Diploid Nuclei Occur throughout the Life Cycles of Pucciniales Fungi.

Within Eukaryotes, fungi are the typical representatives of haplontic life cycles. Basidiomycota fungi are dikaryotic in extensive parts of their life cycle, but diploid nuclei are known to form only in basidia. Among Basidiomycota, the Pucciniales are notorious for presenting the most complex life cycles, with high host specialization, and for their expanded genomes. Using cytogenomic (flow cytometry and cell sorting on propidium iodide-stained nuclei) and cytogenetic (FISH with rDNA probe) approaches, we report the widespread occurrence of replicating haploid and diploid nuclei (i.e., 1C, 2C and a small proportion of 4C nuclei) in diverse life cycle stages (pycnial, aecial, uredinial, and telial) of all 35 Pucciniales species analyzed, but not in sister taxa. These results suggest that the Pucciniales life cycle is distinct from any cycle known, i.e., neither haplontic, diplontic nor haplodiplontic, corroborating patchy and disregarded previous evidence. However, the biological basis and significance of this phenomenon remain undisclosed. IMPORTANCE Within Eukaryotes, fungi are the typical representatives of haplontic life cycles, contrasting with plants and animals. As such, fungi thus contain haploid nuclei throughout their life cycles, with sexual reproduction generating a single diploid cell upon karyogamy that immediately undergoes meiosis, thus resuming the haploid cycle. In this work, using cytogenetic and cytogenomic tools, we demonstrate that a vast group of fungi presents diploid nuclei throughout their life cycles, along with haploid nuclei, and that both types of nuclei replicate. Moreover, haploid nuclei are absent from urediniospores. The phenomenon appears to be transversal to the organisms in the order Pucciniales (rust fungi) and it does not occur in neighboring taxa, but a biological explanation or function for it remains elusive.

Exploring the crop epigenome: a comparison of DNA methylation profiling techniques.

Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.

Genome-wide identification, phylogeny, and gene duplication of the epigenetic regulators in Fagaceae.

Epigenetic regulators are proteins involved in controlling gene expression. Information about the epigenetic regulators within the Fagaceae, a relevant family of trees and shrubs of the northern hemisphere ecosystems, is scarce. With the intent to characterize these proteins in Fagaceae, we searched for orthologs of DNA methyltransferases (DNMTs) and demethylases (DDMEs) and Histone modifiers involved in acetylation (HATs), deacetylation (HDACs), methylation (HMTs), and demethylation (HDMTs) in Fagus, Quercus, and Castanea genera. Blast searches were performed in the available genomes, and freely available RNA-seq data were used to de novo assemble transcriptomes. We identified homologs of seven DNMTs, three DDMEs, six HATs, 11 HDACs, 32 HMTs, and 21 HDMTs proteins. Protein analysis showed that most of them have the putative characteristic domains found in these protein families, which suggests their conserved function. Additionally, to elucidate the evolutionary history of these genes within Fagaceae, paralogs were identified, and phylogenetic analyses were performed with DNA and histone modifiers. We detected duplication events in all species analyzed with higher frequency in Quercus and Castanea and discuss the evidence of transposable elements adjacent to paralogs and their involvement in gene duplication. The knowledge gathered from this work is a steppingstone to upcoming studies concerning epigenetic regulation in this economically important family of Fagaceae.

Epigenetics at the crossroads of secondary growth regulation.

The development of plant tissues and organs during post-embryonic growth occurs through the activity of both primary and secondary meristems. While primary meristems (root and shoot apical meristems) promote axial plant growth, secondary meristems (vascular and cork cambium or phellogen) promote radial thickening and plant axes strengthening. The vascular cambium forms the secondary xylem and phloem, whereas the cork cambium gives rise to the periderm that envelops stems and roots. Periderm takes on an increasingly important role in plant survival under climate change scenarios, but it is also a forest product with unique features, constituting the basis of a sustainable and profitable cork industry. There is established evidence that epigenetic mechanisms involving histone post-translational modifications, DNA methylation, and small RNAs play important roles in the activity of primary meristem cells, their maintenance, and differentiation of progeny cells. Here, we review the current knowledge on the epigenetic regulation of secondary meristems, particularly focusing on the phellogen activity. We also discuss the possible involvement of DNA methylation in the regulation of periderm contrasting phenotypes, given the potential impact of translating this knowledge into innovative breeding programs.

The Dynamics of Flower Development in Castanea sativa Mill.

The sweet chestnut tree (Castanea sativa Mill.) is one of the most significant Mediterranean tree species, being an important natural resource for the wood and fruit industries. It is a monoecious species, presenting unisexual male catkins and bisexual catkins, with the latter having distinct male and female flowers. Despite the importance of the sweet chestnut tree, little is known regarding the molecular mechanisms involved in the determination of sexual organ identity. Thus, the study of how the different flowers of C. sativa develop is fundamental to understand the reproductive success of this species and the impact of flower phenology on its productivity. In this study, a C. sativa de novo transcriptome was assembled and the homologous genes to those of the ABCDE model for floral organ identity were identified. Expression analysis showed that the C. sativa B- and C-class genes are differentially expressed in the male flowers and female flowers. Yeast two-hybrid analysis also suggested that changes in the canonical ABCDE protein-protein interactions may underlie the mechanisms necessary to the development of separate male and female flowers, as reported for the monoecious Fagaceae Quercus suber. The results here depicted constitute a step towards the understanding of the molecular mechanisms involved in unisexual flower development in C. sativa, also suggesting that the ABCDE model for flower organ identity may be molecularly conserved in the predominantly monoecious Fagaceae family.

Cork cells in cork oak periderms undergo programmed cell death and proanthocyanidin deposition.

Vascular plants with secondary growth develop a periderm mostly composed of dead suberized cork cells to face environmental hostile conditions. Cork oak has a highly active and long-living phellogen forming a remarkably thick periderm that is periodically debarked for industrial purposes. This wounding originates the quick formation of a new traumatic periderm, making cork oak an exceptional model to study the first periderm differentiation during normal development in young sprigs and traumatic (wound) periderm formation after debarking. Here, we studied the poorly known first periderm differentiation steps that involve cell wall suberization, polyphenolic accumulation and programmed cell death (PCD) by combining transmission electron microscopy, histochemical and molecular methods in periderms from young sprigs. These processes were further compared with traumatic periderms formed after wounding using molecular and histochemical techniques, such as the polyphenolic accumulation. In the first periderms from young sprigs, four distinct differentiation stages were defined according to the presence of PCD morphological features. First young and traumatic periderms showed an upregulation of genes related to suberin biosynthesis, proanthocyanidins biosynthesis and transport, autophagy, and PCD. Traumatic periderms revealed an overall upregulation of these genes, likely resulting from ontogeny differences and distinct phellogen origin associated with a faster metabolism, highlighting the impact of wounding on phellogen activity after debarking. First periderms from young sprigs showed gradual accumulation of proanthocyanidins in the vacuoles throughout PCD stages until total filled lumens, whereas in traumatic periderms, these compounds were found cell wall linked in already empty cells. This work enabled a comprehensive overview of the cork cells differentiation processes contributing to deepening the knowledge of the fundamental ontogenic program of this protective tissue, which is also a unique forest product, constituting the basis of a sustainable and profitable industry.

Genome-Wide Identification of Epigenetic Regulators in Quercus suber L.

Modifications of DNA and histones, including methylation and acetylation, are critical for the epigenetic regulation of gene expression during plant development, particularly during environmental adaptation processes. However, information on the enzymes catalyzing all these modifications in trees, such as Quercus suber L., is still not available. In this study, eight DNA methyltransferases (DNA Mtases) and three DNA demethylases (DDMEs) were identified in Q. suber. Histone modifiers involved in methylation (35), demethylation (26), acetylation (8), and deacetylation (22) were also identified in Q. suber. In silico analysis showed that some Q. suber DNA Mtases, DDMEs and histone modifiers have the typical domains found in the plant model Arabidopsis, which might suggest a conserved functional role. Additional phylogenetic analyses of the DNA and histone modifier proteins were performed using several plant species homologs, enabling the classification of the Q. suber proteins. A link between the expression levels of each gene in different Q. suber tissues (buds, flowers, acorns, embryos, cork, and roots) with the functions already known for their closest homologs in other species was also established. Therefore, the data generated here will be important for future studies exploring the role of epigenetic regulators in this economically important species.

ChIP-Seq reveals that QsMYB1 directly targets genes involved in lignin and suberin biosynthesis pathways in cork oak (Quercus suber).

BACKGROUND: Gene activity is largely controlled by transcriptional regulation through the action of transcription factors and other regulators. QsMYB1 is a member of the R2R3-MYB transcription factor family related to secondary growth, and in particular, with the cork development process. In order to identify the putative gene targets of QsMYB1 across the cork oak genome we developed a ChIP-Seq strategy. RESULTS: Results provide direct evidence that QsMY1B targets genes encoding for enzymes involved in the lignin and suberin pathways as well as gene encoding for ABCG transporters and LTPs implicated in the transport of monomeric suberin units across the cellular membrane. These results highlight the role of QsMYB1 as a regulator of lignin and suberin biosynthesis, transport and assembly. CONCLUSION: To our knowledge, this work constitutes the first ChIP-Seq experiment performed in cork oak, a non-model plant species with a long-life cycle, and these results will contribute to deepen the knowledge about the molecular mechanisms of cork formation and differentiation.

Cork Oak Young and Traumatic Periderms Show PCD Typical Chromatin Patterns but Different Chromatin-Modifying Genes Expression.

Plants are subjected to adverse conditions being outer protective tissues fundamental to their survival. Tree stems are enveloped by a periderm made of cork cells, resulting from the activity of the meristem phellogen. DNA methylation and histone modifications have important roles in the regulation of plant cell differentiation. However, studies on its involvement in cork differentiation are scarce despite periderm importance. Cork oak periderm development was used as a model to study the formation and differentiation of secondary protective tissues, and their behavior after traumatic wounding (traumatic periderm). Nuclei structural changes, dynamics of DNA methylation, and posttranslational histone modifications were assessed in young and traumatic periderms, after cork harvesting. Lenticular phellogen producing atypical non-suberized cells that disaggregate and form pores was also studied, due to high impact for cork industrial uses. Immunolocalization of active and repressive marks, transcription analysis of the corresponding genes, and correlations between gene expression and cork porosity were investigated. During young periderm development, a reduction in nuclei area along with high levels of DNA methylation occurred throughout epidermis disruption. As cork cells became more differentiated, whole nuclei progressive chromatin condensation with accumulation in the nuclear periphery and increasing DNA methylation was observed. Lenticular cells nuclei were highly fragmented with faint 5-mC labeling. Phellogen nuclei were less methylated than in cork cells, and in lenticular phellogen were even lower. No significant differences were detected in H3K4me3 and H3K18ac signals between cork cells layers, although an increase in H3K4me3 signals was found from the phellogen to cork cells. Distinct gene expression patterns in young and traumatic periderms suggest that cork differentiation might be under specific silencing regulatory pathways. Significant correlations were found between QsMET1, QsMET2, and QsSUVH4 gene expression and cork porosity. This work evidences that DNA methylation and histone modifications play a role in cork differentiation and epidermis induced tension-stress. It also provides the first insights into chromatin dynamics during cork and lenticular cells differentiation pointing to a distinct type of remodeling associated with cell death.

Differential DNA Methylation Patterns Are Related to Phellogen Origin and Quality of Quercus suber Cork.

DNA methylation is thought to influence Quercus suber cork quality, which is the main constraint for its economic valorisation. However, a deep knowledge of the cytosine methylation patterns disclosing the epigenetic variability of trees with different cork quality types is totally missing. This study investigates the hypothesis that variations in DNA methylation contribute to differences in cork cellular characteristics directly related to original or traumatic phellogen activity. We used MSAPs (Methylation Sensitive Amplified Polymorphism) to assess DNA methylation patterns of cork and leaf tissues of Q. suber adult trees growing in three cork oak stands. The relationship between the detected polymorphisms and the diversity of cork quality traits was explored by a marker-trait analysis focusing on the most relevant quality characteristics. Populations differed widely in cork quality, but only slightly in degree of epigenetic differentiation. Four MSAP markers (1.3% of the total) were significantly associated with the most noteworthy quality traits: wood inclusions (nails) and porosity. This evidence supports the potential role of cytosine methylation in the modulation of differential phellogen activity either involved in localized cell death or in pore production, resulting in different cork qualities. Although, the underlying basis of the methylation polymorphism of loci affecting cork quality traits remain unclear, the disclosure of markers statistically associated with cork quality strengthens the potential role of DNA methylation in the regulation of these traits, namely at the phellogen level.

Advancing Eucalyptus Genomics: Cytogenomics Reveals Conservation of Eucalyptus Genomes.

The genus Eucalyptus encloses several species with high ecological and economic value, being the subgenus Symphyomyrtus one of the most important. Species such as E. grandis and E. globulus are well characterized at the molecular level but knowledge regarding genome and chromosome organization is very scarce. Here we characterized and compared the karyotypes of three economically important species, E. grandis, E. globulus, and E. calmadulensis, and three with ecological relevance, E. pulverulenta, E. cornuta, and E. occidentalis, through an integrative approach including genome size estimation, fluorochrome banding, rDNA FISH, and BAC landing comprising genes involved in lignin biosynthesis. All karyotypes show a high degree of conservation with pericentromeric 35S and 5S rDNA loci in the first and third pairs, respectively. GC-rich heterochromatin was restricted to the 35S rDNA locus while the AT-rich heterochromatin pattern was species-specific. The slight differences in karyotype formulas and distribution of AT-rich heterochromatin, along with genome sizes estimations, support the idea of Eucalyptus genome evolution by local expansions of heterochromatin clusters. The unusual co-localization of both rDNA with AT-rich heterochromatin was attributed mainly to the presence of silent transposable elements in those loci. The cinnamoyl CoA reductase gene (CCR1) previously assessed to linkage group 10 (LG10) was clearly localized distally at the long arm of chromosome 9 establishing an unexpected correlation between the cytogenetic chromosome 9 and the LG10. Our work is novel and contributes to the understanding of Eucalyptus genome organization which is essential to develop successful advanced breeding strategies for this genus.

The Quest for Molecular Regulation Underlying Unisexual Flower Development.

The understanding of the molecular mechanisms responsible for the making of a unisexual flower has been a long-standing quest in plant biology. Plants with male and female flowers can be divided mainly into two categories: dioecious and monoecious, and both sexual systems co-exist in nature in ca of 10% of the angiosperms. The establishment of male and female traits has been extensively described in a hermaphroditic flower and requires the interplay of networks, directly and indirectly related to the floral organ identity genes including hormonal regulators, transcription factors, microRNAs, and chromatin-modifying proteins. Recent transcriptomic studies have been uncovering the molecular processes underlying the establishment of unisexual flowers and there are many parallelisms between monoecious, dioecious, and hermaphroditic individuals. Here, we review the paper entitled "Comparative transcriptomic analysis of male and female flowers of monoecious Quercus suber" published in 2014 in the Frontiers of Plant Science (volume 5 |Article 599) and discussed it in the context of recent studies with other dioecious and monoecious plants that utilized high-throughput platforms to obtain transcriptomic profiles of male and female unisexual flowers. In some unisexual flowers, the developmental programs that control organ initiation fail and male or female organs do not form, whereas in other species, organ initiation and development occur but they abort or arrest during different species-specific stages of differentiation. Therefore, a direct comparison of the pathways responsible for the establishment of unisexual flowers in different species are likely to reveal conserved modules of gene regulatory hubs involved in stamen or carpel development, as well as differences that reflect the different stages of development in which male and/or female organ arrest or loss-of-function occurs.

Comparative transcriptomic analysis of male and female flowers of monoecious Quercus suber.

Monoecious species provide a comprehensive system to study the developmental programs underlying the establishment of female and male organs in unisexual flowers. However, molecular resources for most monoecious non-model species are limited, hampering our ability to study the molecular mechanisms involved in flower development of these species. The objective of this study was to identify differentially expressed genes during the development of male and female flowers of the monoecious species Quercus suber, an economically important Mediterranean tree. Total RNA was extracted from different developmental stages of Q. suber flowers. Non-normalized cDNA libraries of male and female flowers were generated using 454 pyrosequencing technology producing a total of 962,172 high-quality reads with an average length of 264 nucleotides. The assembly of the reads resulted in 14,488 contigs for female libraries and 10,438 contigs for male libraries. Comparative analysis of the transcriptomes revealed genes differentially expressed in early and late stages of development of female and male flowers, some of which have been shown to be involved in pollen development, in ovule formation and in flower development of other species with a monoecious, dioecious, or hermaphroditic sexual system. Moreover, we found differentially expressed genes that have not yet been characterized and others that have not been previously shown to be implicated in flower development. This transcriptomic analysis constitutes a major step toward the characterization of the molecular mechanisms involved in flower development in a monoecious tree with a potential contribution toward the knowledge of conserved developmental mechanisms in other species.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Molecular organization of the 25S-18S rDNA IGS of Fagus sylvatica and Quercus suber: a comparative analysis.

The 35S ribosomal DNA (rDNA) units, repeated in tandem at one or more chromosomal loci, are separated by an intergenic spacer (IGS) containing functional elements involved in the regulation of transcription of downstream rRNA genes. In the present work, we have compared the IGS molecular organizations in two divergent species of Fagaceae, Fagus sylvatica and Quercus suber, aiming to comprehend the evolution of the IGS sequences within the family. Self- and cross-hybridization FISH was done on representative species of the Fagaceae. The IGS length variability and the methylation level of 18 and 25S rRNA genes were assessed in representatives of three genera of this family: Fagus, Quercus and Castanea. The intergenic spacers in Beech and Cork Oak showed similar overall organizations comprising putative functional elements needed for rRNA gene activity and containing a non-transcribed spacer (NTS), a promoter region, and a 5'-external transcribed spacer. In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak. The 5'-ETS is the region with the higher similarity, having nonetheless different lengths. FISH with the NTS-5'-ETS revealed fainter signals in cross-hybridization in agreement with the divergence between genera. The diversity of IGS lengths revealed variants from ∼ 2 kb in Fagus, and Quercus up to 5.3 kb in Castanea, and a lack of correlation between the number of variants and the number of rDNA loci in several species. Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea. These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.

Corky, a gypsy-like retrotransposon is differentially transcribed in Quercus suber tissues.

BACKGROUND: Transposable elements (TEs) make up a large part of eukaryotic genomes. Due to their repetitive nature and to the fact that they harbour regulatory signals, TEs can be responsible for chromosomal rearrangements, movement of gene sequences and evolution of gene regulation and function. Retrotransposon ubiquity raises the question about their function in genomes and most are transcriptionally inactive due to rearrangements that compromise their activity. However, the activity of TEs is currently considered to have been one of the major processes in genome evolution. FINDINGS: We report on the characterization of a transcriptionally active gypsy-like retrotransposon (named Corky) from Quercus suber, in a comparative and quantitative study of expression levels in different tissues and distinct developmental stages through RT-qPCR. We observed Corky's differential transcription levels in all the tissues analysed. CONCLUSIONS: These results document that Corky's transcription levels are not constant. Nevertheless, they depend upon the developmental stage, the tissue analysed and the potential occurring events during an individuals' life span. This modulation brought upon by different developmental and environmental influences suggests an involvement of Corky in stress response and during development.

Genomic organization and dynamics of repetitive DNA sequences in representatives of three Fagaceae genera.

Oaks, chestnuts, and beeches are economically important species of the Fagaceae. To understand the relationship between these members of this family, a deep knowledge of their genome composition and organization is needed. In this work, we have isolated and characterized several AFLP fragments obtained from Quercus rotundifolia Lam. through homology searches in available databases. Genomic polymorphisms involving some of these sequences were evaluated in two species of Quercus, one of Castanea, and one of Fagus with specific primers. Comparative FISH analysis with generated sequences was performed in interphase nuclei of the four species, and the co-immunolocalization of 5-methylcytosine was also studied. Some of the sequences isolated proved to be genus-specific, while others were present in all the genera. Retroelements, either gypsy-like of the Tat/Athila clade or copia-like, are well represented, and most are dispersed in euchromatic regions of these species with no DNA methylation associated, pointing to an interspersed arrangement of these retroelements with potential gene-rich regions. A particular gypsy-sequence is dispersed in oaks and chestnut nuclei, but its confinement to chromocenters in beech evidences genome restructuring events during evolution of Fagaceae. Several sequences generated in this study proved to be good tools to comparatively study Fagaceae genome organization.

Epigenetic marks in the mature pollen of Quercus suber L. (Fagaceae).

We have analysed the distribution of epigenetic marks for histone modifications at lysine residues H3 and H4, and DNA methylation, in the nuclei of mature pollen cells of the Angiosperm tree Quercus suber; a monoecious wind pollinated species with a protandrous system, and a long post-pollination period. The ultrasonic treatment developed for the isolation of pollen nuclei proved to be a fast and reliable method, preventing the interference of cell wall autofluorescence in the in situ immunolabelling assays. In contrast with previous studies on herbaceous species with short progamic phases, our results are consistent with a high level of silent (5-mC and H3K9me2) epigenetic marks on chromatin of the generative nucleus, and the prevalence of active marks (H3K9me3 and H4Kac) in the vegetative nucleus. The findings are discussed in terms of the pollination/fertilization timing strategy adopted by this plant species.

Transcriptionally active heterochromatin in rye B chromosomes.

B chromosomes (Bs) are dispensable components of the genomes of numerous species. Thus far, there is a lack of evidence for any transcripts of Bs in plants, with the exception of some rDNA sequences. Here, we show that the Giemsa banding-positive heterochromatic subterminal domain of rye (Secale cereale) Bs undergoes decondensation during interphase. Contrary to the heterochromatic regions of A chromosomes, this domain is simultaneously marked by trimethylated H3K4 and by trimethylated H3K27, an unusual combination of apparently conflicting histone modifications. Notably, both types of B-specific high copy repeat families (E3900 and D1100) of the subterminal domain are transcriptionally active, although with different tissue type-dependent activity. No small RNAs were detected specifically for the presence of Bs. The lack of any significant open reading frame and the highly heterogeneous size of mainly polyadenylated transcripts indicate that the noncoding RNA may function as structural or catalytic RNA.

Genomic analysis of Grapevine Retrotransposon 1 (Gret 1) in Vitis vinifera.

The complete sequence of the first retrotransposon isolated in Vitis vinifera, Gret 1, was used to design primers that permitted its analysis in the genome of grapevine cultivars. This retroelement was found to be dispersed throughout the genome with sites of repeated insertions. Fluorescent in situ hybridization indicated multiple Gret 1 loci distributed throughout euchromatic portions of chromosomes. REMAP and IRAP proved to be useful as molecular markers in grapevine. Both of these techniques showed polymorphisms between cultivars but not between clones of the same cultivar, indicating differences in Gret 1 distribution between cultivars. The combined cytological and molecular results suggest that Gret 1 may have a role in gene regulation and in explaining the enormous phenotypic variability that exists between cultivars.