Genome Elimination Research Articles

Controlled Induction of Parthenogenesis in Transgenic Rice via Post-translational Activation of PsASGR-BBML.

Modern plant breeding programs rely heavily on the generation of homozygous lines, with the traditional process requiring the inbreeding of a heterozygous cross for five to six generations. Doubled haploid (DH) technology, a process of generating haploid plants from an initial heterozygote, followed by chromosome doubling, reduces the process to two generations. Currently established in vitro methods of haploid induction include androgenesis and gynogenesis, while in vivo methods are based on uni-parental genome elimination. Parthenogenesis, embryogenesis from unfertilized egg cells, presents another potential method of haploid induction. PsASGR-BABY BOOM-like, an AP2 transcription factor, induces parthenogenesis in a natural apomictic species, Pennisetum squamulatum (Cenchrus squamulatus) and PsASGR-BBML transgenes promote parthenogenesis in several crop plants, including rice, maize, and pearl millet. The dominant nature of PsASGR-BBML transgenes impedes their use in DH technology. Using a glucocorticoid-based post-translational regulation system and watering with a 100 μM DEX solution before anthesis, PsASGR-BBML can be regulated at the flowering stage to promote parthenogenesis. Conditional expression presents a novel opportunity to use parthenogenetic genes in DH production technology and to elucidate the molecular mechanism underlying parthenogenetic embryogenesis.

Gene-rich X chromosomes implicate intragenomic conflict in the evolution of bizarre genetic systems

Haplodiploidy and paternal genome elimination (HD/PGE) are common in invertebrates, having evolved at least two dozen times, all from male heterogamety (i.e., systems with X chromosomes). However, why X chromosomes are important for the evolution of HD/PGE remains debated. The Haploid Viability Hypothesis posits that X-linked genes promote the evolution of male haploidy by facilitating purging recessive deleterious mutations. The Intragenomic Conflict Hypothesis holds that conflict between genes drives genetic system turnover; under this model, X-linked genes could promote the evolution of male haploidy due to conflicts with autosomes over sex ratios and genetic transmission. We studied lineages where we can distinguish these hypotheses: species with germline PGE that retain an XX/X0 sex determination system (gPGE+X). Because evolving PGE in these cases involves changes in transmission without increases in male hemizygosity, a high degree of X linkage in these systems is predicted by the Intragenomic Conflict Hypothesis but not the Haploid Viability Hypothesis. To quantify the degree of X linkage, we sequenced and compared 7 gPGE+X species’ genomes with 11 related species with typical XX/XY or XX/X0 genetic systems, representing three transitions to gPGE. We find highly increased X linkage in both modern and ancestral genomes of gPGE+X species compared to non-gPGE relatives and recover a significant positive correlation between percent X linkage and the evolution of gPGE. These empirical results substantiate longstanding proposals for a role for intragenomic conflict in the evolution of genetic systems such as HD/PGE.

Haploidy and aneuploidy in switchgrass mediated by misexpression of CENH3.

Cross bred species such as switchgrass may benefit from advantageous breeding strategies requiring inbred lines. Doubled haploid production methods offer several ways that these lines can be produced that often involve uniparental genome elimination as the rate limiting step. We have used a centromere-mediated genome elimination strategy in which modified CENH3 is expressed to induce the process. Transgenic tetraploid switchgrass lines coexpressed Cas9, a poly-cistronic tRNA-gRNA tandem array containing eight guide RNAs that target two CENH3 genes, and different chimeric versions of CENH3 with alterations to the N-terminal tail region. Genotyping of CENH3 genes in transgenics identified edits including frameshift mutations and deletions in one or both copies of the two CENH3 genes. Flow cytometry of T1 seedlings identified two T0 lines that produced five haploid individuals representing an induction rate of 0.5% and 1.4%. Eight different T0 lines produced aneuploids at rates ranging from 2.1 to 14.6%. A sample of aneuploid lines were sequenced at low coverage and aligned to the reference genome, revealing missing chromosomes and chromosome arms.

Sexual antagonism in haplodiploids.

Females and males may face different selection pressures, such that alleles conferring a benefit in one sex may be deleterious in the other. Such sexual antagonism has received a great deal of theoretical and empirical attention, almost all of which has focused on diploids. However, a sizeable minority of animals display an alternative haplodiploid mode of inheritance, encompassing both arrhenotoky, whereby males develop from unfertilized eggs, and paternal genome elimination (PGE), whereby males receive but do not transmit a paternal genome. Alongside unusual genetics, haplodiploids often exhibit social ecologies that modulate the relative value of females and males. Here, we develop a series of evolutionary-genetic models of sexual antagonism for haplodiploids, incorporating details of their molecular biology and social ecology. We find that: (1) PGE promotes female-beneficial alleles more than arrhenotoky, and to an extent determined by the timing of elimination-and degree of silencing of-the paternal genome; (2) sib-mating relatively promotes female-beneficial alleles, as do other forms of inbreeding including limited male-dispersal, oedipal-mating, and the pseudo-hermaphroditism of Icerya purchasi; (3) resource competition between related females inhibits the invasion of female-beneficial alleles; and (4) sexual antagonism foments conflicts between parents and offspring, endosymbionts and hosts, and maternal- and paternal-origin genes.

Epigenetically mismatched parental centromeres trigger genome elimination in hybrids.

Wide crosses result in postzygotic elimination of one parental chromosome set, but the mechanisms that result in such differential fate are poorly understood. Here, we show that alterations of centromeric histone H3 (CENH3) lead to its selective removal from centromeres of mature Arabidopsis eggs and early zygotes, while wild-type CENH3 persists. In the hybrid zygotes and embryos, CENH3 and essential centromere proteins load preferentially on the CENH3-rich centromeres of the wild-type parent, while CENH3-depleted centromeres fail to reconstitute new CENH3-chromatin and the kinetochore and are frequently lost. Genome elimination is opposed by E3 ubiquitin ligase VIM1. We propose a model based on cooperative binding of CENH3 to chromatin to explain the differential CENH3 loading rates. Thus, parental CENH3 polymorphisms result in epigenetically distinct centromeres that instantiate a strong mating barrier and produce haploids.

A CENH3 mutation promotes meiotic exit and restores fertility in SMG7-deficient Arabidopsis.

Meiosis in angiosperm plants is followed by mitotic divisions to form multicellular haploid gametophytes. Termination of meiosis and transition to gametophytic development is, in Arabidopsis, governed by a dedicated mechanism that involves SMG7 and TDM1 proteins. Mutants carrying the smg7-6 allele are semi-fertile due to reduced pollen production. We found that instead of forming tetrads, smg7-6 pollen mother cells undergo multiple rounds of chromosome condensation and spindle assembly at the end of meiosis, resembling aberrant attempts to undergo additional meiotic divisions. A suppressor screen uncovered a mutation in centromeric histone H3 (CENH3) that increased fertility and promoted meiotic exit in smg7-6 plants. The mutation led to inefficient splicing of the CENH3 mRNA and a substantial decrease of CENH3, resulting in smaller centromeres. The reduced level of CENH3 delayed formation of the mitotic spindle but did not have an apparent effect on plant growth and development. We suggest that impaired spindle re-assembly at the end of meiosis limits aberrant divisions in smg7-6 plants and promotes formation of tetrads and viable pollen. Furthermore, the mutant with reduced level of CENH3 was very inefficient haploid inducer indicating that differences in centromere size is not the key determinant of centromere-mediated genome elimination.

Delete and survive: strategies of programmed genetic material elimination in eukaryotes.

ABSTRACTGenome stability is a crucial feature of eukaryotic organisms because its alteration drastically affects the normal development and survival of cells and the organism as a whole. Nevertheless, some organisms can selectively eliminate part of their genomes from certain cell types during specific stages of ontogenesis. This review aims to describe the phenomenon of programmed DNA elimination, which includes chromatin diminution (together with programmed genome rearrangement or DNA rearrangements), B and sex chromosome elimination, paternal genome elimination, parasitically induced genome elimination, and genome elimination in animal and plant hybrids. During programmed DNA elimination, individual chromosomal fragments, whole chromosomes, and even entire parental genomes can be selectively removed. Programmed DNA elimination occurs independently in different organisms, ranging from ciliate protozoa to mammals. Depending on the sequences destined for exclusion, programmed DNA elimination may serve as a radical mechanism of dosage compensation and inactivation of unnecessary or dangerous genetic entities. In hybrids, genome elimination results from competition between parental genomes. Despite the different consequences of DNA elimination, all genetic material destined for elimination must be first recognised, epigenetically marked, separated, and then removed and degraded.

Understanding and exploiting uniparental genome elimination in plants: insights from Arabidopsis thaliana

Vacuolar storage of iron (Fe) is important for Fe homeostasis in plants. When sufficient, the excess Fe could be stored in vacuoles for remobilization in case of Fe deficiency. Although the mechanism of Fe remobilization from vacuoles is critical for crop development under low Fe stress, the transporters that mediate vacuolar Fe translocation into the cytosol in rice remains unknown. Here, we showed that under higher Fe 2+ concentrations, the Δccc1 yeast mutant transformed with rice natural resistance-associated macrophage protein 2 (OsNRAMP2) became more sensitive to Fe toxicity. In rice protoplasts and transgenic plants expressing Pro35S: OsNRAMP2-GFP, OsNRAMP2 was localized to tonoplast. Vacuolar Fe contents in osnramp2 knockdown lines were higher than in the wild-type, while the growth of osnramp2 knockdown plants was significantly influenced by Fe deficiency. Furthermore, the germination of osnramp2 knockdown plants was arrested. Inversely, the vacuolar Fe contents of Pro35S: OsNRAMP2-GFP lines were significantly lower than in the wild-type, and overexpression of OsNRAMP2 increased shoot biomass under Fe deficiency. Taken together, we propose that OsNRAMP2 transports Fe from the vacuole to the cytosol and plays a pivotal role in seed germination.

Rare instances of haploid inducer DNA in potato dihaploids and ploidy-dependent genome instability.

In cultivated tetraploid potato (Solanum tuberosum), reduction to diploidy (dihaploidy) allows for hybridization to diploids and introgression breeding and may facilitate the production of inbreds. Pollination with haploid inducers (HIs) yields maternal dihaploids, as well as triploid and tetraploid hybrids. Dihaploids may result from parthenogenesis, entailing the development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and the loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of HI DNA in some dihaploids. We characterized the genomes of 919 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three HIs: IVP35, IVP101, and PL-4. Whole-chromosome or segmental aneuploidy was observed in 76 dihaploids, with karyotypes ranging from 2n = 2x - 1 = 23 to 2n = 2x + 3 = 27. Of the additional chromosomes in 74 aneuploids, 66 were from the non-inducer parent and 8 from the inducer parent. Overall, we detected full or partial chromosomes from the HI parent in 0.87% of the dihaploids, irrespective of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny, correlating instability to sperm ploidy and to haploid induction. The residual HI DNA discovered in the progeny is consistent with genome elimination as the mechanism of haploid induction.

Sex-specific expression and DNA methylation in a species with extreme sexual dimorphism and paternal genome elimination.

Phenotypic differences between sexes are often mediated by differential expression and alternative splicing of genes. However, the mechanisms that regulate these expression and splicing patterns remain poorly understood. The mealybug, Planococcus citri, displays extreme sexual dimorphism and exhibits an unusual instance of sex-specific genomic imprinting, paternal genome elimination (PGE), in which the paternal chromosomes in males are highly condensed and eliminated from the sperm. Planococcus citri has no sex chromosomes and both sexual dimorphism and PGE are predicted to be under epigenetic control. We recently showed that P.citri females display a highly unusual DNA methylation profile for an insect species, with the presence of promoter methylation associated with lower levels of gene expression. Here, we therefore decided to explore genome-wide differences in DNA methylation between male and female P.citri using whole-genome bisulphite sequencing. We identified extreme differences in genome-wide levels and patterns between the sexes. Males display overall higher levels of DNA methylation which manifest as more uniform low levels across the genome. Whereas females display more targeted high levels of methylation. We suggest these unique sex-specific differences are due to chromosomal differences caused by PGE and may be linked to possible ploidy compensation. Using RNA-Seq, we identify extensive sex-specific gene expression and alternative splicing, but we find no correlation with cis-acting DNA methylation.

Males That Silence Their Father's Genes: Genomic Imprinting of a Complete Haploid Genome.

Genetic conflict is considered a key driver in the evolution of reproductive systems with non-Mendelian inheritance, where parents do not contribute equally to the genetic makeup of their offspring. One of the most extraordinary examples of non-Mendelian inheritance is paternal genome elimination (PGE), a form of haplodiploidy which has evolved repeatedly across arthropods. Under PGE, males are diploid but only transmit maternally inherited chromosomes, while the paternally inherited homologues are excluded from sperm. This asymmetric inheritance is thought to have evolved through an evolutionary arms race between the paternal and maternal genomes over transmission to future generations. In several PGE clades, such as the mealybugs (Hemiptera: Pseudococcidae), paternal chromosomes are not only eliminated from sperm, but also heterochromatinized early in development and thought to remain inactive, which could result from genetic conflict between parental genomes. Here, we present a parent-of-origin allele-specific transcriptome analysis in male mealybugs showing that expression is globally biased toward the maternal genome. However, up to 70% of somatically expressed genes are to some degree paternally expressed, while paternal genome expression is much more restricted in the male reproductive tract, with only 20% of genes showing paternal contribution. We also show that parent-of-origin-specific gene expression patterns are remarkably similar across genotypes, and that genes with completely biparental expression show elevated rates of molecular evolution. Our results provide the clearest example yet of genome-wide genomic imprinting in insects and enhance our understanding of PGE, which will aid future empirical tests of evolutionary theory regarding the origin of this unusual reproductive strategy.

Uniparental Genome Elimination in Australian Carp Gudgeons.

Metazoans usually reproduce sexually, blending the unique identity of parental genomes for the next generation through functional crossing-over and recombination in meiosis. However, some metazoan lineages have evolved reproductive systems where offspring are either full (clonal) or partial (hemiclonal) genetic replicas. In the latter group, the process of uniparental genome elimination selectively eliminates either the maternal or paternal genome from germ cells, and only one parental genome is selected for transmission. Although fairly common in plants, hybridogenesis (i.e., clonal haploidization via chromosome elimination) remains a poorly understood process in animals. Here, we explore the proximal cytogenomic mechanisms of somatic and germ cell chromosomes in sexual and hybrid genotypes of Australian carp gudgeons (Hypseleotris) by tracing the fate of each set during mitosis (in somatic tissues) and meiosis (in gonads). Our comparative study of diploid hybrid and sexual individuals revealed visually functional gonads in male and female hybrid genotypes and generally high karyotype variability, although the number of chromosome arms remains constant. Our results delivered direct evidence for classic hybridogenesis as a reproductive mode in carp gudgeons. Two parental sets with integral structure in the hybrid soma (the F1 constitution) contrasted with uniparental chromosomal inheritance detected in gonads. The inheritance mode happens through premeiotic genome duplication of the parental genome to be transmitted, whereas the second parental genome is likely gradually eliminated already in juvenile individuals. The role of metacentric chromosomes in hybrid evolution is also discussed.

Hybridogenesis in the Water Frogs from Western Russian Territory: Intrapopulation Variation in Genome Elimination.

Hybridogenesis in an interspecific hybrid frog is a coupling mechanism in the gametogenic cell line that eliminates the genome of one parental species with endoduplication of the remaining genome of the other parental species. It has been intensively investigated in the edible frog Pelophylax kl. esculentus (RL), a natural hybrid between the marsh frog P. ridibundus (RR) and the pool frog P. lessonae (LL). However, the genetic mechanisms involved remain unclear. Here, we investigated the water frogs in the western Russian territory. In three of the four populations, we genetically identified 16 RL frogs living sympatrically with the parental LL species, or with both parental species. In addition, two populations contained genome introgression with another species, P. bedriagae (BB) (a close relative of RR). In the gonads of 13 RL frogs, the L genome was eliminated, producing gametes of R (or R combined with the B genome). In sharp contrast, one RL male eliminated the L or R genome, producing both R and L sperm. We detected a variation in genome elimination within a population. Based on the genetic backgrounds of RL frogs, we hypothesize that the introgression of the B genome resulted in the change in choosing a genome to be eliminated.

Natural and Artificial Mechanisms of Mitochondrial Genome Elimination.

The generally accepted theory of the genetic drift of mitochondrial alleles during mammalian ontogenesis is based on the presence of a selective bottleneck in the female germline. However, there is a variety of different theories on the pathways of genetic regulation of mitochondrial DNA (mtDNA) dynamics in oogenesis and adult somatic cells. The current review summarizes present knowledge on the natural mechanisms of mitochondrial genome elimination during mammalian development. We also discuss the variety of existing and developing methodologies for artificial manipulation of the mtDNA heteroplasmy level. Understanding of the basics of mtDNA dynamics will shed the light on the pathogenesis and potential therapies of human diseases associated with mitochondrial dysfunction.

Generation of Doubled Haploid Barley by Interspecific Pollination with Hordeum bulbosum.

The generation of doubled haploid barley plants by means of the so-called "Bulbosum" method has been practiced for meanwhile five decades. It rests upon the pollination of barley by its wild relative Hordeum bulbosum. This can result in the formation of hybrid embryos whose further development is typically associated with the loss of the pollinator's chromosomes. In recent years, this principle has, however, only rarely been used owing to the availability of efficient methods of anther and microspore culture. On the other hand, immature pollen-derived embryogenesis is to some extent prone to segregation bias in the resultant populations of haploids, which is due to its genotype dependency. Therefore, the principle of uniparental genome elimination has more recently regained increasing interest within the plant research and breeding community. The development of the present protocol relied on the use of the spring-type barley cultivar Golden Promise. The protocol is the result of a series of comparative experiments, which have addressed various methodological facets. The most influential ones included the method of emasculation, the temperature at flowering and early embryo development, the method, point in time and concentration of auxin administration for the stimulation of caryopsis development, the developmental stage at embryo dissection, as well as the nutrient medium used for embryo rescue. The present protocol allows the production of haploid barley plants at an efficiency of ca. 25% of the pollinated florets.

Influence of reproductive biology on establishment capacity in introduced Hymenoptera species

Introduced species face numerous biological barriers before they can establish in a new environment. Understanding how they overcome these obstacles is crucial for the development of effective risk assessment and regulation. Reproductive biology is known to influence establishment capacity in plants and is widely used for risk assessment. This biological field should receive more attention, and particularly in the case of insects, as they display a wide range of reproductive traits and have a great impact on the economy and environment. Among insects, the order Hymenoptera is of interest for its diversity, both in terms of reproductive traits and introduction history, as invasive species and biological control agents. We review the main reproductive strategies of Hymenoptera, spanning parthenogenesis, sex determination, reproductive parasites and mating strategies, and evaluate their effect on invasive potential. For instance, thelytoky could decrease the strength of Allee effects while Arrhenotoky could increase adaptive potential. A species with complementary sex determination could be more affected by inbreeding than other species, while paternal genome elimination could lead to high levels of homozygosity. Finally, some reproductive behaviours could decrease inbreeding, facilitate mate location or adaptation by encouraging admixture. The two invasive species Apis mellifera scutellata and Leptocybe invasa and the biocontrol agent Aphidius ervi serve as case studies to illustrate the effect of reproductive traits on species capacities to become established in a new area.

A chromosome-level genome assembly provides new insights into paternal genome elimination in the cotton mealybug Phenacoccus solenopsis.

Mealybugs (Hemiptera: Pseudococcidae) are economically important agricultural pests with several compelling biological phenomena including paternal genome elimination (PGE). However, limited high-quality genome assemblies of mealybugs hinder a full understanding of this striking and unusual biological phenomenon. Here, we generated a chromosome-level genome assembly of cotton mealybug, Phenacoccus solenopsis, by combining Illumina short reads, PacBio long reads and Hi-C scaffolding. The assembled genome was 292.54Mb with a contig N50 of 489.8kb and a scaffold N50 of 49.0Mb. Hi-C scaffolding assigned 84.42% of the bases to five chromosomes. A total of 110.75Mb (37.9%) repeat sequences and 11,880 protein-coding genes were predicted. The completeness of the genome assembly was estimated to be 95.5% based on BUSCO genes. In addition, 27,086 (95.3%) full-length PacBio transcripts were uniquely mapped to the assembled scaffolds, suggesting the high quality of the genome assembly. We showed that cotton mealybugs lack differentiated sex chromosomes by analysing genome resequencing data of males and females. DAPI staining confirmed that one chromosome set in males becomes heterochromatin at an early embryo stage. Chromatin immunoprecipitation assays with sequencing analysis demonstrated that the epigenetic modifications H3K9me3 and H3K27me3 are distributed across the whole genome in males, suggesting that these two modifications might be involved in maintaining heterochromatin status. Both markers were more likely to be distributed in repeat regions, while H3K27me3 had higher overall enrichment. Our results provide a valuable genomic resource and shed new light on the genomic and epigenetic basis of PGE in cotton mealybugs.

Haploid Bio-Induction in Plant through Mock Sexual Reproduction.

SummaryHaploidization is invaluable for basic genetic research and crop breeding. The haploid bio-induction principle is an important topic that remains largely unexplored. In this study, both CenH3 RNAi and in vitro inhibition were used to simulate and induce haploids in allopolyploid crop. Notably, in vitro CenH3 inhibition showed that the results were much the same to that of RNAi in phenotype, chromosome behavior, microspore production, and haploid induction. Cytological analyses of RNAi and inhibitor-treated progenies revealed elimination of chromosomes, defective microspores with empty nuclei, thereby giving rise to pseudo male gametes, and haploid parthenogenesis induction. We found distinct defective empty microspores that were positively correlated with the decrease of CenH3 during RNAi manipulation. Investigation through both in vivo and in vitro studies revealed that haploidization was induced through the pseudo male gamete-mediated mock sexual reproduction. The present results provide insights for the haploid parthenogenesis induction process.

Site-directed mutagenesis in bread and durum wheat via pollination by cas9/guide RNA-transgenic maize used as haploidy inducer.

Site-directed mutagenesis in bread and durum wheat via pollination by cas9/guide RNA-transgenic maize used as haploidy inducer.

Micronuclei in germ cells of hybrid frogs from Pelophylax esculentus complex contain gradually eliminated chromosomes

In most organisms, cells typically maintain genome integrity, as radical genome reorganization leads to dramatic consequences. However, certain organisms, ranging from unicellular ciliates to vertebrates, are able to selectively eliminate specific parts of their genome during certain stages of development. Moreover, partial or complete elimination of one of the parental genomes occurs in interspecies hybrids reproducing asexually. Although several examples of this phenomenon are known, the molecular and cellular processes involved in selective elimination of genetic material remain largely undescribed for the majority of such organisms. Here, we elucidate the process of selective genome elimination in water frog hybrids from the Pelophylax esculentus complex reproducing through hybridogenesis. Specifically, in the gonads of diploid and triploid hybrids, but not those of the parental species, we revealed micronuclei in the cytoplasm of germ cells. In each micronucleus, only one centromere was detected with antibodies against kinetochore proteins, suggesting that each micronucleus comprises a single chromosome. Using 3D-FISH with species-specific centromeric probe, we determined the role of micronuclei in selective genome elimination. We found that in triploid LLR hybrids, micronuclei preferentially contain P. ridibundus chromosomes, while in diploid hybrids, micronuclei preferentially contain P. lessonae chromosomes. The number of centromere signals in the nuclei suggested that germ cells were aneuploid until they eliminate the whole chromosomal set of one of the parental species. Furthermore, in diploid hybrids, misaligned P. lessonae chromosomes were observed during the metaphase stage of germ cells division, suggesting their possible elimination due to the inability to attach to the spindle and segregate properly. Additionally, we described gonocytes with an increased number of P. ridibundus centromeres, indicating duplication of the genetic material. We conclude that selective genome elimination from germ cells of diploid and triploid hybrids occurs via the gradual elimination of individual chromosomes of one of the parental genomes, which are enclosed within micronuclei.