Epigenetic histone H3 phosphorylation marks discriminate between univalent- and bivalent-forming chromosomes during canina asymmetrical meiosis.

BACKGROUND AND AIMS: Dogroses (Rosa sect. Caninae) are mostly pentaploid, bearing 2n = 5x = 35 chromosomes in somatic cells. They evolved a unique form of asymmetrical meiosis characterized by two types of chromosomes: (1) chromosomes forming bivalents and distributed in the normal sexual way; and (2) chromosomes occurring as univalents and transferred by a female gamete only. In the mature pollen of pentaploid species, seven bivalent-derived chromosomes are transmitted to offspring, and 21 unpaired univalent chromosomes are eliminated during microsporogenesis. To discriminate between bivalent- and univalent-forming chromosomes, we studied histone H3 phosphorylation patterns regulating meiotic chromosome condensation and segregation. METHODS: We analysed histone modification patterns during male canina meiosis in two representative dogrose species, 5x Rosa canina and 5x Rosa rubiginosa, by immunohistochemical and molecular cytogenetics approaches. Immunostaining of meiotic cells included α-tubulin, histone H3 phosphorylation (H3S10p, H3S28p and H3T3p) and methylation (H3K4me3 and H3K27me3) marks. In addition, fluorescent in situ hybridization was carried out with an 18S rDNA probe. KEY RESULTS: In the first meiotic division, univalent chromosomes underwent equational division into chromatids, while homologues in bivalents were segregated as regular dyads. In diakinesis, bivalent chromosomes displayed strong H3 phosphorylation signals in proximal regions, spreading to the rest of the chromosome. In contrast, in univalents, the H3 phosphorylation signals were weaker, occurring mostly outside proximal regions largely overlapping with the H3K4me3 signals. Reduced phosphorylation was associated with relative under-condensation of the univalent chromosomes, particularly at early diakinesis. CONCLUSIONS: We hypothesize that the absence of pairing and/or recombination in univalent chromosomes negatively affects the histone H3 phosphorylation of their chromatin and perhaps the loading of meiotic-specific cohesins. This apparently destabilizes cohesion of sister chromatids, leading to their premature split in the first meiotic division.

Untangling the hedge: Genetic diversity in clonally and sexually transmitted genomes of European wild roses, Rosa L.

While European wild roses are abundant and widely distributed, their morphological taxonomy is complicated and ambiguous. In particular, the polyploid Rosa section Caninae (dogroses) is characterised by its unusual meiosis, causing simultaneous clonal and sexual transmission of sub-genomes. This hemisexual reproduction, which often co-occurs with vegetative reproduction, defies the standard definition of species boundaries. We analysed seven highly polymorphic microsatellite loci, scored for over 2 600 Rosa samples of differing ploidy, collected across Europe within three independent research projects. Based on their morphology, these samples had been identified as belonging to 21 dogrose and five other native rose species. We quantified the degree of clonality within species and at individual sampling sites. We then compared the genetic structure within our data to current rose morpho-systematics and searched for hemisexually co-inherited sets of alleles at individual loci. We found considerably fewer copies of identical multi-locus genotypes in dogroses than in roses with regular meiosis, with some variation recorded among species. While clonality showed no detectable geographic pattern, some genotypes appeared to be more widespread. Microsatellite data confirmed the current classification of subsections, but they did not support most of the generally accepted dogrose microspecies. Under canina meiosis, we found co-inherited sets of alleles as expected, but could not distinguish between sexually and clonally inherited sub-genomes, with only some of the detected allele combinations being lineage-specific.

Population genetics and plant growth experiments as prerequisite for conservation measures of the rare European aquatic plant Luronium natans (Alismataceae).

Information provided by population genetic studies is often necessary to effectively protect endangered species. In general, such data is scarce for aquatic plants and this holds also for Luronium natans, an aquatic macrophyte endemic to northwestern and western Europe. It is threatened across its whole distribution range due to human influences, in particular due to eutrophication and intensive fish farming. In spite of habitat protection populations continue to decline and re-introductions are one possibility to prevent the species' extinction. Therefore, insights in genetic diversity and relatedness of source populations is warranted. Thus, we performed Amplified Fragment-Length Polymorphism (AFLP) on two large populations in Saxony, Germany (Großenhainer Pflege and Niederspree), complemented with numerous additional occurrences from Europe. In addition, we conducted experiments on plant growth to assess optimal conditions for ex-situ cultivation taking water temperature, water level and substrate into account. We revealed considerably high levels of genetic diversity within populations (Shannon Indices ranged from 0.367 to 0.416) implying that populations are not restricted to clonal growth only but reproduce also by open-pollinated flowers. Remarkably, the two geographically close Saxon populations were genetically distant to each other but subpopulations within a locality were completely intermingled. Concerning optimal cultivation conditions, longest roots were obtained at temperatures >14°C and saturated, but not submerging water levels. Thus, our findings advocate for a re-introduction scheme from nearby source populations and provide detailed information on successful ex-situ cultivation.

Phylogeography of Artemisia frigida (Anthemideae, Asteraceae) based on genotyping-by-sequencing and plastid DNA data: Migration through Beringia.

Artemisia frigida is a temperate grassland species that has the largest natural range among its genus, with occurrences across the temperate grassland biomes of Eurasia and North America. Despite its wide geographic range, we know little about the species' distribution history. Hence, we conducted a phylogeographical study to test the hypothesis that the species' distribution pattern is related to a potential historical migration over the 'Bering land bridge'. We applied two molecular approaches: genotyping-by-sequencing (GBS) and Sanger sequencing of the plastid intergenic spacer region (rpl32 - trnL) to investigate genetic differentiation and relatedness among 21 populations from North America, Middle Asia, Central Asia and the Russian Far East. Furthermore, we identified the ploidy level of individuals based on GBS data. Our results indicate that A. frigida originated in Asia, spread northwards to the Far East and then to North America across the Bering Strait. We found a pronounced genetic structuring between Middle and Central Asian populations with mixed ploidy levels, tetraploids in the Far East, and nearly exclusively diploids in North America except for one individual. According to phylogenetic analysis, two populations of Kazakhstan (KZ2 and KZ3) represent the most likely ancestral diploids that constitute the basally branching lineages, and subsequent polyploidization has occurred on several occasions independently. Mantel tests revealed weak correlations between genetic distance and geographical distance and climatic conditions, which indicates that paleoclimatic fluctuations may have more profoundly influenced A. frigida's spatial genetic structure and distribution than the current environment.

Power and Weakness of Repetition - Evaluating the Phylogenetic Signal From Repeatomes in the Family Rosaceae With Two Case Studies From Genera Prone to Polyploidy and Hybridization (Rosa and Fragaria).

Plant genomes consist, to a considerable extent, of non-coding repetitive DNA. Several studies showed that phylogenetic signals can be extracted from such repeatome data by using among-species dissimilarities from the RepeatExplorer2 pipeline as distance measures. Here, we advanced this approach by adjusting the read input for comparative clustering indirectly proportional to genome size and by summarizing all clusters into a main distance matrix subjected to Neighbor Joining algorithms and Principal Coordinate Analyses. Thus, our multivariate statistical method works as a "repeatomic fingerprint," and we proved its power and limitations by exemplarily applying it to the family Rosaceae at intrafamilial and, in the genera Fragaria and Rosa, at the intrageneric level. Since both taxa are prone to hybridization events, we wanted to show whether repeatome data are suitable to unravel the origin of natural and synthetic hybrids. In addition, we compared the results based on complete repeatomes with those from ribosomal DNA clusters only, because they represent one of the most widely used barcoding markers. Our results demonstrated that repeatome data contained a clear phylogenetic signal supporting the current subfamilial classification within Rosaceae. Accordingly, the well-accepted major evolutionary lineages within Fragaria were distinguished, and hybrids showed intermediate positions between parental species in data sets retrieved from both complete repeatomes and rDNA clusters. Within the taxonomically more complicated and particularly frequently hybridizing genus Rosa, we detected rather weak phylogenetic signals but surprisingly found a geographic pattern at a population scale. In sum, our method revealed promising results at larger taxonomic scales as well as within taxa with manageable levels of reticulation, but success remained rather taxon specific. Since repeatomes can be technically easy and comparably inexpensively retrieved even from samples of rather poor DNA quality, our phylogenomic method serves as a valuable alternative when high-quality genomes are unavailable, for example, in the case of old museum specimens.

Ancient Origin of Two 5S rDNA Families Dominating in the Genus Rosa and Their Behavior in the Canina-Type Meiosis.

The genus Rosa comprises more than 100 woody species characterized by intensive hybridization, introgression, and an overall complex evolutionary history. Besides many diploid species (2n = 2x = 14) polyploids ranging from 3x to 10x are frequently found. Here we analyzed 5S ribosomal DNA in 19 species covering two subgenera and the major sections within subg. Rosa. In addition to diploids and polyploids with regular meiosis, we focused on 5x dogroses (Rosa sect. Caninae), which exhibit an asymmetric meiosis differentiating between bivalent- and univalent-forming chromosomes. Using genomic resources, we reconstructed 5S rDNA units to reveal their phylogenetic relationships. Additionally, we designed locus-specific probes derived from intergenic spacers (IGSs) and determined the position and number of 5S rDNA families on chromosomes. Two major 5S rDNA families (termed 5S_A and 5S_B, respectively) were found at variable ratios in both diploid and polyploid species including members of the early diverging subgenera, Rosa persica and Rosa minutifolia. Within subg. Rosa species of sect. Rosa amplified the 5S_A variant only, while taxa of other sections contained both variants at variable ratios. The 5S_B family was often co-localized with 35S rDNA at the nucleolar organizer regions (NOR) chromosomes, whereas the co-localization of the 5S_A family with NOR was only exceptionally observed. The allo-pentaploid dogroses showed a distinct distribution of 5S rDNA families between bivalent- and univalent-forming chromosomes. In conclusion, two divergent 5S rDNA families dominate rose genomes. Both gene families apparently arose in the early history of the genus, already 30 myrs ago, and apparently survived numerous speciation events thereafter. These observations are consistent with a relatively slow genome turnover in the Rosa genus.

Asymmetrical canina meiosis is accompanied by the expansion of a pericentromeric satellite in non-recombining univalent chromosomes in the genus Rosa.

BACKGROUND AND AIMS: Despite their abundant odd-ploidy (2n = 5x = 35), dogroses (Rosa sect. Caninae) are capable of sexual reproduction due to their unique meiosis. During canina meiosis, two sets of chromosomes form bivalents and are transmitted by male and female gametes, whereas the remaining chromosomes form univalents and are exclusively transmitted by the egg cells. Thus, the evolution of chromosomes is expected to be driven by their behaviour during meiosis. METHODS: To gain insight into differential chromosome evolution, fluorescence in situ hybridization was conducted for mitotic and meiotic chromosomes in four dogroses (two subsections) using satellite and ribosomal DNA probes. By exploiting high-throughput sequencing data, we determined the abundance and diversity of the satellite repeats in the genus Rosa by analysing 20 pentaploid, tetraploid and diploid species in total. KEY RESULTS: A pericentromeric satellite repeat, CANR4, was found in all members of the genus Rosa, including the basal subgenera Hulthemia and Hesperhodos. The satellite was distributed across multiple chromosomes (5-20 sites per mitotic cell), and its genomic abundance was higher in pentaploid dogroses (2.3 %) than in non-dogrose species (1.3 %). In dogrose meiosis, univalent chromosomes were markedly enriched in CANR4 repeats based on both the number and the intensity of the signals compared to bivalent-forming chromosomes. Single-nucleotide polymorphisms and cluster analysis revealed high intragenomic homogeneity of the satellite in dogrose genomes. CONCLUSIONS: The CANR4 satellite arose early in the evolution of the genus Rosa. Its high content and extraordinary homogeneity in dogrose genomes is explained by its recent amplification in non-recombining chromosomes. We hypothesize that satellite DNA expansion may contribute to the divergence of univalent chromosomes in Rosa species with non-symmetrical meiosis.

The fate of ribosomal RNA genes in spontaneous polyploid dogrose hybrids [Rosa L. sect. Caninae (DC.) Ser.] exhibiting non-symmetrical meiosis.

Dogroses represent an exceptional system for studying the effects of genome doubling and hybridization: their asymmetrical meiosis enables recombination in bi-parentally inherited chromosomes but prevents it in maternally inherited ones. We employed fluorescent in situ hybridization, genome skimming, amplicon sequencing of genomic and cDNA as well as conventional cloning of nuclear ribosomal DNA in two phylogenetically distinct pentaploid (2n = 5x = 35) species, Rosa canina and Rosa inodora, and their naturally occurring reciprocal hybrids, Rosa dumalis (5x) and Rosa agrestis (5x, 6x). Both progenitor species differed in composition, meiotic behaviour and expression of rDNA loci: R. canina (five 18S and 5-8 5S loci) was dominated by the Canina ribotypes, but R. inodora (four 18S loci and 7-8 5S loci) by the Rubiginosa ribotype. The co-localized 5S/18S loci occurred on either bivalent-forming (R. canina) or univalent-forming (R. inodora) chromosomes. Ribosomal DNA loci were additively inherited; however, the Canina ribotypes were dominantly expressed, even in genotypes with relatively low copy number of these genes. Moreover, we observed rDNA homogenization towards the paternally transmitted Canina ribotype in 6x R. agrestis. The here-observed variation in arrangement and composition of rDNA types between R. canina and R. inodora suggests the involvement of different genomes in bivalent formation. This results supports the hypothesis that the asymmetrical meiosis arose at least twice by independent ancient hybridization events.