Repeat turnover meets stable chromosomes: repetitive DNA sequences mark speciation and gene pool boundaries in sugar beet and wild beets.

Sugar beet and its wild relatives share a base chromosome number of nine and similar chromosome morphologies. Yet, interspecific breeding is impeded by chromosome and sequence divergence that is still not fully understood. Since repetitive DNAs are among the fastest evolving parts of the genome, we investigated, if repeatome innovations and losses are linked to chromosomal differentiation and speciation. We traced genome and chromosome-wide evolution across 13 beet species comprising all sections of the genera Beta and Patellifolia. For this, we combined short and long read sequencing, flow cytometry, and cytogenetics to build a comprehensive framework that spans the complete scale from DNA to chromosome to genome. Genome sizes and repeat profiles reflect the separation into three gene pools with contrasting evolutionary patterns. Among all repeats, satellite DNAs harbor most genomic variability, leading to fundamentally different centromere architectures, ranging from chromosomal uniformity in Beta and Patellifolia to the formation of patchwork chromosomes in Corollinae/Nanae. We show that repetitive DNAs are causal for the genome expansions and contractions across the beet genera, providing insights into the genomic underpinnings of beet speciation. Satellite DNAs in particular vary considerably between beet genomes, leading to the evolution of distinct chromosomal setups in the three gene pools, likely contributing to the barriers in beet breeding. Thus, with their isokaryotypic chromosome sets, beet genomes present an ideal system for studying the link between repeats, genomic variability, and chromosomal differentiation and provide a theoretical fundament for understanding barriers in any crop breeding effort.

Genomic characterization of a nematode tolerance locus in sugar beet.

BACKGROUND: Infection by beet cyst nematodes (BCN, Heterodera schachtii) causes a serious disease of sugar beet, and climatic change is expected to improve the conditions for BCN infection. Yield and yield stability under adverse conditions are among the main breeding objectives. Breeding of BCN tolerant sugar beet cultivars offering high yield in the presence of the pathogen is therefore of high relevance. RESULTS: To identify causal genes providing tolerance against BCN infection, we combined several experimental and bioinformatic approaches. Relevant genomic regions were detected through mapping-by-sequencing using a segregating F2 population. DNA sequencing of contrasting F2 pools and analyses of allele frequencies for variant positions identified a single genomic region which confers nematode tolerance. The genomic interval was confirmed and narrowed down by genotyping with newly developed molecular markers. To pinpoint the causal genes within the potential nematode tolerance locus, we generated long read-based genome sequence assemblies of the tolerant parental breeding line Strube U2Bv and the susceptible reference line 2320Bv. We analyzed continuous sequences of the potential locus with regard to functional gene annotation and differential gene expression upon BCN infection. A cluster of genes with similarity to the Arabidopsis thaliana gene encoding nodule inception protein-like protein 7 (NLP7) was identified. Gene expression analyses confirmed transcriptional activity and revealed clear differences between susceptible and tolerant genotypes. CONCLUSIONS: Our findings provide new insights into the genomic basis of plant-nematode interactions that can be used to design and accelerate novel management strategies against BCN.

Complete pan-plastome sequences enable high resolution phylogenetic classification of sugar beet and closely related crop wild relatives.

BACKGROUND: As the major source of sugar in moderate climates, sugar-producing beets (Beta vulgaris subsp. vulgaris) have a high economic value. However, the low genetic diversity within cultivated beets requires introduction of new traits, for example to increase their tolerance and resistance attributes - traits that often reside in the crop wild relatives. For this, genetic information of wild beet relatives and their phylogenetic placements to each other are crucial. To answer this need, we sequenced and assembled the complete plastome sequences from a broad species spectrum across the beet genera Beta and Patellifolia, both embedded in the Betoideae (order Caryophyllales). This pan-plastome dataset was then used to determine the wild beet phylogeny in high-resolution. RESULTS: We sequenced the plastomes of 18 closely related accessions representing 11 species of the Betoideae subfamily and provided high-quality plastome assemblies which represent an important resource for further studies of beet wild relatives and the diverse plant order Caryophyllales. Their assembly sizes range from 149,723 bp (Beta vulgaris subsp. vulgaris) to 152,816 bp (Beta nana), with most variability in the intergenic sequences. Combining plastome-derived phylogenies with read-based treatments based on mitochondrial information, we were able to suggest a unified and highly confident phylogenetic placement of the investigated Betoideae species. Our results show that the genus Beta can be divided into the two clearly separated sections Beta and Corollinae. Our analysis confirms the affiliation of B. nana with the other Corollinae species, and we argue against a separate placement in the Nanae section. Within the Patellifolia genus, the two diploid species Patellifolia procumbens and Patellifolia webbiana are, regarding the plastome sequences, genetically more similar to each other than to the tetraploid Patellifolia patellaris. Nevertheless, all three Patellifolia species are clearly separated. CONCLUSION: In conclusion, our wild beet plastome assemblies represent a new resource to understand the molecular base of the beet germplasm. Despite large differences on the phenotypic level, our pan-plastome dataset is highly conserved. For the first time in beets, our whole plastome sequences overcome the low sequence variation in individual genes and provide the molecular backbone for highly resolved beet phylogenomics. Hence, our plastome sequencing strategy can also guide genomic approaches to unravel other closely related taxa.

Repeat Composition of CenH3-chromatin and H3K9me2-marked heterochromatin in Sugar Beet (Beta vulgaris).

BACKGROUND: Sugar beet (Beta vulgaris) is an important crop of temperate climate zones, which provides nearly 30 % of the world's annual sugar needs. From the total genome size of 758 Mb, only 567 Mb were incorporated in the recently published genome sequence, due to the fact that regions with high repetitive DNA contents (e.g. satellite DNAs) are only partially included. Therefore, to fill these gaps and to gain information about the repeat composition of centromeres and heterochromatic regions, we performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) using antibodies against the centromere-specific histone H3 variant of sugar beet (CenH3) and the heterochromatic mark of dimethylated lysine 9 of histone H3 (H3K9me2). RESULTS: ChIP-Seq analysis revealed that active centromeres containing CenH3 consist of the satellite pBV and the Ty3-gypsy retrotransposon Beetle7, while heterochromatin marked by H3K9me2 exhibits heterogeneity in repeat composition. H3K9me2 was mainly associated with the satellite family pEV, the Ty1-copia retrotransposon family Cotzilla and the DNA transposon superfamily of the En/Spm type. In members of the section Beta within the genus Beta, immunostaining using the CenH3 antibody was successful, indicating that orthologous CenH3 proteins are present in closely related species within this section. CONCLUSIONS: The identification of repetitive genome portions by ChIP-Seq experiments complemented the sugar beet reference sequence by providing insights into the repeat composition of poorly characterized CenH3-chromatin and H3K9me2-heterochromatin. Therefore, our work provides the basis for future research and application concerning the sugar beet centromere and repeat-rich heterochromatic regions characterized by the presence of H3K9me2.

Rapid gene identification in sugar beet using deep sequencing of DNA from phenotypic pools selected from breeding panels.

BACKGROUND: The combination of bulk segregant analysis (BSA) and next generation sequencing (NGS), also known as mapping by sequencing (MBS), has been shown to significantly accelerate the identification of causal mutations for species with a reference genome sequence. The usual approach is to cross homozygous parents that differ for the monogenic trait to address, to perform deep sequencing of DNA from F2 plants pooled according to their phenotype, and subsequently to analyze the allele frequency distribution based on a marker table for the parents studied. The method has been successfully applied for EMS induced mutations as well as natural variation. Here, we show that pooling genetically diverse breeding lines according to a contrasting phenotype also allows high resolution mapping of the causal gene in a crop species. The test case was the monogenic locus causing red vs. green hypocotyl color in Beta vulgaris (R locus). RESULTS: We determined the allele frequencies of polymorphic sequences using sequence data from two diverging phenotypic pools of 180 B. vulgaris accessions each. A single interval of about 31 kbp among the nine chromosomes was identified which indeed contained the causative mutation. CONCLUSIONS: By applying a variation of the mapping by sequencing approach, we demonstrated that phenotype-based pooling of diverse accessions from breeding panels and subsequent direct determination of the allele frequency distribution can be successfully applied for gene identification in a crop species. Our approach made it possible to identify a small interval around the causative gene. Sequencing of parents or individual lines was not necessary. Whenever the appropriate plant material is available, the approach described saves time compared to the generation of an F2 population. In addition, we provide clues for planning similar experiments with regard to pool size and the sequencing depth required.

Diversification, evolution and methylation of short interspersed nuclear element families in sugar beet and related Amaranthaceae species.

Short interspersed nuclear elements (SINEs) are non-autonomous non-long terminal repeat retrotransposons which are widely distributed in eukaryotic organisms. While SINEs have been intensively studied in animals, only limited information is available about plant SINEs. We analysed 22 SINE families from seven genomes of the Amaranthaceae family and identified 34 806 SINEs, including 19 549 full-length copies. With the focus on sugar beet (Beta vulgaris), we performed a comparative analysis of the diversity, genomic and chromosomal organization and the methylation of SINEs to provide a detailed insight into the evolution and age of Amaranthaceae SINEs. The lengths of consensus sequences of SINEs range from 113 nucleotides (nt) up to 224 nt. The SINEs show dispersed distribution on all chromosomes but were found with higher incidence in subterminal euchromatic chromosome regions. The methylation of SINEs is increased compared with their flanking regions, and the strongest effect is visible for cytosines in the CHH context, indicating an involvement of asymmetric methylation in the silencing of SINEs.

Exploiting single-molecule transcript sequencing for eukaryotic gene prediction.

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes.

SMRT sequencing only de novo assembly of the sugar beet (Beta vulgaris) chloroplast genome.

BACKGROUND: Third generation sequencing methods, like SMRT (Single Molecule, Real-Time) sequencing developed by Pacific Biosciences, offer much longer read length in comparison to Next Generation Sequencing (NGS) methods. Hence, they are well suited for de novo- or re-sequencing projects. Sequences generated for these purposes will not only contain reads originating from the nuclear genome, but also a significant amount of reads originating from the organelles of the target organism. These reads are usually discarded but they can also be used for an assembly of organellar replicons. The long read length supports resolution of repetitive regions and repeats within the organelles genome which might be problematic when just using short read data. Additionally, SMRT sequencing is less influenced by GC rich areas and by long stretches of the same base. RESULTS: We describe a workflow for a de novo assembly of the sugar beet (Beta vulgaris ssp. vulgaris) chloroplast genome sequence only based on data originating from a SMRT sequencing dataset targeted on its nuclear genome. We show that the data obtained from such an experiment are sufficient to create a high quality assembly with a higher reliability than assemblies derived from e.g. Illumina reads only. The chloroplast genome is especially challenging for de novo assembling as it contains two large inverted repeat (IR) regions. We also describe some limitations that still apply even though long reads are used for the assembly. CONCLUSIONS: SMRT sequencing reads extracted from a dataset created for nuclear genome (re)sequencing can be used to obtain a high quality de novo assembly of the chloroplast of the sequenced organism. Even with a relatively small overall coverage for the nuclear genome it is possible to collect more than enough reads to generate a high quality assembly that outperforms short read based assemblies. However, even with long reads it is not always possible to clarify the order of elements of a chloroplast genome sequence reliantly which we could demonstrate with Fosmid End Sequences (FES) generated with Sanger technology. Nevertheless, this limitation also applies to short read sequencing data but is reached in this case at a much earlier stage during finishing.

Reliable in silico identification of sequence polymorphisms and their application for extending the genetic map of sugar beet (Beta vulgaris).

Molecular markers are a highly valuable tool for creating genetic maps. Like in many other crops, sugar beet (Beta vulgaris L.) breeding is increasingly supported by the application of such genetic markers. Single nucleotide polymorphism (SNP) based markers have a high potential for automated analysis and high-throughput genotyping. We developed a bioinformatics workflow that uses Sanger and 2nd-generation sequence data for detection, evaluation and verification of new transcript-associated SNPs from sugar beet. RNAseq data from one parent of an established mapping population were produced by 454-FLX sequencing and compared to Sanger ESTs derived from the other parent. The workflow established for SNP detection considers the quality values of both types of reads, provides polymorphic alignments as well as selection criteria for reliable SNP detection and allows painless generation of new genetic markers within genes. We obtained a total of 14,323 genic SNPs and InDels. According to empirically optimised settings for the quality parameters, we classified these SNPs into four usability categories. Validation of a subset of the in silico detected SNPs by genotyping the mapping population indicated a high success rate of the SNP detection. Finally, a total of 307 new markers were integrated with existing data into a new genetic map of sugar beet which offers improved resolution and the integration of terminal markers.

Genome-wide identification and characterisation of R2R3-MYB genes in sugar beet (Beta vulgaris).

BACKGROUND: The R2R3-MYB genes comprise one of the largest transcription factor gene families in plants, playing regulatory roles in plant-specific developmental processes, metabolite accumulation and defense responses. Although genome-wide analysis of this gene family has been carried out in some species, the R2R3-MYB genes in Beta vulgaris ssp. vulgaris (sugar beet) as the first sequenced member of the order Caryophyllales, have not been analysed heretofore. RESULTS: We present a comprehensive, genome-wide analysis of the MYB genes from Beta vulgaris ssp. vulgaris (sugar beet) which is the first species of the order Caryophyllales with a sequenced genome. A total of 70 R2R3-MYB genes as well as genes encoding three other classes of MYB proteins containing multiple MYB repeats were identified and characterised with respect to structure and chromosomal organisation. Also, organ specific expression patterns were determined from RNA-seq data. The R2R3-MYB genes were functionally categorised which led to the identification of a sugar beet-specific clade with an atypical amino acid composition in the R3 domain, putatively encoding betalain regulators. The functional classification was verified by experimental confirmation of the prediction that the R2R3-MYB gene Bv_iogq encodes a flavonol regulator. CONCLUSIONS: This study provides the first step towards cloning and functional dissection of the role of MYB transcription factor genes in the nutritionally and evolutionarily interesting species B. vulgaris. In addition, it describes the flavonol regulator BvMYB12, being the first sugar beet R2R3-MYB with an experimentally proven function.

The CHH motif in sugar beet satellite DNA: a modulator for cytosine methylation.

Methylation of DNA is important for the epigenetic silencing of repetitive DNA in plant genomes. Knowledge about the cytosine methylation status of satellite DNAs, a major class of repetitive DNA, is scarce. One reason for this is that arrays of tandemly arranged sequences are usually collapsed in next-generation sequencing assemblies. We applied strategies to overcome this limitation and quantified the level of cytosine methylation and its pattern in three satellite families of sugar beet (Beta vulgaris) which differ in their abundance, chromosomal localization and monomer size. We visualized methylation levels along pachytene chromosomes with respect to small satellite loci at maximum resolution using chromosome-wide fluorescent in situ hybridization complemented with immunostaining and super-resolution microscopy. Only reduced methylation of many satellite arrays was obtained. To investigate methylation at the nucleotide level we performed bisulfite sequencing of 1569 satellite sequences. We found that the level of methylation of cytosine strongly depends on the sequence context: cytosines in the CHH motif show lower methylation (44-52%), while CG and CHG motifs are more strongly methylated. This affects the overall methylation of satellite sequences because CHH occurs frequently while CG and CHG are rare or even absent in the satellite arrays investigated. Evidently, CHH is the major target for modulation of the cytosine methylation level of adjacent monomers within individual arrays and contributes to their epigenetic function. This strongly indicates that asymmetric cytosine methylation plays a role in the epigenetic modification of satellite repeats in plant genomes.

The genome of the recently domesticated crop plant sugar beet (Beta vulgaris).

Sugar beet (Beta vulgaris ssp. vulgaris) is an important crop of temperate climates which provides nearly 30% of the world's annual sugar production and is a source for bioethanol and animal feed. The species belongs to the order of Caryophylalles, is diploid with 2n = 18 chromosomes, has an estimated genome size of 714-758 megabases and shares an ancient genome triplication with other eudicot plants. Leafy beets have been cultivated since Roman times, but sugar beet is one of the most recently domesticated crops. It arose in the late eighteenth century when lines accumulating sugar in the storage root were selected from crosses made with chard and fodder beet. Here we present a reference genome sequence for sugar beet as the first non-rosid, non-asterid eudicot genome, advancing comparative genomics and phylogenetic reconstructions. The genome sequence comprises 567 megabases, of which 85% could be assigned to chromosomes. The assembly covers a large proportion of the repetitive sequence content that was estimated to be 63%. We predicted 27,421 protein-coding genes supported by transcript data and annotated them on the basis of sequence homology. Phylogenetic analyses provided evidence for the separation of Caryophyllales before the split of asterids and rosids, and revealed lineage-specific gene family expansions and losses. We sequenced spinach (Spinacia oleracea), another Caryophyllales species, and validated features that separate this clade from rosids and asterids. Intraspecific genomic variation was analysed based on the genome sequences of sea beet (Beta vulgaris ssp. maritima; progenitor of all beet crops) and four additional sugar beet accessions. We identified seven million variant positions in the reference genome, and also large regions of low variability, indicating artificial selection. The sugar beet genome sequence enables the identification of genes affecting agronomically relevant traits, supports molecular breeding and maximizes the plant's potential in energy biotechnology.

Evolutionary reshuffling in the Errantivirus lineage Elbe within the Beta vulgaris genome.

LTR retrotransposons and retroviruses are closely related. Although a viral envelope gene is found in some LTR retrotransposons and all retroviruses, only the latter show infectivity. The identification of Ty3-gypsy-like retrotransposons possessing putative envelope-like open reading frames blurred the taxonomical borders and led to the establishment of the Errantivirus, Metavirus and Chromovirus genera within the Metaviridae. Only a few plant Errantiviruses have been described, and their evolutionary history is not well understood. In this study, we investigated 27 retroelements of four abundant Elbe retrotransposon families belonging to the Errantiviruses in Beta vulgaris (sugar beet). Retroelements of the Elbe lineage integrated between 0.02 and 5.59 million years ago, and show family-specific variations in autonomy and degree of rearrangements: while Elbe3 members are highly fragmented, often truncated and present in a high number of solo LTRs, Elbe2 members are mainly autonomous. We observed extensive reshuffling of structural motifs across families, leading to the formation of new retrotransposon families. Elbe retrotransposons harbor a typical envelope-like gene, often encoding transmembrane domains. During the course of Elbe evolution, the additional open reading frames have been strongly modified or independently acquired. Taken together, the Elbe lineage serves as retrotransposon model reflecting the various stages in Errantivirus evolution, and allows a detailed analysis of retrotransposon family formation.

The role of a pseudo-response regulator gene in life cycle adaptation and domestication of beet.

Life cycle adaptation to latitudinal and seasonal variation in photoperiod and temperature is a major determinant of evolutionary success in flowering plants. Whereas the life cycle of the dicotyledonous model species Arabidopsis thaliana is controlled by two epistatic genes, FLOWERING LOCUS C and FRIGIDA, three unrelated loci (VERNALIZATION) determine the spring and winter habits of monocotyledonous plants such as temperate cereals. In the core eudicot species Beta vulgaris, whose lineage diverged from that leading to Arabidopsis shortly after the monocot-dicot split 140 million years ago, the bolting locus B is a master switch distinguishing annuals from biennials. Here, we isolated B and show that the pseudo-response regulator gene BOLTING TIME CONTROL 1 (BvBTC1), through regulation of the FLOWERING LOCUS T genes, is absolutely necessary for flowering and mediates the response to both long days and vernalization. Our results suggest that domestication of beets involved the selection of a rare partial loss-of-function BvBTC1 allele that imparts reduced sensitivity to photoperiod that is restored by vernalization, thus conferring bienniality, and illustrate how evolutionary plasticity at a key regulatory point can enable new life cycle strategies.

Palaeohexaploid ancestry for Caryophyllales inferred from extensive gene-based physical and genetic mapping of the sugar beet genome (Beta vulgaris).

Sugar beet (Beta vulgaris) is an important crop plant that accounts for 30% of the world's sugar production annually. The genus Beta is a distant relative of currently sequenced taxa within the core eudicotyledons; the genomic characterization of sugar beet is essential to make its genome accessible to molecular dissection. Here, we present comprehensive genomic information in genetic and physical maps that cover all nine chromosomes. Based on this information we identified the proposed ancestral linkage groups of rosids and asterids within the sugar beet genome. We generated an extended genetic map that comprises 1127 single nucleotide polymorphism markers prepared from expressed sequence tags and bacterial artificial chromosome (BAC) end sequences. To construct a genome-wide physical map, we hybridized gene-derived oligomer probes against two BAC libraries with 9.5-fold cumulative coverage of the 758 Mbp genome. More than 2500 probes and clones were integrated both in genetic maps and the physical data. The final physical map encompasses 535 chromosomally anchored contigs that contains 8361 probes and 22 815 BAC clones. By using the gene order established with the physical map, we detected regions of synteny between sugar beet (order Caryophyllales) and rosid species that involves 1400-2700 genes in the sequenced genomes of Arabidopsis, poplar, grapevine, and cacao. The data suggest that Caryophyllales share the palaeohexaploid ancestor proposed for rosids and asterids. Taken together, we here provide extensive molecular resources for sugar beet and enable future high-resolution trait mapping, gene identification, and cross-referencing to regions sequenced in other plant species.

Survey of sugar beet (Beta vulgaris L.) hAT transposons and MITE-like hATpin derivatives.

Genome-wide analyses of repetitive DNA suggest a significant impact particularly of transposable elements on genome size and evolution of virtually all eukaryotic organisms. In this study, we analyzed the abundance and diversity of the hAT transposon superfamily of the sugar beet (B. vulgaris) genome, using molecular, bioinformatic and cytogenetic approaches. We identified 81 transposase-coding sequences, three of which are part of structurally intact but nonfunctional hAT transposons (BvhAT), in a B. vulgaris BAC library as well as in whole genome sequencing-derived data sets. Additionally, 116 complete and 497 truncated non-autonomous BvhAT derivatives lacking the transposase gene were in silico-detected. The 116 complete derivatives were subdivided into four BvhATpin groups each characterized by a distinct terminal inverted repeat motif. Both BvhAT and BvhATpin transposons are specific for species of the genus Beta and closely related species, showing a localization on B. vulgaris chromosomes predominantely in euchromatic regions. The lack of any BvhAT transposase function together with the high degree of degeneration observed for the BvhAT and the BvhATpin genomic fraction contrasts with the abundance and activity of autonomous and non-autonomous hAT transposons revealed in other plant species. This indicates a possible genus-specific structural and functional repression of the hAT transposon superfamily during Beta diversification and evolution.

Targeted identification of short interspersed nuclear element families shows their widespread existence and extreme heterogeneity in plant genomes.

Short interspersed nuclear elements (SINEs) are non-long terminal repeat retrotransposons that are highly abundant, heterogeneous, and mostly not annotated in eukaryotic genomes. We developed a tool designated SINE-Finder for the targeted discovery of tRNA-derived SINEs. We analyzed sequence data of 16 plant genomes, including 13 angiosperms and three gymnosperms and identified 17,829 full-length and truncated SINEs falling into 31 families showing the widespread occurrence of SINEs in higher plants. The investigation focused on potato (Solanum tuberosum), resulting in the detection of seven different SolS SINE families consisting of 1489 full-length and 870 5' truncated copies. Consensus sequences of full-length members range in size from 106 to 244 bp depending on the SINE family. SolS SINEs populated related species and evolved separately, which led to some distinct subfamilies. Solanaceae SINEs are dispersed along chromosomes and distributed without clustering but with preferred integration into short A-rich motifs. They emerged more than 23 million years ago and were species specifically amplified during the radiation of potato, tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum). We show that tobacco TS retrotransposons are composite SINEs consisting of the 3' end of a long interspersed nuclear element integrated downstream of a nonhomologous SINE family followed by successfully colonization of the genome. We propose an evolutionary scenario for the formation of TS as a spontaneous event, which could be typical for the emergence of SINE families.

Epigenetic profiling of heterochromatic satellite DNA.

Sugar beet (Beta vulgaris) chromosomes consist of large heterochromatic blocks in pericentromeric, centromeric, and intercalary regions comprised of two different highly abundant DNA satellite families. To investigate DNA methylation at single base resolution at heterochromatic regions, we applied a method for strand-specific bisulfite sequencing of more than 1,000 satellite monomers followed by statistical analyses. As a result, we uncovered diversity in the distribution of different methylation patterns in both satellite families. Heavily methylated CG and CHG (H=A, T, or C) sites occur more frequently in intercalary heterochromatin, while CHH sites, with the exception of CAA, are only sparsely methylated, in both intercalary and pericentromeric/centromeric heterochromatin. We show that the difference in DNA methylation intensity is correlated to unequal distribution of heterochromatic histone H3 methylation marks. While clusters of H3K9me2 were absent from pericentromeric heterochromatin and restricted only to intercalary heterochromatic regions, H3K9me1 and H3K27me1 were observed in all types of heterochromatin. By sequencing of a small RNA library consisting of 6.76 million small RNAs, we identified small interfering RNAs (siRNAs) of 24 nucleotides in size which originated from both strands of the satellite DNAs. We hypothesize an involvement of these siRNAs in the regulation of DNA and histone methylation for maintaining heterochromatin.

Analysis of PRODUCTION OF FLAVONOL GLYCOSIDES-dependent flavonol glycoside accumulation in Arabidopsis thaliana plants reveals MYB11-, MYB12- and MYB111-independent flavonol glycoside accumulation.

The flavonol branch of flavonoid biosynthesis is under transcriptional control of the R2R3-MYBs production of flavonol glycoside1 (PFG1/MYB12, PFG2/MYB11 and PFG3/MYB111) in Arabidopsis thaliana. Here, we investigated the influence of specific PFG transcription factors on flavonol distribution in various organs. A combination of genetic and metabolite analysis was used to identify transcription factor gene-metabolite correlations of the flavonol metabolic pathway. Flavonol glycoside accumulation patterns have been analysed in wild-type and multiple R2R3-MYB PFG mutants in an organ- and development-dependent manner using high-performance thin-layer chromatography, supplemented with liquid chromatography-mass spectroscopy metabolite profiling. Our results clearly demonstrate a differential influence of MYB11, MYB12 and MYB111 on the spatial accumulation of specific flavonol derivatives in leaves, stems, inflorescences, siliques and roots. In addition, MYB11-, MYB12- and MYB111-independent flavonol glycoside accumulation was observed in pollen grains and siliques/seeds. The highly complex tissue- and developmental-specific regulation of flavonol biosynthesis in A. thaliana is orchestrated by at least four PFG transcription factors, differentially influencing the spatial accumulation of specific flavonol derivatives. We present evidence that a separate flavonol control mechanism might be at play in pollen.

High-throughput identification of genetic markers using representational oligonucleotide microarray analysis.

We describe a novel approach for high-throughput development of genetic markers using representational oligonucleotide microarray analysis. We test the performance of the method in sugar beet (Beta vulgaris L.) as a model for crop plants with little sequence information available. Genomic representations of both parents of a mapping population were hybridized on microarrays containing in total 146,554 custom made oligonucleotides based on sugar beet bacterial artificial chromosome (BAC) end sequences and expressed sequence tags (ESTs). Oligonucleotides showing a signal with one parental line only, were selected as potential marker candidates and placed onto an array, designed for genotyping of 184 F(2) individuals from the mapping population. Utilizing known co-dominant anchor markers we obtained 511 new dominant markers (392 derived from BAC end sequences, and 119 from ESTs) distributed over all nine sugar beet linkage groups and calculated genetic maps. Further improvements for large-scale application of the approach are discussed and its feasibility for the cost-effective and flexible generation of genetic markers is presented.