BACKGROUND
providing approx.
30 % of the world’s annual sugar demands, beta vulgaris subsp.
vulgaris  is an important crop of the temperate climate zones.
it possesses 2n =  <dig> chromosomes, an estimated genome size of  <dig> megabases   <cit>  and a repeat content of 63 %  <cit> .
the annual or biennial plant belongs to the genus beta, within the order caryophyllales and the family amaranthaceae.
recently, the genome sequence has been published and accounts for 567 mb  <cit> .
the genus beta comprises the three sections beta, corollinae and nanae.
species of the section beta are widely distributed along the mediterranean, central and northern atlantic coastlines  <cit> .
in contrast, nanae is endemic to greece and contains only one species .
species of the sections corollinae are found in the mediterranean area as well as in south-west asia.
the closely related wild beet genus patellifolia  comprises the species patellifolia webbiana, patellifolia procumbens and patellifolia patellaris  <cit> .
while p. webbiana and p. procumbens possess a diploid genome with 2n =  <dig> chromosomes, p. patellaris is tetraploid  <cit> .
long terminal repeat  retrotransposons were the most prominent class of repetitive elements detected in the genome assembly of sugar beet.
in particular, ty3-gypsy retrotransposons were predominantly found in centromeric and pericentromeric regions  <cit> .
the most abundant satellites pbv and pev are only fragmentarily included in the assembly.
both satellites are organized in large arrays and serve as cytogenetic markers for the heterochromatic regions of chromosomes of the genus beta .
technical barriers of second generation sequencing  hamper the complete arrangement of repetitive sequence elements  in the genome assembly.
subsequently, this incompleteness contributes to the deviation of the estimated genome size from the size of the assembled reference sequence.
as a result, heterochromatic regions possessing a high proportion of repetitive elements lack characterization and assignment, which also limits subsequent analysis of epigenetic modifications  <cit> .
heterochromatin is influenced by a variety of epigenetic modifications, including dna methylation, the modification of single histones or the incorporation of specific histone variants .
a histone variant of outstanding interest is the centromere specific histone h <dig> variant, described as cenh <dig> in plants, cid in drosophila, and cenp-a in mammals  <cit> .
cenh <dig> marks centromeric chromatin and is indispensable for proper centromere function.
being responsible for kinetochore formation ⁠ cenh <dig> nucleosomes enable the centromere to act as early guide in cell division during mitosis and meiosis.
thus, cenh <dig> serves as a hallmark for active centromeres in many plant and animal species .
while the cenh <dig> function remains similar in all species studied so far, the protein including its dna binding domain differs even between closely related species  <cit> .
cenh <dig> proteins differ from canonical h <dig> in the n-terminal end and the loop <dig> region, which is part of the c-terminal histone fold domain  <cit> .
in consequence, cenh <dig> antibodies are raised against n-terminal ends of cenh <dig> proteins to produce species-specific cenh <dig> antibodies.
the c-terminus, including the histone fold domain, is more conserved.
for plants it is known that cenh <dig> is preferentially associated with satellite dna often intermingled with ty3-gypsy retrotransposon elements .
next to cenh3-chromatin, pericentromeric-, intercalary- and subtelomeric regions exhibit heterochromatic features  <cit> .
in plants, heterochromatin is mainly characterized by the dimethylation of lysine  <dig> of histone h <dig>  .
h3k9me <dig> is incorporated in genomic regions rich in dna methylation – another hallmark of heterochromatin  <cit> .
furthermore, h3k9me <dig> was detected in genic regions or at transposons  <cit> .
in addition, multiple studies demonstrate that h3k9me2-heterochromatin may also be present at cenh <dig> occupied chromatin  <cit> .
therefore, h3k9me <dig> represents a hallmark of heterochromatin, on the scale of large genomic regions down to smaller sites in genic regions as well as in active centromeres.
sugar beet heterochromatin can be distinguished in large intercalary blocks on both chromosome arms, in pericentromeric and centromeric heterochromatin as well as in smaller subtelomeric and interspersed heterochromatic sites, which are visible as strong dapi staining in metaphase spreads .
fluorescence-in situ-hybridization  experiments demonstrated that the satellite pev and the ty1-copia retrotransposon family cotzilla are largely amplified in intercalary heterochromatin  <cit>  while pericentromeric and centromeric heterochromatin largely consist of the pbv satellite and the ty3-gypsy retrotransposon family beetle  <cit> .
the subtelomeric heterochromatic regions are characterized by the presence of the satellite family pav  <cit> .
small dispersed heterochromatic spots consist of a variety of different repetitive elements including satellite dnas, dna transposons and ltr and non-ltr retrotransposons .
in interphase nuclei it has been shown that sugar beet heterochromatin is characterized by the presence of h3k9me <dig>  very strong signals were detected in intercalary heterochromatin.
pericentromeric and centromeric heterochromatin as well as interspersed heterochromatic sites are characterized by very faint signals of h3k9me <dig> only  <cit> .
furthermore, intercalary, pericentromeric and centromeric heterochromatin is characterized by lower levels of dna methylation compared to adjacent genomic regions.
this might be due to the presence of the large at-rich satellite arrays in which most of the cytosine occur in the asymmetric chh  motif with only low chances to be methylated  <cit> .
the same can be observed for smaller dispersed satellite arrays  <cit> .
interestingly, satellites are transcribed and transcripts may be processed into small rnas mostly  <dig> nucleotides in size which might point to a functional role in heterochromatin maintenance  <cit> .
chromatin immunoprecipitation followed by high-throughput sequencing  is the method of choice for investigating sequence composition of genomic regions associated with specific types of chromatin .
very important work has already been conducted in maize to analyze the dna sequence composition of cenh <dig> chromatin and to investigate the evolution of centromeres and centromeric repeats .
however, the application of chip-seq for the characterization of repeat-rich regions like centromeres is often hampered by the lack of a proper reference, because these regions are only poorly represented in genome assemblies.
an alternative approach introduced by neumann et al.
<cit>  and gong et al.
<cit>  employs the output of graph-based repeat clustering analysis as the reference.
in this method, repeat composition of the genome is first analyzed by clustering of unassembled shotgun genomic reads into groups of frequently overlapping sequences  representing individual families of repetitive elements  <cit> .
the clustering is executed by repeatexplorer, a computational software pipeline that also performs various supplementary analyzes supporting the repeat annotation  <cit> .
the annotated output of the clustering analysis can then be used as a reference for similarity-based mapping of chip-seq reads.
repeats enriched in the selected type of chromatin are identified by elevated proportions of reads from chip versus control.
we employed this approach on cenh3- and h3k9me <dig> chip-seq data to elucidate the complete sequence composition of active centromeres marked by cenh3- and h3k9me2-heterochromatic regions of the sugar beet genome.
RESULTS
clustering of genomic reads gives insights into the total repeat content of the sugar beet genome and provides a reference for chip-seq read mapping
to provide a reference repeat data base to analyze the repeat composition of the chip-seq data, paired-end illumina raw reads originating from the sugar beet sequencing project  <cit>  were used for repeat clustering.
reads were processed and clustered using the repeatexplorer software  <cit> .
clustering of  <dig>  million reads resulted in  <dig>  clusters and  <dig>  single/non-clustered reads .
the  <dig>  clusters represent all repetitive families and subfamilies of the sugar beet genome which account in total for 64 % of the analyzed reads .
for a detailed characterization of the most abundant repeat families, the  <dig> most highly-repetitive clusters were annotated.
each of these clusters represents at least  <dig>  % of the sugar beet genome.
subsequent annotation of chip-seq reads is based on these first  <dig> clusters , which are further referred to as the genomic reference clusters of sugar beet.
for complete cluster information see additional file 1: table a <dig>  within the  <dig> clusters satellites are the most abundant repeat group, followed by plastid dna .
retrotransposons of the ty3-gypsy type are highly abundant occupying  <dig>  % of the genome and ty1-copia retrotransposons make up  <dig>  % of the genome.
ribosomal genes  represent  <dig>  % of the genome while pararetroviruses, telomeric dna, long interspersed nuclear elements  and short interspersed nuclear elements  show genome proportions of ≤1 %.fig.
<dig> genomic clusters of sugar beet.
a summary of the clustering analysis.
illumina reads were integrated into genomic clustering resulting in  <dig>  repeat clusters.
reads in clusters situated left from the red line were analyzed in detail .
b proportions of different repeats in the  <dig> genomic reference clusters
the presented clustering approach enables a description of the repetitive genome content, since it does not depend on any genome assembly.
instead, all related sequences found in the reads are grouped to in silico repeat clusters.
the data obtained by the clustering approach is useful to gain information about sequence composition and genome proportion of repeats which are only fragmentary included in the reference sequence.
therefore, the combination of the repeat annotation of the reference sequence together with the clustering repeat data draws a more realistic picture of the repeat landscape within the sugar beet genome.
in table  <dig>  the clustering approach and results from the genome assembly  are compared.
remarkably, highly abundant repeats such as tandem repeats, e.g.
the satellites pbv and pev, are strongly underrepresented in the genome assembly covering only  <dig>  % and  <dig>  %, respectively.
however, the clustering of genomic reads resulted in a genome proportion of  <dig>  % and  <dig>  % for pbv and pev.
this is a more realistic description of the proportion of these two repeats in the sugar beet genome and is consistent with a renaturation kinetics study which showed that both satellites are the most abundant repeat families in the sugar beet genome.
pbv and pev make up  <dig>  % and  <dig>  %, respectively, of the highly repetitive c0t- <dig> dna fraction demonstrating that both satellites are largely amplified in the genome  <cit> .
the same is observed for selected retrotransposon families, members of the ty3-gypsy retrotransposons beetle and bongo <dig>  and members of ty1-copia retrotransposons.
the cotzilla family  <cit>  is the most prominent example of ty1-copia elements.
cotzilla elements cover  <dig>  mb  of the genome in clustering data, but was described in the genome assembly to make up only  <dig>  mb .
table  <dig> exemplarily shows repeats demonstrated to constitute the sugar beet heterochromatin.
it describes the higher sensitivity of the clustering approach towards repeat proportions and quotes an example to replenish poorly described genome regions within the assembly.
for certain types of repeats, repeatexplorer can produce smaller size estimates compared to the genome assembly annotation.
this can most likely happen to repeats that are relatively less abundant and highly variable in their sequences, and therefore will produce multiple small clusters in the repeatexplorer output which may be below the threshold for annotation.table  <dig> genomic repeat contents in genome assembly and after clustering in comparison
chip-seq data reveal repeat composition of sugar beet cenh3- and h3k9me2-marked chromatin
to study the sugar beet centromeric chromatin and h3k9me2-heterochromatin thoroughly on the level of the underlying dna sequences, we carried out chromatin immunoprecipitation followed by dna sequencing  using antibodies against cenh <dig> and h3k9me <dig>  ten million randomly selected chip-seq and control input reads were mapped to genomic reference clusters using blast.
each read was assigned to maximally one cluster, and the ratio of chip to input read numbers was calculated for all reference clusters.
the chip/input ratio > = <dig>  was chosen as a threshold for considering the corresponding repeat enriched in the chip sample.
this was well above the values obtained for repeats which due to their known positions on chromosomes could be used as controls.
for example, the satellite pev located in intercalary heterochromatin  <cit>  and 45s rdna repeats which are also absent from the centromeres showed no enrichment in cenh <dig> chip experiments .
when using h3k9me <dig> antibody, the chip/input ratio for 45s rdna sequences was  <dig> .
on the other hand, chip-seq data confirmed expected association of the chromovirus beetle <dig>  <cit>  and the satellite pbv  <cit>  with cenh <dig> chromatin and cotzilla and pev repeats  <cit>  with heterochromatin marked by h3k9me <dig> .
for the chromovirus beetle <dig>  <cit>  and the satellite pbv  <cit>  small enrichment factors of  <dig>  and  <dig> – <dig>  were detected, respectively, indicating to a presence of h3k9me <dig> in centromeres.
overall,  <dig> and  <dig> repeat clusters were found enriched in cenh <dig> and h3k9me <dig> chip reads, respectively.
cluster annotation was performed to the level of repeat clades and, if possible, to family level.
the enrichment factor accounts for the specificity of a repetitive element to the specific chromatin region.
in turn, the information about composition and abundance of repetitive sequences in the genomic region is found in the proportion of read counts to individual repeat classes, clades, and families.
the enriched clusters in cenh <dig> chip-seq experiments were divided into four repeat groups , namely satellites , ty3-gypsy retrotransposons , plastid dna  and uncharacterized sequences .
in satellites, which exhibit the most abundant repeat type in centromeric chromatin, only copies of three out of six pbv subfamilies were found .
ty3-gypsy retrotransposons consist exclusively of the beetle family  with beetle <dig> accounting for 58 %.
to exclude the putative existence of wild beet-specific beetle <dig> sequences  <cit> , a similarity search in the sugar beet reference sequence  using the 774 bp ltr sequence of beetle <dig> was conducted confirming the absence of beetle <dig> sequences in sugar beet.
however, because of their assignment to beetle <dig> clusters these sequences were designated beetle2-related.
a very small proportion  of the reads has not been annotated yet and most likely represent either truncated repeating units or unknown centromere-specific repeats in sugar beet .fig.
<dig> sequence composition of cenh3-chromatin .
proportional read counts assigned to repeat groups , and more specifically to clades and families .
the repeat proportions reflect repeat abundance within sugar beet centromeric sequences.
read counts were normalized to the overall read amount introduced into the clustering approach.
a the active centromeres of sugar beet consist mainly of pbv satellite dna, ty3-gypsy retrotransposons and of plastid dna.
only  <dig>  % are uncharacterized repeats, consisting of either putative unknown centromeric sequences or truncated repeats.
b satellites of only the pbv family constitute the active centromeres.
the most abundant subfamily is pbv_iii.
c ty3-gypsy retrotransposons in the active centromeres are exclusively consisting of beetle families with beetle <dig> being the most prominent ty3-gypsy retrotransposon.
note, that sequences assigned to beetle <dig> are most likely sugar beet specific beetle <dig> homologs
to validate the specificity of dna sequences identified by immunoprecipitation towards sugar beet cenh3-chromatin, fish experiments were conducted using probes generated of cenh <dig> chip-dna .
cenh <dig> chip-dna largely hybridized in heterochromatic centromeric and pericentromeric regions of all chromosomes with variable but strong intensity.fig.
<dig> fish using chip-dna and plastid dna probes on sugar beet metaphase chromosomes chromosomes were counterstained with dapi  revealing chromosome morphology.
a and b location of immunoprecipitated dna is shown.
probes were labelled with digoxigenine and detected with antidigoxigenine coupled to fitc .
a cenh <dig> chip-dna probe detects centromeric regions.
bar = 5 μm.
b h3k9me <dig> chip-dna signals are distributed along the chromosome with stronger signals in the major intercalary heterochromatic region of each arm.
note, that signal strength is reduced in centromeric regions.
signals in centromeric regions are due to sequences occurring in association with both, cenh <dig> in the centromere and h3k9me <dig>  e.g.
sequences of beetle <dig>  the signals may be also due to the presence of h3k9me <dig> in active centromeres.
distal euchromatin shows depletion of signals.
bar = 5 μm.
c localization of plastid dna on sugar beet chromosomes using a biotin dutp-labelled plastid dna probe detected by streptavidine-cy <dig> .
plastid dna sequences are found dispersed on all  <dig> chromosomes without restriction to particular chromosome regions.
bar = 5 μm
the clusters enriched in h3k9me <dig> chip-seq data were divided into eight repeat groups.
in fig.
4a the putative composition of h3k9me2-heterochromatin is shown.
similarly to cenh <dig>  satellites are the most abundant repeats in h3k9me2-heterochromatin, comprising 30 %.
at family level, the satellite pev shows remarkably high proportions representing 89 % of all satellites .
both, ty1-copia  and ty3-gypsy  retrotransposons were highly enriched in h3k9me2-heterochromatin.
compared to the results in cenh3-chromatin, ty3-gypsy retrotransposons are more diverse in h3k9me2-heterochromatin.
copies of five repeats are included, of which the env-like clade is the most abundant  and contains the sugar beet specific group elbe  <cit> .
the crm clade is represented in 31 % of all detected ty3-gypsy elements.
similar to cenh <dig>  only members of the families beetle <dig>  beetle <dig> and beetle <dig> are found on crm clade level .
among the ty1-copia retrotransposons, three families were enriched , namely cotzilla, salire, and patty.
repeat clusters related to the cotzilla family of the sire clade represent 85 % of all ty1-copia retrotransposons.
these elements belong to the most abundant retrotransposon families in sugar beet  and were located preferentially in intercalary and pericentromeric heterochromatin  <cit> .
dna transposons are represented with 11 % in h3k9me2-heterochromatin, with the superfamilies en/spm, mutator and helitron identified : the vast majority of transposons belongs to the transposon superfamily en/spm.
about 9 % of the reads were assigned to clusters that were not characterized so far, but still show enrichment in h3k9me <dig> chip-seq data and might be unknown repeats specific for h3k9me2-heterochromatin .
in chip-dna-fish experiments, h3k9me <dig> chip-dna was detected by strong signals in intercalary heterochromatic regions with minor signals in most pericentromeric and centromeric regions as well as minor dispersed signals on all chromosomes .
hybridization to the outermost distal euchromatin  of all chromosomes was not detected.fig.
<dig> sequence composition of h3k9me2-heterochromatin .
proportional read counts assigned to individual sequence clusters are divided into main repeat groups , and subsequently into clades and families .
the repeat proportions reflect their abundances within sugar beet h3k9me2-heterochromatin.
read counts are normalized to the overall read amount introduced into the clustering approach.
a h3k9me2-heterochromatin displays heterogeneity in repeat composition.
satellite dna is the most abundant repeat and is followed by ltr retrotransposons .
b the family pev is the prevalent satellite found in h3k9me2-heterochromatin.
regarding the abundances, the minisatellite bvsat <dig> and the satellites phc, and niobe contribute only partially to h3k9me2-heterochromatin sequences.
c at least four clades of ty3-gypsy retrotransposons are associated with h3k9me2-heterochromatin.
2 % are uncharacterized repeats and represent either putative unknown ty3-gypsy sequences or truncated and/or recombined repeats in h3k9me2-heterochromatin.
d the cotzilla family predominantly contributes to the composition of h3k9me2-heterochromatin in ty1-copia retrotransposons, while 1 % of the ty1-copia sequences have not been characterized to date.
e en/spm is the prevailing dna transposon superfamily followed by mutator elements
in summary, the enriched repeats were more diverse in h3k9me <dig> chip-seq, both with respect to their quality and quantity.
h3k9me <dig> chip-seq detected  <dig> enriched clusters assigned to eight repeat types, while cenh <dig> chip-seq detected  <dig> enriched clusters assigned to four repeat types.
to confirm integration of plastid dna in the genome, hybridizations of plastid dna probes to sugar beet mitotic metaphase chromosomes revealed dispersed signals along the  <dig> chromosomes .
notably, signals of plastid dna were detected on both chromatides of most chromosomes arms with strong signals in subtelomeric and centromeric regions.
immunostaining reveals specificity of the sugar beet cenh <dig> antibody to species of the section beta within the genus beta
immunostaining using the antibody against sugar beet cenh <dig>  on metaphase nuclei resulted in  <dig> distinct and specific anti-bvcenh <dig> double-dot signals in sugar beet .
single cenh <dig> signals  and pbv regions  are clearly distinguishable on a representative metaphase .
the cenh <dig> signals are arranged in pairs of two, corresponding to sister chromatids, while each pair is found in one pbv cluster.
most importantly, fish experiments on interphase nuclei and mitotic chromosomes using the centromere-specific satellite probe pbv revealed a cell cycle stage-independent co-localization of bvcenh <dig> and pbv on sugar beet chromosomes .fig.
<dig> cenh <dig> immunofluorescence on beta nuclei and fish of pbv centromeric satellite on sugar beet.
nuclei were counterstained with 4’,6-diamine-2’-phenylindole-dihydrochloride  showing chromosome morphology.
cenh <dig> binding was detected by the secondary antibody anti-rabbit coupled to fluorescein isothiocyanate .
the pbv probe was labelled with biotin- <dig> utp and detected by streptavidine-cyanine  <dig> .
bar = 5 μm.
a localization of cenh <dig> on sugar beet metaphase chromosomes detected by immuno-fish using the anti-bvcenh <dig> antibody and a centromeric satellite pbv probe as control.
dapi staining of an exemplarily chosen sugar beet metaphase shows chromosome morphology .
anti-bvcenh <dig> binding resulted in  <dig> signals, arranged in pairs corresponding to chromatides .
<dig> pbv signals  are detected and illustrate the centromeres .
overlay of all signals  reveals co-localization of two cenh <dig> signals within one large block of pbv on each chromosome .
b cenh <dig> localization during the sugar beet cell cycle.
anti-bvcenh <dig> binding displays cenh <dig> signals  in all mitotic cell cycle stages.
<dig> chromosomes are identified by anti-bvcenh <dig> binding on all chromosomes.
additional fish using the centromeric satellite pbv probe  exhibits co-localization with cenh <dig> signals throughout the cell cycle.
c cenh <dig> in species of the genera beta and patellifolia.
exemplarily chosen interphase nuclei of different beta species are shown, including sugar beet, b. maritima, and b. patula, as well as p. patellaris belonging to patellifolia.
areas exhibiting brighter dapi staining indicate heterochromatic regions .
distinct anti-bvcenh <dig> signals  were detected in sugar beet, b. maritima and b. patula in heterochromatic regions .
the  <dig> signals observed represent 2n =  <dig> chromosomes which is typical for diploid species of the section beta.
no signals were found on p. patellaris nuclei
the binding of anti-bvcenh <dig> was examined in sugar beet and additionally in related wild species of the genera beta and patellifolia to elucidate cenh <dig> conservation across beta species- and genus borders .
the species investigated included one member each of the same subspecies , of a different species , and of the closely related genus patellifolia .
immunostaining using the anti-bvcenh <dig> on interphase nuclei resulted in  <dig> distinct and specific anti-bvcenh <dig> signals in sugar beet, b. maritima and b. patula, corresponding to the number of chromosomes in these species .
contrarily, for p. patellaris nuclei no bvcenh <dig> signals were observed.
all anti-bvcenh <dig> signals are detected in intensively dapi-stained centromeric heterochromatin, which implies that beta centromeric regions form chromocenters in interphase nuclei .
discussion
the recently published sugar beet reference sequence harbors 567 mb  <cit> , which accounts for approximately 75 % of the estimated genome size .
this difference is mainly due to technical barriers in the assembly of whole genome shotgun data predominantly generated with second generation sequencing technology  <cit> .
the approach regularly fails to assemble highly repetitive genome portions, such as heterochromatic regions or centromeric chromatin harboring large arrays of tandemly repeated sequences and transposable elements.
the clustering and blast approach of genomic and chip-seq reads using the repeatexplorer pipeline applied in this study resulted in a far more realistic description of the repetitive portion in the sugar beet genome.
this procedure has been applied in other species, too, and has been proven to be efficient  <cit> .
strikingly, most repeats identified as enriched in chip-seq experiments  are clearly underrepresented in the sugar beet assembled genome sequence.
as a result, our study complements the constitution of repetitive sequences comprising the cenh3-marked chromatin and h3k9me2-heterochromatin so far underrepresented in the reference sequence.
immunostainings coupled to fish experiments together with cenh <dig> chip-seq data provide evidence on the direct interaction of the pbv satellite and cenh <dig>  pbv was the only satellite family found to be enriched in the centromere.
however, six variants of pbv are known  <cit> .
the genomic abundance reported for the variant pbv_iii  is reflected in cenh <dig> chip-seq experiments, followed by pbv_ii .
the variant pbv_iv possesses the longest monomere size  out of the pbv variants identified in chip-seq data and is hypothesized to originate from pbv_iii  <cit> ⁠.
the occurrence of only three out of six pbv variants in cenh3-chromatin, namely pbv_ii, pbv_iii, and pbv_iv, may be explained with high genome abundances of single variants and/or their individual monomer sizes potentially providing the basis for an optimal packaging of centromeric chromatin  <cit> ⁠.
notably, the pbv variant vi , formerly designated as prv  <cit> ⁠, is not present in cenh3-chromatin.
we conclude, that pbv_vi is not a repeat sequence in active centromeres although it is present in association with other pbv arrays  <cit> ⁠.
as it was detected throughout the mitotic cell cycle, the co-localization of cenh <dig> and pbv suggests an interaction between histone protein and satellite sequence  <cit> ⁠.
in detail, recent studies demonstrated the active impact of long non-coding rnas derived from repeated centromeric dna on centromere function .
it is not the centromere sequence itself, but rather the sequence features which enables the maintenance of an epigenetic environment necessary for proper centromere function  <cit> .
according to cenh <dig> chip-seq data, additionally to pbv, ty3-gypsy retrotransposons of the beetle family constitute the sugar beet centromere, which is in accordance to the statements reported in zakrzewski, weber, and schmidt  based on fish experiments  <cit> .
the appearance of the satellite pbv and the beetle retrotransposon families in sugar beet centromeric heterochromatin is typical for the compositions of plant centromeres  <cit> .
the combination of a satellite repeat in combination with retrotransposons as building blocks of centromeres was detected in many plants to date: e.g.
in monocots .
the existence of beetle <dig> in sugar beet is most unlikely, since fish as well as blot hybridization experiments using a beetle <dig> ltr probe indicated specificity of beetle <dig> sequences exclusively to wild beets  <cit> .
the occurrence of clusters in sugar beet cenh <dig> chip-seq data assigned to beetle <dig> might be explained by the evolution of a species-specific, so far uncharacterized retrotransposon family after separation of sugar beet from wild species.
thus, a similar relation was recently suggested between beetle <dig> from p. procumbens and its homolog in sugar beet, beetle <dig>  it is likely, that beetle <dig> and beetle <dig> share a common ancestor but acquired different ltrs during speciation  <cit> .
based on that knowledge, the annotation of the unknown retrotransposon as beetle <dig> might be the result of high sequence similarity in the coding region between the wild-beet specific beetle <dig> and its homolog in sugar beet.
the characterization of the sugar beet beetle diversity remains subject to future analyzes and might probably reveal an additional beetle family specific for sugar beet centromeres.
another cluster annotated as centromere-specific chromovirus represents the highest enrichment of all repeats in cenh <dig> chromatin .
interestingly, this chromovirus is remarkably specific to the sugar beet centromere, but due to the lack of any ltr sequences could not be annotated to any family so far.
<dig>  % of the enriched centromeric sequences are not annotated yet and remain without further information.
the annotation of these sequences is complicated, since no similarities were reported to any known repeat.
the cenh <dig> function is similar in all species studied to date, but its protein sequence differs even between closely related species  <cit> .
this is also the most likely reason why the anti-bvcenh <dig> antibody which was raised against the sugar beet cenh <dig> sequence failed to detect cenh <dig> in patellifolia species.
in contrast, the anti-rice cenh <dig> antibody recognized successfully cenh <dig> proteins in genera as far remote as maize and oat  <cit> .
sugar beet chromosomes are characterized by high enrichment of h3k9me <dig> in large intercalary heterochromatic blocks on each chromosome arm  <cit> .
these intercalary heterochromatic knobs may constitute the main portion of h3k9me <dig> heterochromatin in sugar beet  <cit> .
h3k9me <dig> chip-seq in sugar beet revealed a considerable diversity of repetitive sequences.
the high proportion of the pev satellite may indicate that the pev satellite may contribute to the establishment and maintenance of intercalary heterochromatin as has been described for satellites  <cit> .
in accordance with the model proposed by picaard et al.
<cit>  it is hypothesized that satellite dna may contribute to the formation of heterochromatin due to epigenetic mechanisms  <cit> : satellites are transcribed by polymerase iv into ssrnas, and subsequently dsrnas are generated by rna-dependent rna polymerase.
the satellite dsrnas are processed into 24-nt sirnas by the activity of dicer-like <dig>  24-nt sirnas from satellite repeats are loaded on argounaute <dig>  which then recognizes polymerase v satellite transcripts complementary to the incorporated 24-nt sirna.
domain rearranged dna methyltransferase <dig> associates to the argounaute complex and induces de novo cytosine methylation at cg, chg  and chh sites at satellite loci homologous to 24-nt sirnas.
furthermore, 24-nt sirnas might be involved in the dimethylation of histone h <dig>  <cit> , subsequently leading to heterochromatization as described in animals  <cit> .
therefore, satellites may serve as optimal sequence platform for the establishment and maintenance of heterochromatin at a large scale due to their simple structure of tandemly repeated monomers and their expansiveness of tandem arrays  <cit> .
similar to the enrichment in cenh <dig> binding sequences, the crm families beetle <dig> and beetle <dig> are also found in h3k9me <dig> data.
several beetle families are located beyond the centromere .
therefore it is likely, that beetle <dig> and beetle  <dig> might tend to integrate beyond the centromere due to several target sites and/or the diversification steps.
based on fish investigations  <cit> ⁠ a similar explanation is proposed for beetle <dig>  which is the centromeric retrotransposon in sugar beet.
it cannot be excluded that these sequences were partially located in pericentromeric heterochromatin or in centromeric chromatin, which may also contain small proportions of h3k9me <dig>  <cit> ⁠.
in accordance with this assumption h3k9me <dig> chip-seq revealed small enrichment factors of  <dig>  for the centromere-specific satellite pbv_iii although the enrichment factor threshold chosen in this study was at least  <dig> .
studies in maize also provided evidence for h3k9me2-heterochromatin in centromeres  <cit> .
in immunostaining experiments very little h3k9me <dig> signals have been detected in sugar beet centromeres  <cit> .
however, this faint signal intensity could also be explained by the inability of antibody or stains to access centromeres during metaphase.
one reason might be the protection of centromeres by a large complex of kinetochore factors that might have not been eliminated by protease treatment of metaphase spreads.
the cotzilla family is an evolutionary young member of the sire clade belonging to the superfamily of ty1-copia retrotransposons, which is remarkably abundant in h3k9me <dig> chip-seq data.
although the ranking of cotzilla enrichment factors might suggest intermediate specificity to h3k9me <dig> heterochromatin, chip-seq data validated that cotzilla elements constitute high proportions of h3k9me <dig> heterochromatic sequences.
this emphasizes the findings of weber etal.
<cit> ⁠, where fish experiments using an ltr-specific cotzilla <dig> probe demonstrated its high abundance in sugar beet intercalary and pericentromeric heterochromatin.
bongo <dig> is a sugar beet ty3-gypsy retrotransposon of the tekay clade of chromoviruses which was found dispersed in small clusters along chromosomes  <cit> .
although the enrichment shows reduced specificity, bongo <dig> is another element associated with h3k9me <dig>
belonging to ty3-gypsy retrotransposons, elbe <dig>  which is a sugar beet specific errantivirus  <cit> , exhibited a wide distribution on all chromosomes in fish experiments.
the sugar beet elbe group includes gag-pol polyproteins of the elements elbe <dig>  elbe <dig>  elbe <dig> and elbe <dig>  no restriction to certain genome regions has been reported  <cit> ⁠.
unlike bongo <dig>  elbe <dig> exhibits higher enrichments in h3k9me <dig> chip-seq experiments indicating a more specific relation between elbe <dig> and h3k9me <dig>  in addition, besides satellite repeats and ltr retrotransposon sequences, the dna transposon superfamily en/spm was remarkably enriched in h3k9me <dig> chip-seq data.
to date, little is known about the precise localization of the transposon superfamily cacta-en/spm in sugar beet  <cit> ⁠.
nevertheless, the detection of en/spm sequences in h3k9me <dig> chip-seq experiments is plausible as h3k9me <dig> is found in association with smaller heterochromatic spots in genic and transposon-rich regions  <cit> ⁠.
interestingly, the most specific  h3k9me <dig> binding sequence is not homologous to known repeats .
thus, the highest enrichment was found for a repeat, which has not been annotated so far.
similar to the unknown sequences in cenh <dig> chip-seq, the characterization has to be performed to reveal the heterochromatin specificity of unknown repeats.
the major findings of this study were integrated in a summarizing figure  based on the results obtained in this study and previously reported fish data ⁠.
the simplified model shows the complexity of cenh3-chromatin and h3k9me2-heterochromatin repeat composition in sugar beet.
while the satellites pev and pbv are mutually exclusive, ty3-gypsy retrotransposons are found in intercalary heterochromatin as well as in the centromere.
for transposable elements of the en/spm superfamily it might be likely that they are preferentially located in smaller dispersed heterochromatic spots beyond intercalary and centromeric heterochromatin.fig.
<dig> simplified model of histone modifications/substitutions in relationship with major repeats in cenh3-chromatin and h3k9me2-heterochromatin.
a typical metacentric sugar beet chromosome composed of two chromatids is illustrated.
a h3k9me <dig> is found in small heterochromatic sites in genic regions, in vicinity to transposons and putatively with retrotransposons, in large clusters in intercalary heterochromatin  and in small proportions in cenh <dig> chromatin .
the underlying sequence is composed of several repeats, but the satellite pev and the ty1-copia retrotransposon cotzilla predominate the sequence composition.
h3k9me <dig> interacts with beetle <dig> and occasionally with beetle <dig> in pericentromeric regions .
active centromeres are defined by cenh <dig>  which preferentially binds to the centromeric satellite pbv and the ty3-gypsy retrotransposon beetle <dig>  beetle <dig> is found in centromeric and presumably also in pericentromeric heterochromatin, in association with both, h3k9me <dig> and cenh <dig>  note, that this figure is not drawn to scale.
plastid dna is not incorporated, due to various integration sites, which need to be further confirmed and analyzed in subsequent studies
remarkably, plastid dna apparently constitutes high proportions of both heterochromatic regions, although it has not been integrated in the genome assembly  <cit> .
however, fish using plastid dna confirms dispersed chromosomal localization.
consequently, in contrast to earlier descriptions, plastid dna shows no major preference for integration into certain chromosomal regions .
the existence of plastid dna in sugar beet chromosomes may be due to multiple plastid dna integration events which have taken place several times independently and therefore could be a frequent event rather than a single incident.
therefore, plastid dna is left out of the summarizing figure as it has to be studied in far more detail which part of the chloroplast dna and how many copies integrated into the sugar beet genome in future studies.
CONCLUSIONS
sugar beet highly repetitive sequences are largely amplified in heterochromatic regions and centromeric chromatin but are only fragmentary included in the genome sequence.
therefore, the repeat composition of sugar beet heterochromatin and centromeric chromatin was annotated and characterized using chip-seq and subsequent sequence clustering approaches via repeatexplorer: centromeric chromatin is hallmarked by the sugar beet cenh <dig>  which binds to pbv satellites and to elements of the ty3-gypsy retrotransposon family beetle <dig>  this finding underpins the typical sequence composition of a typical plant centromere.
heterochromatin in plants is defined by the presence of h3k9me <dig>  it occurs either in clusters  or in smaller heterochromatic spots and is associated with a diverse pattern of repetitive sequences consisting of different families of satellite dnas, ty3-gypsy and ty1-copia retrotransposons as well as dna transposons.
the knowledge of repetitive sequences constituting the only poorly characterized heterochromatin and centromeric chromatin gained within this project provides so far missing data for the understanding of the organization and evolution of the sugar beet genome.
furthermore, it enhances the annotation quality of repetitive sequences, which is potentially useful for future genomic studies.
the presented approach in sugar beet has already been performed in other plants before and is eligible to be applied to further plant genomes for the annotation and characterization of repeats localized in cenh3-chromatin and in heterochromatic blocks, which are not or only fragmentarily included in reference genome sequences.
consequently, the analysis of the complete repeat composition of active plant centromeres and heterochromatin will provide comprehensive knowledge on a neglected but major sequence component of plant genomes.

 providing approx. 30 % of the world’s annual sugar demands, beta vulgaris subsp. vulgaris is an important crop of the temperate climate zones. it possesses 2n =  chromosomes, an estimated genome size of megabases and a repeat content of 63 %. the annual or biennial plant belongs to the genus beta, within the order caryophyllales and the family amaranthaceae. recently, the genome sequence has been published and accounts for 567 mb. the genus beta comprises the three sections beta, corollinae and nanae. species of the section beta are widely distributed along the mediterranean, central and northern atlantic coastlines. in contrast, nanae is endemic to greece and contains only one species. species of the sections corollinae are found in the mediterranean area as well as in south-west asia. the closely related wild beet genus patellifolia comprises the species patellifolia webbiana, patellifolia procumbens and patellifolia patellaris. while p. webbiana and p. procumbens possess a diploid genome with 2n =  chromosomes, p. patellaris is tetraploid. long terminal repeat retrotransposons were the most prominent class of repetitive elements detected in the genome assembly of sugar beet. in particular, ty3-gypsy retrotransposons were predominantly found in centromeric and pericentromeric regions. the most abundant satellites pbv and pev are only fragmentarily included in the assembly. both satellites are organized in large arrays and serve as cytogenetic markers for the heterochromatic regions of chromosomes of the genus beta. technical barriers of second generation sequencing hamper the complete arrangement of repetitive sequence elements in the genome assembly. subsequently, this incompleteness contributes to the deviation of the estimated genome size from the size of the assembled reference sequence. as a result, heterochromatic regions possessing a high proportion of repetitive elements lack characterization and assignment, which also limits subsequent analysis of epigenetic modifications. heterochromatin is influenced by a variety of epigenetic modifications, including dna methylation, the modification of single histones or the incorporation of specific histone variants. a histone variant of outstanding interest is the centromere specific histone h variant, described as cenh in plants, cid in drosophila, and cenp-a in mammals. cenh marks centromeric chromatin and is indispensable for proper centromere function. being responsible for kinetochore formation ⁠ cenh nucleosomes enable the centromere to act as early guide in cell division during mitosis and meiosis. thus, cenh serves as a hallmark for active centromeres in many plant and animal species. while the cenh function remains similar in all species studied so far, the protein including its dna binding domain differs even between closely related species. cenh proteins differ from canonical h in the n-terminal end and the loop region, which is part of the c-terminal histone fold domain. in consequence, cenh antibodies are raised against n-terminal ends of cenh proteins to produce species-specific cenh antibodies. the c-terminus, including the histone fold domain, is more conserved. for plants it is known that cenh is preferentially associated with satellite dna often intermingled with ty3-gypsy retrotransposon elements. next to cenh3-chromatin, pericentromeric-, intercalary- and subtelomeric regions exhibit heterochromatic features. in plants, heterochromatin is mainly characterized by the dimethylation of lysine of histone h. h3k9me is incorporated in genomic regions rich in dna methylation – another hallmark of heterochromatin. furthermore, h3k9me was detected in genic regions or at transposons. in addition, multiple studies demonstrate that h3k9me2-heterochromatin may also be present at cenh occupied chromatin. therefore, h3k9me represents a hallmark of heterochromatin, on the scale of large genomic regions down to smaller sites in genic regions as well as in active centromeres. sugar beet heterochromatin can be distinguished in large intercalary blocks on both chromosome arms, in pericentromeric and centromeric heterochromatin as well as in smaller subtelomeric and interspersed heterochromatic sites, which are visible as strong dapi staining in metaphase spreads. fluorescence-in situ-hybridization experiments demonstrated that the satellite pev and the ty1-copia retrotransposon family cotzilla are largely amplified in intercalary heterochromatin while pericentromeric and centromeric heterochromatin largely consist of the pbv satellite and the ty3-gypsy retrotransposon family beetle. the subtelomeric heterochromatic regions are characterized by the presence of the satellite family pav. small dispersed heterochromatic spots consist of a variety of different repetitive elements including satellite dnas, dna transposons and ltr and non-ltr retrotransposons. in interphase nuclei it has been shown that sugar beet heterochromatin is characterized by the presence of h3k9me very strong signals were detected in intercalary heterochromatin. pericentromeric and centromeric heterochromatin as well as interspersed heterochromatic sites are characterized by very faint signals of h3k9me only. furthermore, intercalary, pericentromeric and centromeric heterochromatin is characterized by lower levels of dna methylation compared to adjacent genomic regions. this might be due to the presence of the large at-rich satellite arrays in which most of the cytosine occur in the asymmetric chh motif with only low chances to be methylated. the same can be observed for smaller dispersed satellite arrays. interestingly, satellites are transcribed and transcripts may be processed into small rnas mostly nucleotides in size which might point to a functional role in heterochromatin maintenance. chromatin immunoprecipitation followed by high-throughput sequencing is the method of choice for investigating sequence composition of genomic regions associated with specific types of chromatin. very important work has already been conducted in maize to analyze the dna sequence composition of cenh chromatin and to investigate the evolution of centromeres and centromeric repeats. however, the application of chip-seq for the characterization of repeat-rich regions like centromeres is often hampered by the lack of a proper reference, because these regions are only poorly represented in genome assemblies. an alternative approach introduced by neumann et al. and gong et al. employs the output of graph-based repeat clustering analysis as the reference. in this method, repeat composition of the genome is first analyzed by clustering of unassembled shotgun genomic reads into groups of frequently overlapping sequences representing individual families of repetitive elements. the clustering is executed by repeatexplorer, a computational software pipeline that also performs various supplementary analyzes supporting the repeat annotation. the annotated output of the clustering analysis can then be used as a reference for similarity-based mapping of chip-seq reads. repeats enriched in the selected type of chromatin are identified by elevated proportions of reads from chip versus control. we employed this approach on cenh3- and h3k9me chip-seq data to elucidate the complete sequence composition of active centromeres marked by cenh3- and h3k9me2-heterochromatic regions of the sugar beet genome.  clustering of genomic reads gives insights into the total repeat content of the sugar beet genome and provides a reference for chip-seq read mapping to provide a reference repeat data base to analyze the repeat composition of the chip-seq data, paired-end illumina raw reads originating from the sugar beet sequencing project were used for repeat clustering. reads were processed and clustered using the repeatexplorer software. clustering of million reads resulted in clusters and single/non-clustered reads. the clusters represent all repetitive families and subfamilies of the sugar beet genome which account in total for 64 % of the analyzed reads. for a detailed characterization of the most abundant repeat families, the most highly-repetitive clusters were annotated. each of these clusters represents at least  % of the sugar beet genome. subsequent annotation of chip-seq reads is based on these first clusters, which are further referred to as the genomic reference clusters of sugar beet. for complete cluster information see additional file 1: table a within the clusters satellites are the most abundant repeat group, followed by plastid dna. retrotransposons of the ty3-gypsy type are highly abundant occupying  % of the genome and ty1-copia retrotransposons make up  % of the genome. ribosomal genes represent  % of the genome while pararetroviruses, telomeric dna, long interspersed nuclear elements and short interspersed nuclear elements show genome proportions of ≤1 %.fig. genomic clusters of sugar beet. a summary of the clustering analysis. illumina reads were integrated into genomic clustering resulting in repeat clusters. reads in clusters situated left from the red line were analyzed in detail. b proportions of different repeats in the genomic reference clusters the presented clustering approach enables a description of the repetitive genome content, since it does not depend on any genome assembly. instead, all related sequences found in the reads are grouped to in silico repeat clusters. the data obtained by the clustering approach is useful to gain information about sequence composition and genome proportion of repeats which are only fragmentary included in the reference sequence. therefore, the combination of the repeat annotation of the reference sequence together with the clustering repeat data draws a more realistic picture of the repeat landscape within the sugar beet genome. in table  the clustering approach and results from the genome assembly are compared. remarkably, highly abundant repeats such as tandem repeats, e.g. the satellites pbv and pev, are strongly underrepresented in the genome assembly covering only  % and  %, respectively. however, the clustering of genomic reads resulted in a genome proportion of  % and  % for pbv and pev. this is a more realistic description of the proportion of these two repeats in the sugar beet genome and is consistent with a renaturation kinetics study which showed that both satellites are the most abundant repeat families in the sugar beet genome. pbv and pev make up  % and  %, respectively, of the highly repetitive c0t- dna fraction demonstrating that both satellites are largely amplified in the genome. the same is observed for selected retrotransposon families, members of the ty3-gypsy retrotransposons beetle and bongo and members of ty1-copia retrotransposons. the cotzilla family is the most prominent example of ty1-copia elements. cotzilla elements cover  mb of the genome in clustering data, but was described in the genome assembly to make up only  mb. table  exemplarily shows repeats demonstrated to constitute the sugar beet heterochromatin. it describes the higher sensitivity of the clustering approach towards repeat proportions and quotes an example to replenish poorly described genome regions within the assembly. for certain types of repeats, repeatexplorer can produce smaller size estimates compared to the genome assembly annotation. this can most likely happen to repeats that are relatively less abundant and highly variable in their sequences, and therefore will produce multiple small clusters in the repeatexplorer output which may be below the threshold for annotation.table genomic repeat contents in genome assembly and after clustering in comparison chip-seq data reveal repeat composition of sugar beet cenh3- and h3k9me2-marked chromatin to study the sugar beet centromeric chromatin and h3k9me2-heterochromatin thoroughly on the level of the underlying dna sequences, we carried out chromatin immunoprecipitation followed by dna sequencing using antibodies against cenh and h3k9me ten million randomly selected chip-seq and control input reads were mapped to genomic reference clusters using blast. each read was assigned to maximally one cluster, and the ratio of chip to input read numbers was calculated for all reference clusters. the chip/input ratio > = was chosen as a threshold for considering the corresponding repeat enriched in the chip sample. this was well above the values obtained for repeats which due to their known positions on chromosomes could be used as controls. for example, the satellite pev located in intercalary heterochromatin and 45s rdna repeats which are also absent from the centromeres showed no enrichment in cenh chip experiments. when using h3k9me antibody, the chip/input ratio for 45s rdna sequences was. on the other hand, chip-seq data confirmed expected association of the chromovirus beetle and the satellite pbv with cenh chromatin and cotzilla and pev repeats with heterochromatin marked by h3k9me. for the chromovirus beetle and the satellite pbv small enrichment factors of and – were detected, respectively, indicating to a presence of h3k9me in centromeres. overall, and repeat clusters were found enriched in cenh and h3k9me chip reads, respectively. cluster annotation was performed to the level of repeat clades and, if possible, to family level. the enrichment factor accounts for the specificity of a repetitive element to the specific chromatin region. in turn, the information about composition and abundance of repetitive sequences in the genomic region is found in the proportion of read counts to individual repeat classes, clades, and families. the enriched clusters in cenh chip-seq experiments were divided into four repeat groups, namely satellites, ty3-gypsy retrotransposons, plastid dna and uncharacterized sequences. in satellites, which exhibit the most abundant repeat type in centromeric chromatin, only copies of three out of six pbv subfamilies were found. ty3-gypsy retrotransposons consist exclusively of the beetle family with beetle accounting for 58 %. to exclude the putative existence of wild beet-specific beetle sequences, a similarity search in the sugar beet reference sequence using the 774 bp ltr sequence of beetle was conducted confirming the absence of beetle sequences in sugar beet. however, because of their assignment to beetle clusters these sequences were designated beetle2-related. a very small proportion of the reads has not been annotated yet and most likely represent either truncated repeating units or unknown centromere-specific repeats in sugar beet.fig. sequence composition of cenh3-chromatin. proportional read counts assigned to repeat groups, and more specifically to clades and families. the repeat proportions reflect repeat abundance within sugar beet centromeric sequences. read counts were normalized to the overall read amount introduced into the clustering approach. a the active centromeres of sugar beet consist mainly of pbv satellite dna, ty3-gypsy retrotransposons and of plastid dna. only  % are uncharacterized repeats, consisting of either putative unknown centromeric sequences or truncated repeats. b satellites of only the pbv family constitute the active centromeres. the most abundant subfamily is pbv_iii. c ty3-gypsy retrotransposons in the active centromeres are exclusively consisting of beetle families with beetle being the most prominent ty3-gypsy retrotransposon. note, that sequences assigned to beetle are most likely sugar beet specific beetle homologs to validate the specificity of dna sequences identified by immunoprecipitation towards sugar beet cenh3-chromatin, fish experiments were conducted using probes generated of cenh chip-dna. cenh chip-dna largely hybridized in heterochromatic centromeric and pericentromeric regions of all chromosomes with variable but strong intensity.fig. fish using chip-dna and plastid dna probes on sugar beet metaphase chromosomes chromosomes were counterstained with dapi revealing chromosome morphology. a and b location of immunoprecipitated dna is shown. probes were labelled with digoxigenine and detected with antidigoxigenine coupled to fitc. a cenh chip-dna probe detects centromeric regions. bar = 5 μm. b h3k9me chip-dna signals are distributed along the chromosome with stronger signals in the major intercalary heterochromatic region of each arm. note, that signal strength is reduced in centromeric regions. signals in centromeric regions are due to sequences occurring in association with both, cenh in the centromere and h3k9me e.g. sequences of beetle the signals may be also due to the presence of h3k9me in active centromeres. distal euchromatin shows depletion of signals. bar = 5 μm. c localization of plastid dna on sugar beet chromosomes using a biotin dutp-labelled plastid dna probe detected by streptavidine-cy. plastid dna sequences are found dispersed on all chromosomes without restriction to particular chromosome regions. bar = 5 μm the clusters enriched in h3k9me chip-seq data were divided into eight repeat groups. in fig. 4a the putative composition of h3k9me2-heterochromatin is shown. similarly to cenh satellites are the most abundant repeats in h3k9me2-heterochromatin, comprising 30 %. at family level, the satellite pev shows remarkably high proportions representing 89 % of all satellites. both, ty1-copia and ty3-gypsy retrotransposons were highly enriched in h3k9me2-heterochromatin. compared to the results in cenh3-chromatin, ty3-gypsy retrotransposons are more diverse in h3k9me2-heterochromatin. copies of five repeats are included, of which the env-like clade is the most abundant and contains the sugar beet specific group elbe. the crm clade is represented in 31 % of all detected ty3-gypsy elements. similar to cenh only members of the families beetle beetle and beetle are found on crm clade level. among the ty1-copia retrotransposons, three families were enriched, namely cotzilla, salire, and patty. repeat clusters related to the cotzilla family of the sire clade represent 85 % of all ty1-copia retrotransposons. these elements belong to the most abundant retrotransposon families in sugar beet and were located preferentially in intercalary and pericentromeric heterochromatin. dna transposons are represented with 11 % in h3k9me2-heterochromatin, with the superfamilies en/spm, mutator and helitron identified: the vast majority of transposons belongs to the transposon superfamily en/spm. about 9 % of the reads were assigned to clusters that were not characterized so far, but still show enrichment in h3k9me chip-seq data and might be unknown repeats specific for h3k9me2-heterochromatin. in chip-dna-fish experiments, h3k9me chip-dna was detected by strong signals in intercalary heterochromatic regions with minor signals in most pericentromeric and centromeric regions as well as minor dispersed signals on all chromosomes. hybridization to the outermost distal euchromatin of all chromosomes was not detected.fig. sequence composition of h3k9me2-heterochromatin. proportional read counts assigned to individual sequence clusters are divided into main repeat groups, and subsequently into clades and families. the repeat proportions reflect their abundances within sugar beet h3k9me2-heterochromatin. read counts are normalized to the overall read amount introduced into the clustering approach. a h3k9me2-heterochromatin displays heterogeneity in repeat composition. satellite dna is the most abundant repeat and is followed by ltr retrotransposons. b the family pev is the prevalent satellite found in h3k9me2-heterochromatin. regarding the abundances, the minisatellite bvsat and the satellites phc, and niobe contribute only partially to h3k9me2-heterochromatin sequences. c at least four clades of ty3-gypsy retrotransposons are associated with h3k9me2-heterochromatin. 2 % are uncharacterized repeats and represent either putative unknown ty3-gypsy sequences or truncated and/or recombined repeats in h3k9me2-heterochromatin. d the cotzilla family predominantly contributes to the composition of h3k9me2-heterochromatin in ty1-copia retrotransposons, while 1 % of the ty1-copia sequences have not been characterized to date. e en/spm is the prevailing dna transposon superfamily followed by mutator elements in summary, the enriched repeats were more diverse in h3k9me chip-seq, both with respect to their quality and quantity. h3k9me chip-seq detected enriched clusters assigned to eight repeat types, while cenh chip-seq detected enriched clusters assigned to four repeat types. to confirm integration of plastid dna in the genome, hybridizations of plastid dna probes to sugar beet mitotic metaphase chromosomes revealed dispersed signals along the chromosomes. notably, signals of plastid dna were detected on both chromatides of most chromosomes arms with strong signals in subtelomeric and centromeric regions. immunostaining reveals specificity of the sugar beet cenh antibody to species of the section beta within the genus beta immunostaining using the antibody against sugar beet cenh on metaphase nuclei resulted in distinct and specific anti-bvcenh double-dot signals in sugar beet. single cenh signals and pbv regions are clearly distinguishable on a representative metaphase. the cenh signals are arranged in pairs of two, corresponding to sister chromatids, while each pair is found in one pbv cluster. most importantly, fish experiments on interphase nuclei and mitotic chromosomes using the centromere-specific satellite probe pbv revealed a cell cycle stage-independent co-localization of bvcenh and pbv on sugar beet chromosomes.fig. cenh immunofluorescence on beta nuclei and fish of pbv centromeric satellite on sugar beet. nuclei were counterstained with 4’,6-diamine-2’-phenylindole-dihydrochloride showing chromosome morphology. cenh binding was detected by the secondary antibody anti-rabbit coupled to fluorescein isothiocyanate. the pbv probe was labelled with biotin- utp and detected by streptavidine-cyanine. bar = 5 μm. a localization of cenh on sugar beet metaphase chromosomes detected by immuno-fish using the anti-bvcenh antibody and a centromeric satellite pbv probe as control. dapi staining of an exemplarily chosen sugar beet metaphase shows chromosome morphology. anti-bvcenh binding resulted in signals, arranged in pairs corresponding to chromatides. pbv signals are detected and illustrate the centromeres. overlay of all signals reveals co-localization of two cenh signals within one large block of pbv on each chromosome. b cenh localization during the sugar beet cell cycle. anti-bvcenh binding displays cenh signals in all mitotic cell cycle stages. chromosomes are identified by anti-bvcenh binding on all chromosomes. additional fish using the centromeric satellite pbv probe exhibits co-localization with cenh signals throughout the cell cycle. c cenh in species of the genera beta and patellifolia. exemplarily chosen interphase nuclei of different beta species are shown, including sugar beet, b. maritima, and b. patula, as well as p. patellaris belonging to patellifolia. areas exhibiting brighter dapi staining indicate heterochromatic regions. distinct anti-bvcenh signals were detected in sugar beet, b. maritima and b. patula in heterochromatic regions. the signals observed represent 2n =  chromosomes which is typical for diploid species of the section beta. no signals were found on p. patellaris nuclei the binding of anti-bvcenh was examined in sugar beet and additionally in related wild species of the genera beta and patellifolia to elucidate cenh conservation across beta species- and genus borders. the species investigated included one member each of the same subspecies, of a different species, and of the closely related genus patellifolia. immunostaining using the anti-bvcenh on interphase nuclei resulted in distinct and specific anti-bvcenh signals in sugar beet, b. maritima and b. patula, corresponding to the number of chromosomes in these species. contrarily, for p. patellaris nuclei no bvcenh signals were observed. all anti-bvcenh signals are detected in intensively dapi-stained centromeric heterochromatin, which implies that beta centromeric regions form chromocenters in interphase nuclei. discussion the recently published sugar beet reference sequence harbors 567 mb, which accounts for approximately 75 % of the estimated genome size. this difference is mainly due to technical barriers in the assembly of whole genome shotgun data predominantly generated with second generation sequencing technology. the approach regularly fails to assemble highly repetitive genome portions, such as heterochromatic regions or centromeric chromatin harboring large arrays of tandemly repeated sequences and transposable elements. the clustering and blast approach of genomic and chip-seq reads using the repeatexplorer pipeline applied in this study resulted in a far more realistic description of the repetitive portion in the sugar beet genome. this procedure has been applied in other species, too, and has been proven to be efficient. strikingly, most repeats identified as enriched in chip-seq experiments are clearly underrepresented in the sugar beet assembled genome sequence. as a result, our study complements the constitution of repetitive sequences comprising the cenh3-marked chromatin and h3k9me2-heterochromatin so far underrepresented in the reference sequence. immunostainings coupled to fish experiments together with cenh chip-seq data provide evidence on the direct interaction of the pbv satellite and cenh pbv was the only satellite family found to be enriched in the centromere. however, six variants of pbv are known. the genomic abundance reported for the variant pbv_iii is reflected in cenh chip-seq experiments, followed by pbv_ii. the variant pbv_iv possesses the longest monomere size out of the pbv variants identified in chip-seq data and is hypothesized to originate from pbv_iii ⁠. the occurrence of only three out of six pbv variants in cenh3-chromatin, namely pbv_ii, pbv_iii, and pbv_iv, may be explained with high genome abundances of single variants and/or their individual monomer sizes potentially providing the basis for an optimal packaging of centromeric chromatin ⁠. notably, the pbv variant vi, formerly designated as prv ⁠, is not present in cenh3-chromatin. we conclude, that pbv_vi is not a repeat sequence in active centromeres although it is present in association with other pbv arrays ⁠. as it was detected throughout the mitotic cell cycle, the co-localization of cenh and pbv suggests an interaction between histone protein and satellite sequence ⁠. in detail, recent studies demonstrated the active impact of long non-coding rnas derived from repeated centromeric dna on centromere function. it is not the centromere sequence itself, but rather the sequence features which enables the maintenance of an epigenetic environment necessary for proper centromere function. according to cenh chip-seq data, additionally to pbv, ty3-gypsy retrotransposons of the beetle family constitute the sugar beet centromere, which is in accordance to the statements reported in zakrzewski, weber, and schmidt based on fish experiments. the appearance of the satellite pbv and the beetle retrotransposon families in sugar beet centromeric heterochromatin is typical for the compositions of plant centromeres. the combination of a satellite repeat in combination with retrotransposons as building blocks of centromeres was detected in many plants to date: e.g. in monocots. the existence of beetle in sugar beet is most unlikely, since fish as well as blot hybridization experiments using a beetle ltr probe indicated specificity of beetle sequences exclusively to wild beets. the occurrence of clusters in sugar beet cenh chip-seq data assigned to beetle might be explained by the evolution of a species-specific, so far uncharacterized retrotransposon family after separation of sugar beet from wild species. thus, a similar relation was recently suggested between beetle from p. procumbens and its homolog in sugar beet, beetle it is likely, that beetle and beetle share a common ancestor but acquired different ltrs during speciation. based on that knowledge, the annotation of the unknown retrotransposon as beetle might be the result of high sequence similarity in the coding region between the wild-beet specific beetle and its homolog in sugar beet. the characterization of the sugar beet beetle diversity remains subject to future analyzes and might probably reveal an additional beetle family specific for sugar beet centromeres. another cluster annotated as centromere-specific chromovirus represents the highest enrichment of all repeats in cenh chromatin. interestingly, this chromovirus is remarkably specific to the sugar beet centromere, but due to the lack of any ltr sequences could not be annotated to any family so far.  % of the enriched centromeric sequences are not annotated yet and remain without further information. the annotation of these sequences is complicated, since no similarities were reported to any known repeat. the cenh function is similar in all species studied to date, but its protein sequence differs even between closely related species. this is also the most likely reason why the anti-bvcenh antibody which was raised against the sugar beet cenh sequence failed to detect cenh in patellifolia species. in contrast, the anti-rice cenh antibody recognized successfully cenh proteins in genera as far remote as maize and oat. sugar beet chromosomes are characterized by high enrichment of h3k9me in large intercalary heterochromatic blocks on each chromosome arm. these intercalary heterochromatic knobs may constitute the main portion of h3k9me heterochromatin in sugar beet. h3k9me chip-seq in sugar beet revealed a considerable diversity of repetitive sequences. the high proportion of the pev satellite may indicate that the pev satellite may contribute to the establishment and maintenance of intercalary heterochromatin as has been described for satellites. in accordance with the model proposed by picaard et al. it is hypothesized that satellite dna may contribute to the formation of heterochromatin due to epigenetic mechanisms: satellites are transcribed by polymerase iv into ssrnas, and subsequently dsrnas are generated by rna-dependent rna polymerase. the satellite dsrnas are processed into 24-nt sirnas by the activity of dicer-like 24-nt sirnas from satellite repeats are loaded on argounaute which then recognizes polymerase v satellite transcripts complementary to the incorporated 24-nt sirna. domain rearranged dna methyltransferase associates to the argounaute complex and induces de novo cytosine methylation at cg, chg and chh sites at satellite loci homologous to 24-nt sirnas. furthermore, 24-nt sirnas might be involved in the dimethylation of histone h, subsequently leading to heterochromatization as described in animals. therefore, satellites may serve as optimal sequence platform for the establishment and maintenance of heterochromatin at a large scale due to their simple structure of tandemly repeated monomers and their expansiveness of tandem arrays. similar to the enrichment in cenh binding sequences, the crm families beetle and beetle are also found in h3k9me data. several beetle families are located beyond the centromere. therefore it is likely, that beetle and beetle might tend to integrate beyond the centromere due to several target sites and/or the diversification steps. based on fish investigations ⁠ a similar explanation is proposed for beetle which is the centromeric retrotransposon in sugar beet. it cannot be excluded that these sequences were partially located in pericentromeric heterochromatin or in centromeric chromatin, which may also contain small proportions of h3k9me ⁠. in accordance with this assumption h3k9me chip-seq revealed small enrichment factors of for the centromere-specific satellite pbv_iii although the enrichment factor threshold chosen in this study was at least. studies in maize also provided evidence for h3k9me2-heterochromatin in centromeres. in immunostaining experiments very little h3k9me signals have been detected in sugar beet centromeres. however, this faint signal intensity could also be explained by the inability of antibody or stains to access centromeres during metaphase. one reason might be the protection of centromeres by a large complex of kinetochore factors that might have not been eliminated by protease treatment of metaphase spreads. the cotzilla family is an evolutionary young member of the sire clade belonging to the superfamily of ty1-copia retrotransposons, which is remarkably abundant in h3k9me chip-seq data. although the ranking of cotzilla enrichment factors might suggest intermediate specificity to h3k9me heterochromatin, chip-seq data validated that cotzilla elements constitute high proportions of h3k9me heterochromatic sequences. this emphasizes the findings of weber etal. ⁠, where fish experiments using an ltr-specific cotzilla probe demonstrated its high abundance in sugar beet intercalary and pericentromeric heterochromatin. bongo is a sugar beet ty3-gypsy retrotransposon of the tekay clade of chromoviruses which was found dispersed in small clusters along chromosomes. although the enrichment shows reduced specificity, bongo is another element associated with h3k9me belonging to ty3-gypsy retrotransposons, elbe which is a sugar beet specific errantivirus, exhibited a wide distribution on all chromosomes in fish experiments. the sugar beet elbe group includes gag-pol polyproteins of the elements elbe elbe elbe and elbe no restriction to certain genome regions has been reported ⁠. unlike bongo elbe exhibits higher enrichments in h3k9me chip-seq experiments indicating a more specific relation between elbe and h3k9me in addition, besides satellite repeats and ltr retrotransposon sequences, the dna transposon superfamily en/spm was remarkably enriched in h3k9me chip-seq data. to date, little is known about the precise localization of the transposon superfamily cacta-en/spm in sugar beet ⁠. nevertheless, the detection of en/spm sequences in h3k9me chip-seq experiments is plausible as h3k9me is found in association with smaller heterochromatic spots in genic and transposon-rich regions ⁠. interestingly, the most specific h3k9me binding sequence is not homologous to known repeats. thus, the highest enrichment was found for a repeat, which has not been annotated so far. similar to the unknown sequences in cenh chip-seq, the characterization has to be performed to reveal the heterochromatin specificity of unknown repeats. the major findings of this study were integrated in a summarizing figure based on the results obtained in this study and previously reported fish data ⁠. the simplified model shows the complexity of cenh3-chromatin and h3k9me2-heterochromatin repeat composition in sugar beet. while the satellites pev and pbv are mutually exclusive, ty3-gypsy retrotransposons are found in intercalary heterochromatin as well as in the centromere. for transposable elements of the en/spm superfamily it might be likely that they are preferentially located in smaller dispersed heterochromatic spots beyond intercalary and centromeric heterochromatin.fig. simplified model of histone modifications/substitutions in relationship with major repeats in cenh3-chromatin and h3k9me2-heterochromatin. a typical metacentric sugar beet chromosome composed of two chromatids is illustrated. a h3k9me is found in small heterochromatic sites in genic regions, in vicinity to transposons and putatively with retrotransposons, in large clusters in intercalary heterochromatin and in small proportions in cenh chromatin. the underlying sequence is composed of several repeats, but the satellite pev and the ty1-copia retrotransposon cotzilla predominate the sequence composition. h3k9me interacts with beetle and occasionally with beetle in pericentromeric regions. active centromeres are defined by cenh which preferentially binds to the centromeric satellite pbv and the ty3-gypsy retrotransposon beetle beetle is found in centromeric and presumably also in pericentromeric heterochromatin, in association with both, h3k9me and cenh note, that this figure is not drawn to scale. plastid dna is not incorporated, due to various integration sites, which need to be further confirmed and analyzed in subsequent studies remarkably, plastid dna apparently constitutes high proportions of both heterochromatic regions, although it has not been integrated in the genome assembly. however, fish using plastid dna confirms dispersed chromosomal localization. consequently, in contrast to earlier descriptions, plastid dna shows no major preference for integration into certain chromosomal regions. the existence of plastid dna in sugar beet chromosomes may be due to multiple plastid dna integration events which have taken place several times independently and therefore could be a frequent event rather than a single incident. therefore, plastid dna is left out of the summarizing figure as it has to be studied in far more detail which part of the chloroplast dna and how many copies integrated into the sugar beet genome in future studies.  sugar beet highly repetitive sequences are largely amplified in heterochromatic regions and centromeric chromatin but are only fragmentary included in the genome sequence. therefore, the repeat composition of sugar beet heterochromatin and centromeric chromatin was annotated and characterized using chip-seq and subsequent sequence clustering approaches via repeatexplorer: centromeric chromatin is hallmarked by the sugar beet cenh which binds to pbv satellites and to elements of the ty3-gypsy retrotransposon family beetle this finding underpins the typical sequence composition of a typical plant centromere. heterochromatin in plants is defined by the presence of h3k9me it occurs either in clusters or in smaller heterochromatic spots and is associated with a diverse pattern of repetitive sequences consisting of different families of satellite dnas, ty3-gypsy and ty1-copia retrotransposons as well as dna transposons. the knowledge of repetitive sequences constituting the only poorly characterized heterochromatin and centromeric chromatin gained within this project provides so far missing data for the understanding of the organization and evolution of the sugar beet genome. furthermore, it enhances the annotation quality of repetitive sequences, which is potentially useful for future genomic studies. the presented approach in sugar beet has already been performed in other plants before and is eligible to be applied to further plant genomes for the annotation and characterization of repeats localized in cenh3-chromatin and in heterochromatic blocks, which are not or only fragmentarily included in reference genome sequences. consequently, the analysis of the complete repeat composition of active plant centromeres and heterochromatin will provide comprehensive knowledge on a neglected but major sequence component of plant genomes. 
chip-seq analysis revealed that active centromeres containing cenh consist of the satellite pbv and the ty3-gypsy retrotransposon beetle while heterochromatin marked by h3k9me exhibits heterogeneity in repeat composition. in members of the section beta within the genus beta, immunostaining using the cenh antibody was successful, indicating that orthologous cenh proteins are present in closely related species within this section. 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, to fill these gaps and to gain information about the repeat composition of centromeres and heterochromatic regions, we performed chromatin immunoprecipitation followed by sequencing using antibodies against the centromere-specific histone h variant of sugar beet and the heterochromatic mark of dimethylated lysine of histone h. h3k9me was mainly associated with the satellite family pev, the ty1-copia retrotransposon family cotzilla and the dna transposon superfamily of the en/spm type. 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 h3k9me 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 are only partially included. sugar beet is an important crop of temperate climate zones, which provides nearly 30 % of the world’s annual sugar needs.
 sugar beet 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 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 using antibodies against the centromere-specific histone h variant of sugar beet and the heterochromatic mark of dimethylated lysine of histone h.  chip-seq analysis revealed that active centromeres containing cenh consist of the satellite pbv and the ty3-gypsy retrotransposon beetle while heterochromatin marked by h3k9me exhibits heterogeneity in repeat composition. h3k9me 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 cenh antibody was successful, indicating that orthologous cenh proteins are present in closely related species within this section.  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 h3k9me electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords centromerecenh3h3k9me2heterochromatinrepeatsbeta vulgarischip-seqhttp://dx.doi.org/ /501100002347bundesministerium für bildung und forschungfkz weisshaar bernd czech science foundationgbp501/12/g090macas jiří http://dx.doi.org/ /501100004240akademie věd České republikyrvo:60077344macas jiří issue-copyright-statement© the author 2016 