Tooth-composite bond failure with a universal and an etch-and-rinse adhesive depending on mode and frequency of application.

OBJECTIVES: To evaluate the effect of an additional layer of universal adhesive on the interfacial enamel/dentin-composite gap formation in relation to application mode and aging, via spectral domain optical coherence tomography (SD-OCT) and scanning electron microscopy (SEM). METHODS: In vitro class V cavities in 114 caries-free premolars were restored by applying one or two layers of a universal adhesive (Scotchbond Universal, SBU) in self-etch (se) and etch-and-rinse (er) mode or the reference adhesive OptiBond FL (OFL-er). The restorations were imaged by SD-OCT (six groups, n = 8) and SEM (n = 3) directly after filling (t1), water storage (t2, 24 h), embedding (t3), and thermo-mechanical loading (t4, TCML). The interfacial gaps were quantified using 26 parameters and analyzed using principal component analysis and linear mixed effect models. RESULTS: Gap formation at enamel and dentin was significantly influenced by the adhesive, the application mode and number of layers (p < 0.001). This was due to the influence of the SBU-er mode (p < 1e-05), which showed significantly more gap formation and a greater range of variation with double application when compared to SBU-se and OFL. The fewest interfacial gaps occurred with one or two applications of OFL-er and one layer of SBU-er. SIGNIFICANCE: Adhesive application mode and the number of adhesive layers are relevant factors in the tooth-composite bond failure. Double application worsened the adaptation of SBU to freshly prepared dentin conditioned with phosphoric acid.

Seed size, endosperm and germination variation in sexual and apomictic Boechera.

Asexual reproduction results in offspring that are genetically identical to the mother. Among apomictic plants (reproducing asexually through seeds) many require paternal genetic contribution for proper endosperm development (pseudogamous endosperm). We examined phenotypic diversity in seed traits using a diverse panel of sexual and apomictic accessions from the genus Boechera. While genetic uniformity resulting from asexual reproduction is expected to reduce phenotypic diversity in seeds produced by apomictic individuals, pseudogamous endosperm, variable endosperm ploidy, and the deviations from 2:1 maternal:paternal genome ratio in endosperm can all contribute to increased phenotypic diversity among apomictic offspring. We characterized seed size variation in 64 diploid sexual and apomictic (diploid and triploid) Boechera lineages. In order to find out whether individual seed size was related to endosperm ploidy we performed individual seed measurements (projected area and mass) using the phenoSeeder robot system and flow cytometric seed screen. In order to test whether individual seed size had an effect on resulting fitness we performed a controlled growth experiment and recorded seedling life history traits (germination success, germination timing, and root growth rate). Seeds with triploid embryos were 33% larger than those with diploid embryos, but no average size difference was found between sexual and apomictic groups. We identified a maternal effect whereby chloroplast lineage 2 had 30% larger seeds than lineage 3, despite having broad and mostly overlapping geographic ranges. Apomictic seeds were not more uniform in size than sexual seeds, despite genetic uniformity of the maternal gametophyte in the former. Among specific embryo/endosperm ploidy combinations, seeds with tetraploid (automomous) endosperm were on average smaller, and the proportion of such seeds was highest in apomicts. Larger seeds germinated more quickly than small seeds, and lead to higher rates of root growth in young seedlings. Seed mass is under balancing selection in Boechera, and it is an important predictor of several traits, including germination probability and timing, root growth rates, and developmental abnormalities in apomictic accessions.

Chasing the Apomictic Factors in the Ranunculus auricomus Complex: Exploring Gene Expression Patterns in Microdissected Sexual and Apomictic Ovules.

Apomixis, the asexual reproduction via seeds, is associated to polyploidy and hybridization. To identify possible signatures of apomixis, and possible candidate genes underlying the shift from sex to apomixis, microarray-based gene expression patterns of live microdissected ovules at four different developmental stages were compared between apomictic and sexual individuals of the Ranunculus auricomus complex. Following predictions from previous work on mechanisms underlying apomixis penetrance and expressivity in the genus, gene expression patterns were classified into three categories based on their relative expression in apomicts compared to their sexual parental ancestors. We found evidence of misregulation and differential gene expression between apomicts and sexuals, with the highest number of differences detected during meiosis progression and emergence of aposporous initial (AI) cells, a key developmental stage in the ovule of apomicts where a decision between divergent reproductive pathways takes place. While most of the differentially expressed genes (DEGs) could not be annotated, gene expression was classified into transgressive, parent of origin and ploidy effects. Genes related to gametogenesis and meiosis demonstrated patterns reflective of transgressive and genome dosage effects, which support the hypothesis of a dominant factor controlling apomixis in Ranunculus and modulated by secondary modifiers. Three genes with probable functions in sporogenesis and gametogenesis development are identified and characterized for future studies.

Holocentromeres in Rhynchospora are associated with genome-wide centromere-specific repeat arrays interspersed among euchromatin.

Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinetochores distributed along almost the entire poleward surface of the chromatids, to which spindle fibers attach. No centromere-specific DNA sequence has been found for any holocentric organism studied so far. It was proposed that centromeric repeats, typical for many monocentric species, could not occur in holocentrics, most likely because of differences in the centromere organization. Here we show that the holokinetic centromeres of the Cyperaceae Rhynchospora pubera are highly enriched by a centromeric histone H3 variant-interacting centromere-specific satellite family designated "Tyba" and by centromeric retrotransposons (i.e., CRRh) occurring as genome-wide interspersed arrays. Centromeric arrays vary in length from 3 to 16 kb and are intermingled with gene-coding sequences and transposable elements. We show that holocentromeres of metaphase chromosomes are composed of multiple centromeric units rather than possessing a diffuse organization, thus favoring the polycentric model. A cell-cycle-dependent shuffling of multiple centromeric units results in the formation of functional (poly)centromeres during mitosis. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromeric chromatin organization among eukaryotes. Thus, different types of holocentromeres exist in different species, namely with and without centromeric repetitive sequences.

Emergence of apospory and bypass of meiosis via apomixis after sexual hybridisation and polyploidisation.

Hybridisation and polyploidy are major forces contributing to plant speciation. Homoploid (2x) and heteroploid (3x) hybrids, however, represent critical stages for evolution due to disturbed meiosis and reduced fertility. Apomixis--asexual reproduction via seeds--can overcome hybrid sterility, but requires several concerted alterations of developmental pathways to result in functional seed formation. Here, we analyse the reproductive behaviours of homo- and heteroploid synthetic hybrids from crosses between sexual diploid and tetraploid Ranunculus auricomus species to test the hypothesis that developmental asynchrony in hybrids triggers the shift to apomictic reproduction. Evaluation of male and female gametophyte development, viability and functionality of gametes shows developmental asynchrony, whereas seed set and germinability indicate reduced fitness in synthetic hybrids compared to sexual parents. We present the first experimental evidence for spontaneous apospory in most hybrids as an alternative pathway to meiosis, and the appearance of functional apomictic seeds in triploids. Bypassing meiosis permits these triploid genotypes to form viable seed and new polyploid progeny. Asynchronous development causes reduced sexual seed set and emergence of apospory in synthetic Ranunculus hybrids. Apomixis is functional in triploids and associated with drastic meiotic abnormalities. Selection acts to stabilise developmental patterns and to tolerate endosperm dosage balance shifts which facilitates successful seed set and establishment of apomictic lineages.

Asexual genome evolution in the apomictic Ranunculus auricomus complex: examining the effects of hybridization and mutation accumulation.

Asexual lineages are thought to be prone to extinction because of deleterious mutation accumulation (Muller's ratchet). Here, we analyse genomic effects of hybridity, polyploidy and allelic divergence in apomictic plants, and identify loci under divergent selection among sexual/apomictic lineages. RNAseq was used to sequence the flower-specific transcriptomes of five genotypes of the Ranunculus auricomus complex, representing three sexual and two apomictic reproductive biotypes. The five sequence libraries were pooled and de novo assembly performed, and the resultant assembly was used as a backbone for a subsequent alignment of each separate library. High-quality single-nucleotide (SNP) and insertion-deletion (indel) polymorphisms were mined from each library. Annotated genes for which open reading frames (ORF) could be determined were analysed for signatures of divergent versus stabilizing selection. A comparison between all genotypes supports the hypothesis of Pleistocene hybrid origin of both apomictic genotypes from R. carpaticola and R. cassubicifolius, with subsequent allelic divergence of apomictic lineages (Meselson effect). Pairwise comparisons of nonsynonymous (dN) to synonymous (dS) substitution rate ratios between apomictic and sexual genotypes for 1231 genes demonstrated similar distributions for all comparisons, although 324 genes demonstrated outlier (i.e. elevated) dN/dS ratios. Gene ontology analyses of these outliers revealed significant enrichment of genes associated with reproduction including meiosis and gametogenesis, following predictions of divergent selection between sexual and apomictic reproduction, although no significant signal of genome-wide mutation accumulation could be identified. The results suggest that gene function should be considered in order to understand effects of mutation accumulation in asexual lineages.

Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera.

BACKGROUND: Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX) derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates. RESULTS: Based on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18), Efalpha1 (Elongation factor 1 alpha), ACT 2 (Actin2), UBQ (polyubiquitin), PEX4 (Peroxisomal ubiquitin conjugating enzyme) and At1g09770.1 (Arabidopsis thaliana cell division cycle 5). Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues) in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEfα1 or BoechUBQ and BoechEfα1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed, different optimal combinations were identified in sexual ovules (BoechUBQ and BoechEfα1), in anthers from both reproductive systems (BoechACT2 and BoechEfα1), in apomictic vegetative tissues (BoechEfα1 and BoechACT2) and sexual vegetative tissues (BoechRPS18 and BoechEfα1). NormFinder ranked BoechACT2 as the most stable in the apomictic plant, while BoechRPS18 was the best in the sexual plant. The subgroups analysis identified the best gene for both apomictic and sexual ovules (BoechRPS18), for anthers from both reproductive system (BoechEfα1) and for apomictic and vegetative tissues (BoechACT2 and BoechRPS18 respectively) CONCLUSIONS: From a total of six tested genes, BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ showed the best stability values. We furthermore provide detailed information for the accurate normalization of specific tissue gene expression analyses of apomictic and sexual Boechera.