Nuclear-cytoplasmic Incompatibility Research Articles

Low copy numbers for mitochondrial DNA moderates the strength of nuclear-cytoplasmic incompatibility in plants.

Plant cells contain only small amounts of mitochondrial DNA (mtDNA), with the genomic information shared among multiple mitochondria. The biological relevance and molecular mechanism underlying this hallmark of plant cells has been unclear. Here, we report that Arabidopsis thaliana plants exhibited significantly reduced growth and mitochondrial dysfunction when the mtDNA copy number was increased to the degree that each mitochondrion possessed DNA. The amounts of mitochondrion-encoded transcripts increased several fold in the presence of elevated mtDNA levels. However, the efficiency of RNA editing decreased with this excess of mitochondrion-encoded transcripts, resulting in impaired assembly of mitochondrial complexes containing mtDNA-encoded subunits, such as respiratory complexes I and IV. These observations indicate the occurrence of nuclear-mitochondrial incompatibility in the cells with increased amounts of mtDNA and provide an initial answer to the fundamental question of why plant cells have much lower mtDNA levels than animal cells. We propose that keeping mtDNA levels low moderates nuclear-mitochondrial incompatibility and that this may be a crucial factor driving plant cells to restrict the copy numbers of mtDNA.

Nuclear Cytoplasmic Conflict in Hybrids of Roach <i>Rutilus rutilus</i> and Bream <i>Abramis brama</i> as a Consequence of the Divergence Species in Body and Genome Sizes

Species divergence in body size is often associated with changes in genome size and the rate of evolution of mitochondrial DNA (mtDNA), which can lead to problems of nuclear-cytoplasmic compatibility and embryonic genome activation and reduce the fitness of hybrids. The bream Abramis brama (L.) is larger than the roach Rutilus rutilus (L.) in body and genome sizes. In the first generation of hybrids according to the ITS1 region of ribosomal DNA, a change in the donor genome of male to the level of the maternal species was previously established, which can affect the inheritance of traits from the male and, in particular, body size. Body length and height, a complex of diagnostic morphological characters, and genotyping (ITS1 rDNA and cyt b mtDNA) of underyearlings and mature individuals of bream, roach, F1 hybrids and underyearlings of backcrosses (Fb) were analyzed. Sexually mature hybrids of both directions of crossing are close in body length to R. rutilus, which indicates a violation of the paternal effect when inheriting the body length of a larger of species. Violation of the inheritance of bream body length in hybrids of cross R. rutilus × A. brama (♀ ♂, RA) is considered as a developmental deviation, which, obviously, can affect the adaptation of hybrids and determines the rarity of this variant in nature. At the same time, alloplasmic ARR backcrosses restore the body length of the bream even in the presence of the roach nuclear genome, which indicates the influence of mitochondrial genes on the development of this trait. The observed decrease in the fitness of first-generation hybrids with roach mtDNA may be associated with to an insufficient level of transcription of ribosomal genes due to a decrease in the number and variability of copies of the donor bream rDNA. Moreover, the high level of changes of mtDNA roach indicate a suboptimal mitochondrial-nuclear correspondence of respiratory a complexes in hybrids RA, which negatively affects key physiological processes, including growth and development of a large body size. Presumably, the development of large body size, as a complex trait with high aerobic fitness, is blocked in RA hybrids for energy reasons. The paper shows that differences families of repeated sequences rDNA and mtDNA in terms of the number and variability of copies in genome of R. rutilus and A. brama can lead to regulatory nuclear-cytoplasmic incompatibility of genomes and affect the fitness of hybrids already in the first generation.

Variation in cytonuclear expression accommodation among allopolyploid plants.

Cytonuclear coevolution is a common feature among plants, which coordinates gene expression and protein products between the nucleus and organelles. Consequently, lineage-specific differences may result in incompatibilities between the nucleus and cytoplasm in hybrid taxa. Allopolyploidy is also a common phenomenon in plant evolution. The hybrid nature of allopolyploids may result in cytonuclear incompatibilities, but the massive nuclear redundancy created during polyploidy affords additional avenues for resolving cytonuclear conflict (i.e. cytonuclear accommodation). Here we evaluate expression changes in organelle-targeted nuclear genes for 6 allopolyploid lineages that represent 4 genera (i.e. Arabidopsis, Arachis, Chenopodium, and Gossypium) and encompass a range in polyploid ages. Because incompatibilities between the nucleus and cytoplasm could potentially result in biases toward the maternal homoeolog and/or maternal expression level, we evaluate patterns of homoeolog usage, expression bias, and expression-level dominance in cytonuclear genes relative to the background of noncytonuclear expression changes and to the diploid parents. Although we find subsets of cytonuclear genes in most lineages that match our expectations of maternal preference, these observations are not consistent among either allopolyploids or categories of organelle-targeted genes. Our results indicate that cytonuclear expression evolution may be subtle and variable among genera and genes, likely reflecting a diversity of mechanisms to resolve nuclear-cytoplasmic incompatibilities in allopolyploid species.

Asymmetric hybridization of roach Rutilus rutilus and common bream Abramis brama in controlled backcrosses: Genetic and morphological patterns

In most cases in natural environments, hybrids of roach Rutilus rutilus L. and bream Abramis brama L. possess mitochondrial DNA of bream. Presumably, the genetic basis for unidirectional hybridization of roach and bream is the high level of divergence in the genes of the mitochondrial electron transport chain (cytochrome b and cytochrome c oxidase subunits I, III). Disruption of the interaction of the products of these genes leads to nuclear-cytoplasmic incompatibility of alien genomes, what is shown in a decrease of viability and developmental abnormalities in hybrids. In the present work we studied the viability and morphology of hybrid underyearlings obtained by crossing of hybrid females of first generation (RA and AR) with males of roach R. rutilus and bream A. brama. The method of genotyping (ITS1 ribosomal DNA, cytochrome b mtDNA) and comparative analysis of the complex of 23 plastic and meristic characteristics of backcrossed hybrids are used. All progenies showed an increase in morphological variability compared to parental species and F1 hybrids. In progenies with introgression of mtDNA, a violation of associations between traits and the formation of transgressive phenotypes that go beyond the parental populations were found. In RAA backcrosses (combining mtDNA of R. rutilus and nuclear genome of A. brama) a decrease in viability with impaired recovery of external traits of bream was found. Conversely, ARR backcrosses (combining mtDNA of A. brama and the nuclear genome of R. rutilus) have a high viability and completely restore the morphotype of roach, which indicates the stable development of hybrids when they include alien genetic material. The differences in viability and morphology between backcrossed hybrids with the mtDNA of R. rutilus and A. brama evidence varying degrees of nuclear-cytoplasmic compatibility of the genomes of roach and bream. The complete interaction between mitochondrial and nuclear DNA from different species (wild-type-like) happens in direction of introgression mtDNA of A. brama, the less polymorphic of the two parental genomes. In the direction of introgression of highly polymorphic mtDNA of R. rutilus the formation of a reproductive barrier occurs. Our results show that the main reason for the asymmetry of hybridization of R. rutilus and A. brama is unequal rates of mitochondrial evolution and the priority of the introgression of mtDNA belongs a species with a lower rate of changes in mtDNA.

Plastome sequences of the subgenus Passiflora reveal highly divergent genes and specific evolutionary features.

Phylogenetic aspects, hotspots of nucleotide divergence, highly divergent genes, and specific RNA editing sites have been identified and characterized in the plastomes of the subgenus Passiflora. The genus Passiflora comprises more than 500 species across five subgenera: Astrophea, Decaloba, Deidamioides, Passiflora, and Tetrapathea. The most economically relevant species belong to the subgenus Passiflora, whose genetic pool and diversity among wild species remain poorly characterized. Similarly, little is known about the interspecific relationships within the subgenus Passiflora and the genetic causes of nuclear-cytoplasmic incompatibility observed in interspecific hybrids. Here, we report the complete nucleotide sequences of six plastomes belonging to species of the subgenus Passiflora, with the aim of better understanding the evolution of the plastome in this subgenus. Complete plastome sequences revealed five hotspots of nucleotide polymorphism: three intergenic regions and two coding sequences. Moreover, among 70 RNA editing sites predicted in our analysis for the subgenus Passiflora, 38 were not shared by all analyzed species, highlighting their species-specific occurrence. Furthermore, phylogenies based on plastid sequences accurately resolved most relationships between species and suggested a non-monophyletic origin of three super-sections of the subgenus Passiflora, previously defined solely based on morphological traits. Finally, our findings identified putative candidates, including predicted RNA editing sites and the coding sequences of accD and clpP genes, responsible for nuclear-cytoplasmic incompatibility in the interspecific hybrids of Passiflora.

Allelic Diversity of Acetyl Coenzyme A Carboxylase accD/bccp Genes Implicated in Nuclear-Cytoplasmic Conflict in the Wild and Domesticated Pea (Pisum sp.).

Reproductive isolation is an important component of species differentiation. The plastid accD gene coding for the acetyl-CoA carboxylase subunit and the nuclear bccp gene coding for the biotin carboxyl carrier protein were identified as candidate genes governing nuclear-cytoplasmic incompatibility in peas. We examined the allelic diversity in a set of 195 geographically diverse samples of both cultivated (Pisum sativum, P. abyssinicum) and wild (P. fulvum and P. elatius) peas. Based on deduced protein sequences, we identified 34 accD and 31 bccp alleles that are partially geographically and genetically structured. The accD is highly variable due to insertions of tandem repeats. P. fulvum and P. abyssinicum have unique alleles and combinations of both genes. On the other hand, partial overlap was observed between P. sativum and P. elatius. Mapping of protein sequence polymorphisms to 3D structures revealed that most of the repeat and indel polymorphisms map to sequence regions that could not be modeled, consistent with this part of the protein being less constrained by requirements for precise folding than the enzymatically active domains. The results of this study are important not only from an evolutionary point of view but are also relevant for pea breeding when using more distant wild relatives.

Nuclear-cytoplasmic conflict in pea (Pisum sativum L.) is associated with nuclear and plastidic candidate genes encoding acetyl-CoA carboxylase subunits.

In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized.

Wild peas vary in their cross-compatibility with cultivated pea (Pisum sativum subsp. sativum L.) depending on alleles of a nuclear-cytoplasmic incompatibility locus.

Divergent wild and endemic peas differ in hybrid sterility in reciprocal crosses with cultivated pea depending on alleles of a nuclear 'speciation gene' involved in nuclear-cytoplasmic compatibility. In hybrids between cultivated and wild peas, nuclear-cytoplasmic conflict frequently occurs. One of the nuclear genes involved, Scs1, was earlier mapped on Linkage Group III. In reciprocal crosses of seven divergent pea accessions with cultivated P. sativum, some alleles of Scs1 manifested incompatibility with an alien cytoplasm as a decrease in pollen fertility to about 50 % in the heterozygotes and lack of some genotypic classes among F2 segregants. Earlier, we defined monophyletic evolutionary lineages A, B, C and D of pea according to allelic state of three markers, from nuclear, plastid and mitochondrial genomes. All tested representatives of wild peas from the lineages A and C exhibited incompatibility due to Scs1 deleterious effects in crosses with testerlines of P. sativum subsp. sativum (the common cultivated pea) at least in one direction. A wild pea from the lineage B and a cultivated pea from the lineage D were compatible with the testerline in both directions. The tested accession of cultivated P. abyssinicum (lineage A) was partially compatible in both directions. The Scs1 alleles of some pea accessions even originating from the same geographic area were remarkably different in their compatibility with cultivated Pisum sativum cytoplasm. Variability of a gene involved in reproductive isolation is of important evolutionary role and nominate Scs1 as a speciation gene.

Reduced expression of BjRCE1 gene modulated by nuclear-cytoplasmic incompatibility alters auxin response in cytoplasmic male-sterile Brassica juncea.

The signal from organelle to nucleus, namely retrograde regulation of nuclear gene expression, was largely unknown. Due to the nuclear-cytoplasmic incompatibility in cytoplasmic male-sterile (CMS) plants, we employed CMS Brassica juncea to investigate the retrograde regulation of nuclear gene expression in this study. We studied how reduced BjRCE1 gene expression caused by the nuclear-cytoplasmic incompatibility altered the auxin response in CMS of B. juncea. We isolated the BjRCE1 gene that was located in the nucleus from B. juncea. Over-expression of BjRCE1 enhanced auxin response in transgenic Arabidopsis. The expression of BjRCE1 was significantly reduced in CMS compared with its maintainer fertile (MF) line of B. juncea. There were fewer lateral roots in CMS than MF under normal and treatment of indole-3-acetic acid (IAA) conditions. Expression patterns of several auxin-related genes together with their phenotypes indicated a reduced auxin response in CMS compared to MF. The phenotypes of auxin response and auxin-related gene expression pattern could be mimicked by inhibiting mitochondrial function in MF. Taken together, we proposed reduced expression of BjRCE1 gene modulated by nuclear-cytoplasmic incompatibility alters auxin response in CMS B. juncea. This may be an important mechanism of retrograde regulation of nuclear gene expression in plants.

Inheritance and genetic mapping of two nuclear genes involved in nuclear-cytoplasmic incompatibility in peas (Pisum sativum L.).

Genetic analysis was performed to finely map and assess the mode of inheritance of two unlinked nuclear genes Scs1 and Scs2 involved in incompatibility of the nuclear genome of the cultivated pea Pisum sativum subsp. sativum with the cytoplasm of the wild pea of the subspecies P. sativum subsp. elatius, accession VIR320. Based on the segregation of genotypes in the progeny of the test-crosses, we concluded that if the cytoplasm was inherited from the wild pea VIR320, the Scs1 allele from the cultivated pea was gametophyte lethal and sporophyte recessive lethal. The Scs2 allele from the cultivated pea reduced male gametophyte viability. In homozygote, Scs2 from cultivated parent brought about nuclear-cytoplasmic conflict manifested as chlorophyll deficiency, reduction of blade organs, and low pollen fertility of about 20%. In heterozygote, Scs1 and Scs2 genes reduced pollen fertility by ca 50 and 30%, respectively. The Scs1 and Scs2 genes involved in nuclear-cytoplasmic incompatibility were genetically mapped. The distance between the markers bordering Scs1 comprised about 2.5cM on linkage group III. The map distance between the bordering markers in the neighborhood of Scs2 varied substantially from cross to cross in the range of 2.0-15.1cM on linkage group V.

Comparative Proteomic Analysis of Liver Mitochondrial Proteins Derived from Cloned Adult Pigs Reconstructed with Meishan Pig Fibroblast Cells and European Pig Enucleated Oocytes

Somatic cell nuclear transfer (SCNT) has been exploited in efforts to clone and propagate valuable animal lineages. However, in many instances, recipient oocytes are obtained from sources independent of donor cell populations. As such, influences of potential nuclear-cytoplasmic incompatibility, post SCNT, are largely unknown. In the present study, alterations in mitochondrial protein levels were investigated in adult SCNT pigs produced by microinjection of Meishan pig fetus fibroblast cells into enucleated matured oocytes (maternal Landrace genetic background). Mitochondrial fractions were prepared from liver samples by mechanical homogenization and differential centrifugation. Liver mitochondria were then subjected to two-dimensional difference gel electrophoresis (2-D DIGE). Protein expression changes were confirmed with a volume ratio greater than 2 fold (P<0.05). 2-D DIGE analysis further revealed differential expression of three proteins between the Meishan (n=3) and Landrace (n=3) breeds. Differential expression patterns of 16 proteins were detected in SCNT pig liver tissue (n=3) when compared with Meishan control samples. However, none of the 16 proteins correlated with the three differentially expressed Meishan and Landrace liver mitochondrial proteins. In summary, alteration of mitochondrial protein expression levels was observed in adult SCNT pigs that did not reflect the breed difference of the recipient oocytes. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.

Nuclear-Cytoplasmic Incompatibility in Inter-Species Cytoplasm Hybrid Embryos: Implications for Inter-Species Nuclear Transfer Hybrid Embryo.

Interspecies somatic cell nuclear transfer (iSCNT) embryos reconstructed between other than closely related species rarely develop to the blastocyst stage and beyond. One explanation for this lack of development is an inability to reprogram the somatic genome. To explore this possibility we evaluated whether using a karyoplast that would require less extensive reprogramming such as an embryonic blastomere or the meiotic spindle from metaphase II oocytes would provide additional insight into the development of iSCNT embryos. Our results showed that karyoplasts of embryonic or oocyte origin are no different from somatic cells; all interspecies nuclear transfer embryos independent of karyoplast origin failed to develop to the blastocyst stage. We speculated that apart from faulty epigenetic reprogramming, nuclear-cytoplasmic incompatibility could be another reason for failure of embryonic development from interspecies nuclear transfer. To address nuclear-cytoplasmic incompatibility we used pig-mouse interspecies cytoplasmic hybrids as a model. Fertilized murine zygotes were reconstructed by fusing with porcine cytoplasts of varying cytoplasmic volumes (1/5 (small) and 1/3 (large) total volume of mouse zygote) produced from activated oocytes. The presence of pig cytoplasm significantly reduced in vitro development of mouse zygotes to the blastocyst stage as compared to control embryos at 120 h Post hCG (41% vs 6% vs 94%, P<0.05), (1/5, 1/3, control respectively). While copy numbers of the mitochondrial genome (mtDNA) remained relatively unchanged in cytoplasmic hybrids and control mouse embryos, genes responsible for mitochondrial replication (Tfam, PlogG, and PlogG2) mitochondrial fusion (mfn2) glucose transport (Glut 3 and Glut1) anti apopotosis (Bcl2 and hspb1) and pluripotency and lineage segregation (Oct4, Nanog, cdx2, Gata3, Tcfap2c) were significantly down regulated in cytoplasmic hybrid embryos. Methylation at promoter regions of Oct4 in cytoplasmic hybrids was slightly increased, however, methylation at LINE1 was not different from control embryos. We speculate that abnormal gene expression in cytoplasmic hybrids may be a consequence of the presence of foreign cytoplasmic components. RNA binding proteins, microRNAS and maternal transcripts of pig cytoplasmic origin may have adversely interfered with precise regulation of maternal murine transcripts and post-transcriptional and/or translation regulation of nascent mRNA synthesized from the mouse embryonic genome. These results demonstrate that the presence of even a small amount of porcine cytoplasm is detrimental to murine embryo development and suggest that a range of factors are likely to contribute to the failure of interspecies nuclear transfer embryos. (poster)

Nuclear–cytoplasmic incompatibility and inefficient development of pig–mouse cytoplasmic hybrid embryos

Inter-species somatic cell nuclear transfer (iSCNT) embryos usually fail to develop to the blastocyst stage and beyond due to incomplete reprogramming of donor cell. We evaluated whether using a karyoplast that would require less extensive reprogramming such as an embryonic blastomere or the meiotic spindle from metaphase II oocytes would provide additional insight into the development of iSCNT embryos. Our results showed that karyoplasts of embryonic or oocyte origin are no different from somatic cells; all iSCNT embryos, irrespective of karyoplast origin, were arrested during early development. We hypothesized that nuclear-cytoplasmic incompatibility could be another reason for failure of embryonic development from iSCNT. We used pig-mouse cytoplasmic hybrids as a model to address nuclear-cytoplasmic incompatibility in iSCNT embryos. Fertilized murine zygotes were reconstructed by fusing with porcine cytoplasts of varying cytoplasmic volumes (1/10 (small) and 1/5 (large) total volume of mouse zygote). The presence of pig cytoplasm significantly reduced the development of mouse zygotes to the blastocyst stage compared with control embryos at 120 h post-human chorionic gondotropin (41 vs 6 vs 94%, P<0.05; 1/10, 1/5, control respectively). While mitochondrial DNA copy numbers remained relatively unchanged, expression of several important genes namely Tfam, Polg, Polg2, Mfn2, Slc2a3 (Glut3), Slc2a1 (Glut1), Bcl2, Hspb1, Pou5f1 (Oct4), Nanog, Cdx2, Gata3, Tcfap2c, mt-Cox1 and mt-Cox2 was significantly reduced in cytoplasmic hybrids compared with control embryos. These results demonstrate that the presence of even a small amount of porcine cytoplasm is detrimental to murine embryo development and suggest that a range of factors are likely to contribute to the failure of inter-species nuclear transfer embryos.

Interspecific hybridization in Lonicera caerulea and Lonicera gracilipes: The occurrence of green/albino plants by reciprocal crossing

Lonicera caerulea L. var. emphyllocalyx (Maxim.) Nakai is a berry crop cultivated in cold regions. So far, commercial cultivars have been mainly introduced from selection of wild plants. Therefore, fruit traits and other agricultural characteristics have been limited. In this study, interspecific crosses between L. caerulea var. emphyllocalyx and Lonicera gracilipes var. glabra Miquel were examined to increase genetic variability of L. caerulea var. emphyllocalyx. Seedlings were obtained from reciprocal crosses between L. caerulea var. emphyllocalyx and L. gracilipes var. glabra. The hybrid nature of seedlings was confirmed with random amplified polymorphic DNA analysis. Viable green plants were obtained efficiently from L. gracilipes var. glabra × L. caerulea var. emphyllocalyx. In contrast, all plants produced from L. caerulea var. emphyllocalyx × L. gracilipes var. glabra were albino. These albino plants were very weak and only survived in culture condition. The chlorophyll deficiency was unilaterally observed, suggesting the occurrence of nuclear–cytoplasmic incompatibility. Viable F 1 hybrids obtained from L. gracilipes var. glabra × L. caerulea var. emphyllocalyx are amphidiploid (2 n = 4 x = 36) as showing same to both parents. The hybrid plants are expected to increase the variability of fruit traits, and may have heat tolerance from L. gracilipes var. glabra.

56 EFFECTS OF PHYLOGENIC GENERA OF RECIPIENT CYTOPLASTS ON DEVELOPMENT AND VIABILITY OF CANADA LYNX (LYNX CANADENSIS) CLONED EMBRYOS

Canada lynx (Lynx canadensis; CL) once occupied 16 states in the Unites States of America, but small populations remain in only 3 states. Interspecies-somatic cell nuclear transfer (Is-SCNT) offers the possibility of preventing their extinction; however, developmental constraints on Is-SCNT embryos are proportional to the phylogenetic distance between the donor cell and the recipient oocyte. Mitochondrial DNA (mtDNA) heteroplasmy may be involved in nuclear-cytoplasmic incompatibilities, thus inhibiting development of cloned embryos at the time of genomic activation. Minimizing the phylogenetic distance between the donor cell and recipient oocyte may enhance development of clone embryos. Caracal (Caracal caracal) may be suitable as an oocyte donor for SCNT and a recipient of CL cloned embryos because caracals hybridize with other felid species and share physical characteristics with the lynx family, marked by being previously classified in the lynx genera and having similar gestational length. To ensure compatibilities between the donor nuclei of the CL and the mitochondria of recipient oocytes, we (1) compared in vitro development of CL cloned embryos reconstructed with domestic cat (Felis catus; DSH) or caracal cytoplasts, (2) examined the mtDNA genotypes in CL cloned embryos, and (3) evaluated in vivo developmental competence of CL cloned embryos after transfer into caracal recipients. A total of 160 and 217 preovulatory oocytes were collected by laparoscopy from gonadotropin-treated caracals (n = 8) and DSH (n = 10) and used as recipient cytoplasts for reconstructing CL embryos. Results indicated that the phylogenetic genera of recipient cytoplasts did not affect embryo cleavage at Day 2 (caracal 50/55, 91% v. DSH 63/65, 97%), but development of CL cloned embryos to the blastocyst stage was higher when caracal oocytes were used as recipient cytoplasts (15/50; 30%) than with DSH cytoplasts (9/63, 14%; P &lt; 0.05). The extent of mtDNA homoplasmy or heteroplasmy in CL cloned embryos was calculated by the number of single nucleotide polymorphisms (SNP) derived from the DSH or caracal oocyte donors and from the somatic cell donor CL. DNA was isolated from 25 and 35 CL cloned embryos reconstructed with caracal or DSH cytoplasts, respectively. All amplified products after PCR were sequenced and SNP analyzed. All CL embryos reconstructed with DSH cytoplasts were homoplasmic, carrying mtDNA only from the DSH oocyte donor (n = 35; SNP DSH = 2-6). Embryos reconstructed with caracal cytoplasts were homoplasmic for CL mtDNA (n = 9; SNPCL = 10-12) or heteroplasmic (caracal × CL, n = 17; SNPCL = 7-9; SNP caracal = 2-3). A total of 69 (mean = 34.5 ± 4.9 per caracal) and 70 (mean = 35.0 ± 9.8 per caracal) CL cloned embryos reconstructed with caracal and DSH cytoplasts, respectively, were transferred into 4 caracal recipients; however, no pregnancies were established. In summary, Is-SCNT between 2 phylogenetically closer species favors retention of the donor’s mitochondria, which might lead to a better nucleo-cytoplasmic interaction for reprogramming of donor nucleus.

Meiotic abnormalities as expression of nuclear-cytoplasmic incompatibility in crosses of Pisum sativum subspecies

Meiosis in anthers and mitosis in somatic cells were studied in reciprocal F1 hybrids of the accession VIR320, which belonged to wild Pisum sativum ssp. elatius (Bieb.) Schmal., and the laboratory line Sprint-1. When VIR320 was used as a maternal form, the hybrids displayed nuclear-cytoplasmic conflict, which caused chlorophyll defects and meiotic abnormalities. One or two chromosomes lagged in the equatorial region during chromosome segregation to the poles, distorting cytokinesis and yielding abnormal microspores. Chlorophyll defects were not observed, and meiotic abnormalities were far less frequent in reciprocal hybrids and in the case of an abnormal paternal inheritance of plastids from Sprint-1. Mitosis lacked overt abnormalities in all of the hybrids.

Karyoplast exchange between strontium- and 6-DMAP-parthenogenetically activated zygotes of cattle

Ooplasmic factors drive nuclear organization after fertilization and are also important for re-programming in nuclear transfer procedures, in which artificial activation is essential for reconstructed embryos to progress in development. The present research evaluated the effect of pronuclear transfer (PT) between zygotes parthenogenetically activated with ionomycin followed by strontium (S) or 6-DMAP (D) on early embryonic development. PT was performed in the same zygote to obtain embryos in control groups (S-PT and D-PT) and between zygotes activated with S and D to achieve embryos with differentially activated cytoplasm (C) and nucleus (N) (SCDN and DCSN). PT procedure did not affect cleavage and blastocyst rates, respectively, in PT control groups compared to non-manipulated control (S-PT: 73.6% and 7.3% compared with S-Control: 77.9% and 7.8%; and D-PT: 73.3% and 31.7% compared with D-Control: 83.1% and 41.5%). Cleavage, eight-cell, and blastocyst rates, respectively, were similar between SCDN (76.5%, 36.4%, and 6.8%) and DCSN (69.5%, 25.0%, and 4.9%) embryos. Developmental rates in SCDN were similar to S-PT, but inferior to D-PT. Developmental arrest up to eight-cell stage was greater in SCDN and DCSN than in S-PT and D-PT. In conclusion, karyoplast exchange between parthenogenetic zygotes activated with strontium and 6-DMAP can lead to nuclear–cytoplasmic incompatibilities and affect embryonic development to the eight-cell and blastocyst stages.

Evidence of restoration cost in the annual gynodioecious Phacelia dubia

A negative pleiotropic effect on fitness of nuclear sex-determining genes (cost of restoration) could explain nuclear-cytoplasmic gynodioecy but rarely has been demonstrated empirically. In a gynodioecious Phacelia dubia population, maternal lineages produce only hermaphroditic progenies irrespective of the pollen parent (N) or can segregate females (S). Natural progenies of N maternal plants had lower seed viability than that of S. Full-sib progenies of unrelated hermaphrodites from all possible matings between N and S lineages had similar pollen filling but differed in sporophyte performance, mainly at seed germination stage. A discrete multivariate analysis reveals that the performance of N(female symbol) x S(male symbol) progeny at early stages of development was significantly lower than that of the other three types of mating in agreement with the silent-cost-of-restoration hypothesis, affecting the sporophyte. The restoration cost and male sterility appear to be dominant and consequence of nuclear-cytoplasmic incompatibilities that may maintain nuclear-cytoplasmic polymorphism by frequency-dependent selection.

Genetic analysis of nuclear-cytoplasmic incompatibility in pea associated with cytoplasm of an accession of wild subspecies Pisum sativum subsp. elatius (Bieb.) Schmahl.

The genetic basis of nuclear-cytoplasmic incompatibility was examined using the wild pea (Pisum sativum subsp. elatius) accession VIR320. When this accession is used as the female parent in crosses with domesticated peas (Pisum sativum subsp. sativum) the F(1) is highly sterile and displays chlorophyll deficiency, chlorophyll variegation, reduction of leaflets and stipulae while the reciprocal cross produces hybrids that appear normal. A mapping recombinant inbred line (RIL) population was established based on a cross in a compatible direction of a tester line WL1238 with VIR320. The ability to cause nuclear-cytoplasmic conflict was analysed by crossing individual RIL plants as pollen parents with VIR320 as donor of cytoplasm and scoring each F(1) for major signs of the conflict. It is concluded that two unlinked nuclear genes are involved in the genetic control of the observed incompatibility. One of the genes, denoted as Scs1, is closely linked to the PhlC gene on linkage group III and the other, denoted as Scs2, is closely linked to the gp gene on linkage group V. Alleles of both genes in WL1238 are dominant and appear to be lethal in the homozygous condition in the VIR320 cytoplasm background.

Telophase-Stage Host Ooplasts Support Complete Reprogramming of Roscovitine-Treated Somatic Cell Nuclei in Cattle

Nuclear-cytoplasmic incompatibilities are known to play a significant role in the developmental outcome of embryos produced by nuclear transfer, particularly when metaphase arrested oocytes are used as hosts for interphase donor nuclei. To further our understanding of how cell cycle coordination affects somatic cell cloning, somatic cells at different stages of the cell cycle were fused to host oocytes either before (metaphase II, M-II) or after (telophase II, T-II) activation. To obtain cells at different stages of the cell cycle, fetal fibroblast (FF) and granulosa cells (GC) were treated with roscovitine, an inhibitor of cyclin-dependent kinases (CDKs) resulting in a large percentage of cells in S/G(2)-phase. In contrast to the M-II group, which did better with confluent cells, embryos reconstructed with T-II cytoplasts resulted in higher rates of blastocyst formation when fused to cells recovered at 16-24 h after passage. Embryos reconstructed with FF treated with roscovitine and T-II cytoplasts (Rosc/T-II) resulted in similar blastocyst rate compared to those produced with confluent cells and M-II cytoplasts (Conf/M-II). Transfer of blastocysts to surrogate heifers resulted pregnancies and birth of healthy calves from Rosc/T-II and Conf/M-II reconstructed embryos. These results indicate that, when combined with nuclear donor cells at specific cell cycle stages, M-II and T-II bovine oocytes are similarly effective in supporting the reprogramming of somatic cell nuclei.