage and mean seed weight. In culture, paternal effects were detected for cotyledon size and embryo weight. Our results demonstrate that both maternal and paternal elements affect embryo growth. The fact that maternal effects are greater than paternal effects on embryo development in culture may result from cytoplasmic elements or maternal nuclear genes. Embryo performance in vivo compared to that in vitro varied among maternal plants. The interaction between an embryo and its endosperm and maternal tissues may be either positive or negative, depending upon the maternal plant and the embryo's developmental stage.     

Genetic control of Endosperm Balance Number (EBN): three additive loci in a threshold-like system

Summary The genetic control of Endosperm Balance Number (EBN) was investigated by a complete diallel of four exceptional diploid Solanum commersonii-S. chacoense hybrids (1 1/2 EBN) and backcrosses to their species parents, S. commersonii (1 EBN) and S. chacoense (2 EBN). Crosses in which the female parent had a higher EBN value than the male, S. chacoense (2 EBN)XF₁ (11/2 EBN) and F₁ (11/2 EBN)X S. commersonii (1 EBN), produced viable seed to aborted seed ratios of 11.1 and 11.3, respectively, and had average to small sized viable seed. Crosses in which the female parent had a lower EBN value than the male, S. commersonii (1 EBN)XF₁ (11/2 EBN) and F₁ (11/2 EBN)XS. chacoense (2 EBN), produced viable seed to aborted seed ratios of 1 7.9 and 1 6.7, respectively, and had average to large sized viable seeds. The results of these crosses appear to be consistent with the relative EBN values of the male and female parent. A model is proposed for the system regulating endosperm development. The assumptions of this model are: (1) three unlinked loci control the system; (2) the loci are homozygous within a species; (3) the genes have additive effects and are of equal strength within a species; (4) the genes within S. chacoense have twice the effect with respect to endosperm regulation as those within S. commersonii; and (5) a slight excess maternal dosage will produce the qualitative effect of small but viable seed. This model, in which quantitative genes operate in a dosage dependent system bears many similarities to classical, threshold-type genetic models.     

Exogenous selection rather than cytonuclear incompatibilities shapes asymmetrical fitness of reciprocal Arabidopsis hybrids

Reciprocal crosses between species often display an asymmetry in the fitness of F₁ hybrids. This pattern, referred to as isolation asymmetry or Darwin's corollary to Haldane's rule, is a general feature of reproductive isolation in plants, yet factors determining its magnitude and direction remain unclear. We evaluated reciprocal species crosses between two naturally hybridizing diploid species of Arabidopsis to assess the degree of isolation asymmetry at different postmating life stages. We found that pollen from Arabidopsis arenosa will usually fertilize ovules from Arabidopsis lyrata; the reverse receptivity being less complete. Maternal A. lyrata parents set more F₁ hybrid seed, but germinate at lower frequency, reversing the asymmetry. As predicted by theory, A. lyrata (the maternal parent with lower seed viability in crosses) exhibited accelerated chloroplast evolution, indicating that cytonuclear incompatibilities may play a role in reproductive isolation. However, this direction of asymmetrical reproductive isolation is not replicated in natural suture zones, where delayed hybrid breakdown of fertility at later developmental stages, or later‐acting selection against A. arenosa maternal hybrids (unrelated to hybrid fertility, e.g., substrate adaptation) may be responsible for an excess of A. lyrata maternal hybrids. Exogenous selection rather than cytonuclear incompatibilities thus shapes the asymmetrical postmating isolation in nature.     

REPRODUCTIVE ISOLATION OF TRITICUM BOEOTICUM AND TRITICUM URARTU AND THE ORIGIN OF THE TETRAPLOID WHEATS

The diploid wheats Triticum boeoticum and T. urartu are sympatric with one another throughout the geographic range of the wild tetraploids. Reciprocal crosses between ecogeographic types within each diploid species gave viable seed, but interspecific crosses consistently gave viable seed only when T. boeoticum was the female parent. Apparently urartu cytoplasm in combination with the boeoticum genome resulted in nonviable seed. The endosperm failed to develop normally despite regular endosperm fertilization. The F₁ plants obtained were completely self sterile although they showed regular intergenomic pairing (7II) at meiosis. Presumably the accumulation of cryptic differences between the two closely related genomes under reproductive isolation accounts for this sterility. The same accumulated cryptic differences could largely account for the preferential diploid pairing in the tetrapolid wheats which presumably were derived from such hybrids by chromosome doubling. The behavior of reciprocal crosses between the diploids and tetraploids suggested that T. boeoticum contributed the cytoplasm to both of the wild tetraploid species.