Overcoming the species hybridization barrier by ploidy manipulation in the genus Oryza

In most eudicot and monocot species, interspecific and interploidy crosses generally display abnormalities in the endosperm that are the major cause of a post‐zygotic hybridization barrier. In some eudicot species, however, this type of hybridization barrier can be overcome by the manipulation of ploidy levels of one parental species, suggesting that the molecular mechanisms underlying the species hybridization barrier can be circumvented by genome dosage. We previously demonstrated that endosperm barriers in interspecific and interploidy crosses in the genus Oryza involve overlapping but different mechanisms. This result contrasts with those in the genus Arabidopsis, which shows similar outcomes in both interploidy and interspecific crosses. Therefore, we postulated that an exploration of pathways for overcoming the species hybridization barrier in Oryza endosperm, by manipulating the ploidy levels in one parental species, might provide novel insights into molecular mechanisms. We showed that fertile hybrid seeds could be produced by an interspecific cross of female tetraploid Oryza sativa and male diploid Oryza longistaminata. Although the rate of nuclear divisions did not return to normal levels in the hybrid endosperm, the timing of cellularization, nucellus degeneration and the accumulation of storage products were close to normal levels. In addition, the expression patterns of the imprinted gene MADS87 and YUCCA11 were changed when the species barrier was overcome. These results suggest that the regulatory machinery for developmental transitions and imprinted gene expression are likely to play a central role in overcoming species hybridization barriers by genome dosage in the genus Oryza.     

Embryonic lethality leads to hybrid male inviability in hybrids between Drosophila melanogaster and D. santomea

The study of the morphological defects unique to interspecific hybrids can reveal which developmental pathways have diverged between species. Drosophila melanogaster and D. santomea diverged more than 10 million years ago, and when crossed produce sterile adult females. Adult hybrid males are absent from all interspecific crosses. We aimed to determine the fate of these hybrid males. To do so, we tracked the development of hybrid females and males using classic genetic markers and techniques. We found that hybrid males die predominantly as embryos with severe segment‐specification defects while a large proportion of hybrid females embryos hatch and survive to adulthood. In particular, we show that most male embryos show a characteristic abdominal ablation phenotype, not observed in either parental species. This suggests that sex‐specific embryonic developmental defects eliminate hybrid males in this interspecific cross. The study of the developmental abnormalities that occur in hybrids can lead to the understanding of cryptic molecular divergence between species sharing a conserved body plan.     

An interpretation of genomic balance in seed formation in interspecific hybrids of Solanum

Summary To investigate the mechanisms of seed failure in intraspecific and interspecific crosses of Solanum two diploid, S. commersonii and Group Phureja, and one tetraploid species, S. acaule, species were crossed and the seeds were analyzed for embryo and endosperm development. Many seeds of certain crosses observed seven days after pollinations were found to contain abnormal embryos and degenerating endosperms. In some cases seeds contained an embryo with no endosperm, or an endosperm with no embryo. Other interspecific crosses which were predicted to fail actually produced seeds with normally developed embryos and endosperms. To further characterize the intra- and interspecific embryos and endosperms the nuclear DNA was measured. There are several ways to explain the ploidy levels of embryos and endosperms among the crosses, the occurrence of unreduced gametes in some cases and genomic instability in other cases. The latter resulted in chromosome loss at meiotic and mitotic divisions. Genomic balance in interspecific seeds is critical to both embryo and endosperm development.Scientific Journal Series Article No. 14636 of the Minnesota Experiment Station     

Genotype effects of Brassica napus on its reproductive behavior after pollination with B. juncea

To investigate the cause of variation in the interspecific crossability of Brassica napus, three different genotypes were studied in respect of their reproductive behavior after pollination with B. juncea. There were great differences among maternal genotypes in allowing foreign pollen to germinate on and penetrate into their stigmas, leading to a wide diversity of interspecific fertilization. The division of the hybrid primary endosperm nucleus and zygote appeared normal in all combinations of crosses. While the abundant free nuclei of the endosperm developed properly and never became cellular, the embryos degenerated as early as 10 days after pollination when the cultivar Rucabo, which had the highest fertilization record with species of B. juncea, was involved. When 81007 was used as female parent, the endosperm grew a little but the embryo halted at the heart-torpedo stage. Lack of nourishment might be responsible for the observed embryo abortion. Among the sic hybrid combinations, the cross 84014A x Changyang hunagjie was the only one where endosperm tissue was observable and an abnormal embryo occurred prior to cellular endosperm formation. Apart from the three typical embryological features, significant variation was also demonstrated among each of the cross combinations. Genetic diversity appears to exist not only between varieties, but also within cultivars. In addition, methods for developing interspecific crossable lines are discussed.     

The progamic phase, embryo and endosperm development in an intraspecific Tulipa gesneriana L. cross and in the incongruent interspecific cross T. gesneriana × T. agenensis DC.

 The development of the embryo and endosperm has been investigated in an intraspecific Tulipa gesneriana cross and in the incongruent cross T. gesneriana ×T. agenensis at intervals of 10 days, from 12 to 82 days after pollination (DAP). In both tulip crosses, the zygote gives rise to an apparently undifferentiated cell mass, the proembryonal cell mass, on which a suspensor then develops. Subsequently, a globular embryo is formed on top of the suspensor. This embryo finally elongates, giving rise to a spindle-shaped embryo. The cellular endosperm fills the whole embryo sac in mature seeds, except for a region immediately around the embryo. In both crosses, aberrant developments were found. In the intraspecific T. gesneriana cross, the pollen tubes did not open in a number of ovules. In other ovules, the pollen tubes seemed to have opened, but an embryo or endosperm was not found or only endosperm was observed. In the cross T. gesneriana ×T. agenensis, fewer pollen tubes entered the ovules than in the intraspecific T. gesneriana cross. The ovules with embryo and endosperm formation of the incongruent interspecific cross showed, in general, retarded development in comparison with the intraspecific T. gesneriana cross. The first globular embryos and spindle-shaped embryos were found at the later fixation dates and the relatively lower number of spindle-shaped embryos at 82 DAP had a shorter average length. The number of ovules with deformations in embryo and/or endosperm development was also higher in the cross T. gesneriana × T. agenensis in comparison with the intraspecific T. gesneriana cross. Between 87% and 100% of the ovules with embryo and endosperm development showed normal development in the intraspecific T. gesneriana cross, while in the incongruent interspecific cross, from 22 DAP, between 17% and 56% of the ovules showed normal development. Of those ovules with aberrations in embryo and/or endosperm formation, about 80% had a deformed endosperm, of which more than 50% also contained a deformed embryo. Embryos of the incongruent cross might be saved by the application of embryo rescue techniques. Received: 10 December 1996 / Revision accepted: 23 April 1997     

LOSS OF HYBRID LETHALITY DURING BACKCROSS PROGRAMS INVOLVING CHLAMYDOMONAS EUGAMETOS AND CHLAMYDOMONAS MOEWUSII (CHLOROPHYCEAE)1

Wild-type strains of the interfertile species Chlamydomonas eugametos (UTEX 9 and 10) and Chlamydomonas moewusii (UTEX 96 and 97) male readily and reciprocally; however, considerable lethality occurs among F₁ hybrid meiotic products. We prepared two hybrid backcross lineages using C. eugametos and C. moewusii. One lineage began with the cross C. eugametos mating-type-plus (mt⁺) × C. moewusii mating-type-minus (mt^?). An F₁ mt⁺ hybrid from this cross was back-crossed to C. moewusii mt^?, and a B₁ mt⁺ hybrid was recovered. The B₁ hybrid was again backcrossed to C. moewusii mt^?, and this process was repeated through the fifth backcross. The other backcross lineage began with the reciprocal cross C. moewusii mt⁺× C. eugametos mt^? and employed C. eugametos as the recurring mt^? parent. This lineage also was continued through the fifth backcross. Meiotic product survival in the reciprocal interspecific crosses was less than 10%. In successive back-cross generations associated with both lineages, this value increased progressively to a maximum of 85–90%, the level observed for the intraspecific crosses. These results are consistent with the hypothesis that multiple genetic differences exist between C. eugametos and C. moewusii and that these are the major source of meiotic product lethality associated with the interspecific crosses. The inheritance of chloroplast genetic markers for resistance to streptomycin (sr-2) and for resistance to erythromycin (er-nM1) was also scored w the interspecific crosses and in the backcrosses. Most hybrid zygospores transmitted the resistance markers of the mt⁺ parent only, or of both parents, with the former zygospore type being more common. Although the intraspecific C. eugametos and C. moewusii crosses differ conspicuously with respect to the fraction of zygospores which transmit chloroplast genetic markers of both parents, the inheritance of chloroplast genetic markers in the interspecific crosses and backcrosses at' scribed here failed to clarify the genetic basis for this difference.     

Suppression of OsMADS7 in rice endosperm stabilizes amylose content under high temperature stress

High temperature significantly alters the amylose content of rice, resulting in mature grains with poor eating quality. However, only few genes and/or quantitative trait loci involved in this process have been isolated and the molecular mechanisms of this effect remain unclear. Here, we describe a floral organ identity gene, OsMADS7, involved in stabilizing rice amylose content at high temperature. OsMADS7 is greatly induced by high temperature at the early filling stage. Constitutive suppression of OsMADS7 stabilizes amylose content under high temperature stress but results in low spikelet fertility. However, rice plants with both stable amylose content at high temperature and normal spikelet fertility can be obtained by specifically suppressing OsMADS7 in endosperm. GBSSI is the major enzyme responsible for amylose biosynthesis. A low filling rate and high expression of GBSSI were detected in OsMADS7 RNAi plants at high temperature, which may be correlated with stabilized amylose content in these transgenic seeds under high temperature. Thus, specific suppression of OsMADS7 in endosperm could improve the stability of rice amylose content at high temperature, and such transgenic materials may be a valuable genetic resource for breeding rice with elite thermal resilience.     

Differentiation of a Miniature Inverted Transposable Element （MITE） System in Asian Rice Cultivars and Its Inference for a Diphyletic Origin of Two Subspecies of Asian Cultivated Rice

In the present study, we report a survey on a Miniature Inverted Transposable Element （MITE） system known as mPing in 102 varieties of Asian cultivated rice （Oryza sativa L.）. We found that mPing populations could be generalized Into two families, mPing-1 and mPing-2, according to their sequence structures. Further analysis showed that these two families of mPing had significant bias in their distribution pattern in two subspecies of rice, namely O. sativa ssp. japonica and indica. 0. sativa japonica has a higher proportion of mPing-1 as a general trait, whereas 0. sativa indica has a higher proportion of roPing-2. We also examined the mPing system In a doubled haploid （DH） cross-breeding population of jingxi 17 （japonica） and zhaiyeqing 8 （indica） varieties and observed that the mPing system was not tightly linked to major subspecies-determining genes. Furthermore, we checked the mPing system in 28 accessions of Asian common wild rice O. rufipogon and found the roPing system in 0. rufipogon. The distribution pattern of the roPing system in O. rufipogon indicated a diphyletlc origin of the Asian cultivated rice O. sativa species. We did not find the mPing system in another 20 Oryza species. These results substantiated a previous hypothesis that O. ruflpogon and O. nivara species were the closest relatives of O. sativa and that the two extant subspecies of O. sativa were evolved independently from corresponding ecotypes of O. ruflpogon.     

High-resolution genetic mapping and candidate gene identification of the <Emphasis Type="Italic">SLP1</Emphasis> locus that controls glume development in rice

Stunted lemma palea 1 (slp1) is a rice mutant that displays dwarfism, shortened inflorescence lengths, severely degenerated lemmas/paleas, and sterility. The SLP1 locus was mapped between markers RM447 and D275 in the distal region of the long arm of chromosome 8, using the F2 progeny derived from the cross between the Slp1/slp1 mutant (Oryza sativa subsp. japonica) and the variety Taichung Native 1 (TN1, O. sativa subsp. indica). The SLP1 locus was further delimited to a 46.4-kb genomic region containing three putative genes: OsSPL16, OsMADS45, and OsMADS37. Comparisons of the sequence variations and expression levels of the three candidate genes between wild-type plants and homozygous slp1 mutants suggested that a missense mutation in the sixth amino acid of the OsSPL16 protein was likely responsible for the slp1 mutant phenotypes.     

A statistical analysis of some genetical,physiological and anatomical parameters of the development of in situ- and in vitro-grown ovules from intra- and interspecific crosses in the genus Beta

Summary Structural observations on in situ- and in vitro-grown ovules from different intra- and interspecific crosses in the genus Beta were tested using computer registration and statistical analysis. In intra- as well as interspecific crosses embryo development occurs with the same rate independent of growth conditions. Therefore, with respect to growth rate, embryo development seems to be highly autonomous, provided that nutrient influx into the ovule is sufficient. The endosperm develops significantly more rapidly during in vitro culture than in situ. A large degree of variability with respect to structural changes in the ovule tissue during in vitro culture is observed in and between the crosses. In general, the interspecific cross responds more rapidly on in vitro culture. In all crosses the embryo, although genetically identical to the suspensor, shows a higher degree of response to in vitro culture than the embryo itself. Early suspensor degeneration in the interspecific cross is the only observed difference between the crosses, which could explains the lack of root formation in seedlings of interspecific hybrids. The use of statistical analysis on the anatomical parameters used to compare treatment as well as crosses has proven to be an efficient and novel approach to plant reproduction biology.     

Rice SNF2 family helicase ENL1 is essential for syncytial endosperm development

The endosperm of cereal grains represents the most important source of human nutrition. In addition, the endosperm provides many investigatory opportunities for biologists because of the unique processes that occur during its ontogeny, including syncytial development at early stages. Rice endospermless 1 (enl1) develops seeds lacking an endosperm but carrying a functional embryo. The enl1 endosperm produces strikingly enlarged amoeboid nuclei. These abnormal nuclei result from a malfunction in mitotic chromosomal segregation during syncytial endosperm development. The molecular identification of the causal gene revealed that ENL1 encodes an SNF2 helicase family protein that is orthologous to human Plk1‐Interacting Checkpoint Helicase (PICH), which has been implicated in the resolution of persistent DNA catenation during anaphase. ENL1‐Venus (enhanced yellow fluorescent protein (YFP)) localizes to the cytoplasm during interphase but moves to the chromosome arms during mitosis. ENL1‐Venus is also detected on a thread‐like structure that connects separating sister chromosomes. These observations indicate the functional conservation between PICH and ENL1 and confirm the proposed role of PICH. Although ENL1 dysfunction also affects karyokinesis in the root meristem, enl1 plants can grow in a field and set seeds, indicating that its indispensability is tissue‐dependent. Notably, despite the wide conservation of ENL1/PICH among eukaryotes, the loss of function of the ENL1 ortholog in Arabidopsis (CHR24) has only marginal effects on endosperm nuclei and results in normal plant development. Our results suggest that ENL1 is endowed with an indispensable role to secure the extremely rapid nuclear cycle during syncytial endosperm development in rice.     

Developmental trajectories and breakdown in F1 interpopulation hybrids of Tribolium castaneum

When hybrid inviability is an indirect by‐product of local adaptation, we expect its degree of severity between pairs of populations to vary and to be sensitive to the environment. While complete reciprocal hybrid inviability is the outcome of the gradual process of local adaptation, it is not representative of the process of accumulation of incompatibility. In the flour beetle, Tribolium castaneum, some pairs of populations exhibit complete, reciprocal F1 hybrid incompatibility while other pairs are fully or partially compatible. We characterize this naturally occurring variation in the degree and timing of expression of the hybrid incompatible phenotype to better understand the number of genes or developmental processes contributing to speciation. We assessed the morphological and developmental variation in four Tribolium castaneum populations and their 12 possible F1 hybrids at each life‐history stage from egg to adult. We find that the rate of hybrid larval development is affected in all interpopulation crosses, including those eventually producing viable, fertile adults. Hybrid incompatibility manifests early in development as changes in the duration of instars and diminished success in the transition between instars are relative to the parent populations. Parent populations with similar developmental profiles may produce hybrids with disrupted development. The degree and timing of expression of hybrid inviability depends upon populations crossed, direction of the cross, and environment in which hybrids are raised. Our findings suggest that the coordinated expression of genes involved in transitional periods of development is the underlying cause of hybrid incompatibility in this species.     

Parent‐of‐origin growth effects and the evolution of hybrid inviability in dwarf hamsters

Mammalian hybrids often show abnormal growth, indicating that developmental inviability may play an important role in mammalian speciation. Yet, it is unclear if this recurrent phenotype reflects a common genetic basis. Here, we describe extreme parent‐of‐origin‐dependent growth in hybrids from crosses between two species of dwarf hamsters, Phodopus campbelli and Phodopus sungorus. One cross type resulted in massive placental and embryonic overgrowth, severe developmental defects, and maternal death. Embryos from the reciprocal cross were viable and normal sized, but adult hybrid males were relatively small. These effects are strikingly similar to patterns from several other mammalian hybrids. Using comparative sequence data from dwarf hamsters and several other hybridizing mammals, we argue that extreme hybrid growth can contribute to reproductive isolation during the early stages of species divergence. Next, we tested if abnormal growth in hybrid hamsters was associated with disrupted genomic imprinting. We found no association between imprinting status at several candidate genes and hybrid growth, though two interacting genes involved in embryonic growth did show reduced expression in overgrown hybrids. Collectively, our study indicates that growth‐related hybrid inviability may play an important role in mammalian speciation but that the genetic underpinnings of these phenotypes remain unresolved.     

Dwarfism and male sterility in interspecific hybrids of Epilobium

Summary Reciprocal differences for male sterility, dwarfism and morphological traits have been studied in intra- and interspecific crosses of five Epilobium species. Male sterility occurred in two interspecific hybrids with E. montanum as the male parent while dwarfism has been found to varying degrees in three interspecific crosses with E. watsonni. In contrast to transient differences in plant height and leaf morphology in reciprocal hybrids of the cross between E. hirsutum and E. parviflorum, male sterility and dwarfism persistently occur as reciprocally different traits which may be influenced by determinants of the cytoplasm. The molecular characterization of the plastid DNA of the parental lines and the F₁ hybrids indicate that the plastome of male sterile and dwarf plants is identical to that of the female parents. Furthermore, in spite of these developmental disturbances, the expression of plastid genes coding for polypeptides of thylakoid-membrane complexes is unchanged. Thus, it seems unlikely that the genetic compartement of the plastids is responsible for the expression of the male sterile or the dwarfed phenotype.     

The influence of temperature on chromosome elimination during embryo development in crosses involving Hordeum spp., wheat (Triticum aestivum L.) and rye (Secale cereale L.)

Summary Several interspecific and intergeneric crosses involving five Hordeum species, Triticum aestivum and Secale cereale were carried out to investigate the influence of two contrasting temperatures on chromosome elimination during embryo development. In four of the interspecific Hordeum crosses, chromosome elimination was significantly increased at the higher of the two temperatures, resulting in greater proportions of haploid plant progenies. However, there was no significant effect of temperature in the other interspecific cross between H. lechleri x H. bulbosum nor in the two intergeneric crosses between H. vulgare x S. cereale and T. aestivum x H. bulbosum whose progeny were exclusively hybrid and haploid, respectively.     

Effects of Parental Embryo and Endosperm Mitotic Cycle Times on Development of Hybrids Between Barley and Rye

Seven different barley/rye crosses were made using genotypeshaving close (predicted compatible) or dissimilar (predictedincompatible) mean cell doubling times. The relative successof the crosses was determined by a cytological study of earlyhybrid seed development and by the yield of 16-day-old hybridembryos. The results support the hypothesis that parental developmentalrates must be similar for successful hybridization. The degenerationof the hybrid endosperm occurred earlier in the predicted incompatiblecrosses than in the more compatible ones. Fewer hybrid embryoswere harvested at day 16 from predicted incompatible crossesthan from compatible crosses. We conclude that development ofhybrid embryos depends on the early stages of endosperm developmentand that mitotic rates in parental endosperms are more importantthan in embryos. Hordeum vulgare L., Secale cereale L., barley, rye, hybrid, mean cell doubling time, embryo, endosperm