BACKGROUND
repetitive dnas, including transposable elements and tandem repeats, are the major components of the genomic sequence and the most important factors in genome size variations in higher eukaryotes  <cit> .
based on the mechanism of transposition, transposable elements can be divided into two classes, transposons and retrotransposons.
the majority of these elements in plant genome are long terminal repeat  retrotransposons and most of them are dispersed throughout all chromosomes  <cit> .
tandem repeats consist of large number of repeat units and are usually found in centromeres, pericentromeres or telomeres  <cit> .
tandem repeats are good cytogenetic markers for chromosome identification and molecular karyotyping  <cit> .
centromeres are specialized regions on chromosomes where centromeric protein and spindle microtubules attach via the kinetochore and typically contain large arrays of satellite repeats and/or retrotransposon-related repetitive sequences in eukaryotes  <cit> .
they are essential for proper chromosome segregation during mitosis and meiosis.
although the function of centromeres is conserved in organisms, centromeric repeats appear to evolve rapidly  <cit> .
satellite repeats go through rapid evolution and significant variation between closely related species or even among different chromosomes of the same species  <cit> .
centromeric regions are comprised of repetitive sequences in most species, suggesting that those sequences play important roles in centromere function  <cit> .
centromeres are universally marked by the presence of a centromere-specific histone h <dig> , that replaces canonical histone h <dig> in centromeric nucleosomes to form functional centromeres  <cit> .
cenh <dig> is a good marker to identify the core centromeric sequences by chromatin immunoprecipitation  with an anti-cenh <dig> antibody  <cit> .
radish , belonging to the family cruciferae, is an important vegetable crop especially in eastern asia.
radish root contains various nutritions which play a part in strengthening immunity  <cit> .
radish is a healthy vegetable and is popular in many dishes.
although radish is a significant vegetable crop, it still lacks cytogenetic analysis.
location of 5s rdna loci and 45s rdna loci were confirmed via fish mapping  <cit> .
these two sequences are located at the pericentromeric heterochromatin regions.
a few studies of the radish repetitive dnas were previously reported.
first an alphoid-like satellite repeat in radish was found in  <dig>  <cit> .
it was a big step to get the draft sequences of the japanese radish ‘aokubi’, with a long and thick root, for the study of repetitive elements.
it has been estimated that the genome size of the radish is 530 mb  <cit>  and about  <dig> % of the genome is made of various dna repeats.
the transposons and retrotransposons were characterized  <cit> .
nevertheless, up to now, understanding of the repetitive sequences of radish is still not sufficient, especially for the tandem repeats.
in this study, 5gb of sequence data was used to analyze the repetitive elements of radish.
we found three types of tandem repeats  in the radish genome.
an integrated metaphase chromosome karyotype was established using tandem repeats , along with rdnas as probes.
the coding sequence of cenh <dig> of radish was identified.
immunostaining and chromatin immunoprecipitation tests demonstrated that both cr-radish and cl <dig> are associated with rscenh <dig> proteins in radish.
RESULTS
composition of the repetitive sequences in the radish genome
5gb sequencing data, which amounts to  <dig> × coverage of the radish genome, was obtained from the hiseq <dig> platform.
repeatexplorer, a graph-based clustering and characterization of repetitive sequence utilities was used for analyzing repetitive elements of the genome.
<dig> clusters were generated with cluster size threshold of  <dig> %, and clusters which were annotated putative mitochondrial and plastid contaminations were removed.
finally,  <dig> clusters were used for calculating genome proportions .
the genome proportions of each type of repetitive dna are shown in table  <dig>  about  <dig> % of the genome is repetitive dnas.
according to our results, it has different repetitive dna types: retrotransposons , transposons , rdna and satellites.
satellite repeats, which occupy  <dig> % of the genome, make up the most dominant part of the repetitive dnas in radish.
the majority of retrotransposons are ty1/copia and ty3/gypsy retrotransposons, with genome proportions of  <dig> % and  <dig> %, respectively.
the genome proportion of transposons is only  <dig> % and the most abundant transposon is hat, with a  <dig> % genome proportion.
estimation of rdna elements abundance showed that they comprise  <dig> % of the genome.table  <dig>
repeat elements and their proportions in radish
elements
gp
retrotransposon
<dig>
copia
<dig>
gypsy
<dig>
line/sine
<dig>
unclassified ltr
<dig>
transposon
<dig>
hat
<dig>
mutator
<dig>
dna/cmc-enspm
<dig>
tc1-mariner
<dig>
rdna
<dig>
satellite
<dig>
unclassified
<dig>
total
<dig>
identification of subtelomeric repeats and centromeric repeats in radish
in addition to 5s rdna and 45s rdna, three tandemly organized repeats  were identified by bioinformatics analysis of the sequencing data.
the cl <dig>  cl <dig> and cl <dig> repeats were estimated to make up  <dig> %,  <dig> %, and  <dig> % of the genome, respectively.
cl <dig> is a telomeric repeat, consisting of a 7 bp monomer , located at both ends of chromosomes .
pcr of cl <dig> and cl <dig> resulted in a ladder like pattern for tandemly organized repetitive units.
to ascertain the size of the monomers of cl <dig> and cl <dig>  specific primers were designed for amplifying these two repeats and then sequenced.
according to sequencing results, cl <dig> consists of ~177 bp monomers , which is almost exactly the same as the size of the alphoid-like satellite repeat reported by grellet  <cit> .
searching genbank and plantsat databases revealed high similarities to centromeric tandem repeats centbr <dig> and centbr <dig> from brassica species   <cit>  and satellite sequences from sinapis alba   <cit> .
the cl <dig> repeat is characterized by a ~348 bp monomer unit  and is a newly found satellite.
similar to cl <dig>  the cl <dig> sequences shared high similarities to brassica species .
in addition, a small part  of the cl <dig> sequence present in the c. elegans.figure  <dig> consensus sequence of cl <dig> and cl <dig> repeats.
the black rectangle outlines the positions in the sequence that share similarities to c. elegans.
fish result showed that cl <dig> is located at the main primary constrictions and cl <dig> appears at the subtelomeric regions .
on account of cl <dig> sharing a high similarity to brassica species, we speculated that cl <dig> should have a specific distribution pattern in these species.
fish mapping of cl <dig> repeats was performed on metaphase chromosomes of several brassica species, including b. rapa , b. nigra , b. oleracea , and b. napus , which are close relatives to radish .
overall, cl <dig> appeared at subtelomeric regions for all the detected species, although different species have various numbers and varied intensities of signals.
intensities of signals are relatively weak in b. rapa, and strong in b. oleracea.
a different distribution pattern was detected in b. nigra with strong signals on  <dig> pairs of chromosomes and weak signals on  <dig> pairs of chromosomes.
therefore the cl <dig> repeat is an ancient repeat which appeared before the differentiation of tribe brassiceae and radish.
it has maintained its subtelomeric positions in all detected cruciferae species.figure  <dig> fish mapping of cl <dig> and cl <dig> repeats.
fish mapping of cl <dig> in radish;  fish mapping of cl25;  radish;
b. rapa;
b. nigra;
b. oleracea;
b. napus.
bars = 5 μm.
karyotyping analyses of radish
given the lack of dna markers for fish analysis, detailed molecular karyotype analyses of radish have not yet been conducted.
repeats identified in this study provide good markers for karyotyping analysis.
sequential fish using repetitive dna sequences  as probes were performed to identify radish chromosomes .
the cl <dig> signals appeared at the middle of all of the chromosomes with varied intensities.
the cl <dig> signals were located at one arm of chromosomes  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig>  and both arms of chromosome  <dig> with one pairs of signals.
signals were large and strong on chromosomes  <dig> and  <dig>  but weak on chromosomes  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  two pairs of 5s rdna signals were detected, and one pair of strong signals were located at the peri-centromeric region of the short arm of chromosome  <dig>  and the other pair of weak signals appeared at the peri-centromeric region of the short arm of chromosome  <dig>  interestingly, we detected  <dig> pairs of 45s rdna signals in early generations, which is the same as koo’s results  <cit> ; however, only  <dig> pairs of the signals were detected  <dig> generations later .
seeds from the new generation were used for karyotyping analysis .
in our study, the signals of 45s rdna were located at the long arms of chromosomes  <dig> and  <dig>  using satellite repeats  combined with rdnas as fish probes, we distinctly identified individual somatic chromosome by the position and intensity of their signals .
an integrated ideogram of radish metaphase chromosomes is shown in figure 3e.figure  <dig> the karyotype and ideograph for radish mitotic metaphase chromosomes.
the mitotic metaphase chromosomes  were counterstained with dapi and pseudocolored in red;  fish with the probe of cl <dig>  and 5s rdna ; .
the same spread was reprobed with the probe of cl <dig>  and 45s rdna ;  individual chromosomes were separated from figure  and listed according to their order;  ideogram showing the position and intensity of cl <dig>  and 5s rdna , cl <dig>  and 45s rdna .
bars = 5 μm.
cloning of cenh3
to identity cenh <dig> in radish, we searched ncbi using the blastn program  with the brcenh <dig> complementary cdna sequence  as the query.
two radish cenp-a gene sequences  were identified.
these two putative cenh <dig> open reading frames share high similarity with a small gap and some snps.
based on these two sequences, specific primers were designed to isolate the complete rscenh <dig> coding region from radish plants.
according to cdna sequencing results, three transcripts were detected: a 635 bp length of transcript , a 513 bp length of transcript , and the majority 537 bp length of transcript .
to analyze the intron/exon structure of rscenh <dig>  the full length of genomic dna sequence of rscenh <dig> was amplified using the same primers.
on the basis of genomic dna results, only one type of dna sequence was found, which has a total length of 1415 bp.
this sequence shares 100% identity to the ab <dig>  and is comprised of nine exons and eight introns.
by comparison with the full length genomic dna sequence, a 635 bp length of transcript transformed from the third intron into an exon, a 513 bp length of the transcript has a deletion from part of the forth exon, and the major transcript is 537 bp.
considering the translation, alignment to other plant cenh3s, and the proportion of these transcripts, we deemed that the small number of transcripts were produced by mis-splicing from the same loci and the cenh <dig> comprises an open reading frame  of length 537 bp encoding a predicted 178-amino acid  protein.
multiple sequence alignment revealed that rscenh <dig> shares high identities with cenh <dig> from brassica species, 77% identity with brcenh <dig>  64% with bncenh <dig>  and 74% with bocenh <dig>  several prominent features of the deduced rscenh <dig> in comparison with those cenh3s and canonical histone h <dig> are as follows .
a longer and more divergent n-terminal tail is present in the deduced rscenh <dig> sequence  that is not alignable to brh <dig> .
each of the predicted proteins encoded a histone fold domain with similarities to histone h <dig>  the loop  <dig> region in the histone fold domain is longer than that of canonical histone h3s .
all of these findings demonstrate that the sequence identified is an authentic cenh <dig> homolog in radish.figure  <dig> a multiple alignment of cenh <dig> sequences.
a multiple alignment of radish , brassica rapa , brassica oleracea , brassica nigra  homologs and brassica rapa h <dig> .
a black rectangle indicates the position of loop <dig> region.
the right side of the vertical bar is the n-tail region and left side is the histone fold domain.
dna sequences associated with rscenh3
a b. rapa -derived cenh <dig> antibody  was previously used to confirm cenh3-associated centromeric sequences in different brassica species  <cit> .
based on the similarities of cenh3’s sequence between radish and brassica species, we speculated b. rapa -derived cenh <dig> antibody should recognize the rscenh <dig> protein at core centromeres in radish.
to confirm whether it recognizes the rscenh <dig> protein, we applied an immunofluorescence assay on somatic cells of radish with the anti-brcenh <dig> antibody.
signals appeared at the centromeric regions of all  <dig> metaphase chromosomes .
in interphase cells, rscenh <dig> signals were located at the edge of the dapi intensively stained heterochromatic regions .
it showed that the antibody also could recognize rscenh <dig> figure  <dig> anti-brcenh <dig> antibody staining in mitotic cells of radish.
rscenh <dig> localization  on somatic interphase cell  and metaphase chromosomes .
bars = 5 μm.
it has been reported that crb  is a core centromeric sequences of brassica species  <cit> .
we also detected a crb-like retrotransposon cl <dig>  which represents  <dig> % of the genome and was named cr-radish in radish.
to verify if the centromere-specific retrotransposon cr-radish and the 177-bp satellite repeat cl <dig> were associated with rscenh <dig> protein in radish, we performed an immunofluorescence assay followed by fish on the same set of cells to detect the co-localization of brcenh <dig> and centromeric dna repeats.
the size of rscenh <dig> immuno-signals were relatively uniform among kinetochores while the size of cl <dig> signals were uneven among different chromosomes .
cl <dig> signals were overlapped with the rscenh <dig> immuno-signals, although they were significantly larger than the rscenh <dig> immuno-signals.
these results suggest that only a limited part of the cl <dig> sequences are associated with the kinetochore complex.
we also conducted anti-brcenh <dig> immunostaining followed by fish of the cr-radish retrotransposon .
different from that of cl <dig>  cr-radish signals were smeared and weak.
as expected, the fish signals overlapped with most of the immuno-signals.
therefore, we propose that the rscenh <dig> protein is also associated with cr-radish.
dual-color fish showed most signals of cr-radish and cl <dig> were co-localized, while the signals of cl <dig> were more concentrated than cr-radish signals .figure  <dig> sequential localization of the anti-brcenh <dig> antibody and centromeric repeats on radish.
rscenh <dig> localization at mitotic metaphase chromosomes of radish;  the same cell was hybridized with cl1;  merged fluorescence signals from a and b;  merged fluorescence signals from c and chromosomes;  rscenh <dig> localization at mitotic metaphase chromosomes of radish;  the same cell was hybridized with cr-radish;  merged fluorescence signals from d and e;  merged fluorescence signals from g and chromosomes;  cr-radish localization at mitotic metaphase chromosomes of radish;  the same cell was probed with cl1;  merged fluorescence signals from g and h;  merged fluorescence signals from h and chromosomes.
bars = 5 μm.
to further confirm our immunostaining results, chip tests with the anti-brcenh <dig> antibody were conducted to assess the association of cl <dig> and cr-radish with rscenh <dig>  fish using the chiped dna as a probe showed high enhanced signals in the centromere regions of all radish chromosomes.
in contrast, using mocked dna as a probe showed no obvious signal  which indicates that the centromere sequences were specifically pulled down by the anti-brcenh <dig> antibody in chip.
the chip-qpcr was performed to verify the enrichment of putative centromeric repeats .
two specific primers, designed from different regions of cl <dig>  cl <dig> and cr-radish, were used to detect each fragment.
the chip-qpcr was repeated three times using cl <dig> as extra-centromeric control.
rfe value for cl25- <dig> was set at  <dig>  and the rfe value of each sequence was normalized using the cl25- <dig> as a reference.
the rfe of the non-centromeric control cl25- <dig>  5s rdna, and 45s rdna were low and similar to each other at  <dig>  ±  <dig> ,  <dig>  ±  <dig> , and  <dig>  ±  <dig> , respectively .
in contrast, the rfe of the cr-radish fragments were as high as  <dig>  ±  <dig>  and  <dig>  ±  <dig> , respectively.
similarly, the rfe of the cl <dig> fragments were  <dig>  ±  <dig>  and  <dig>  ±  <dig> , respectively.
these results indicate that cr-radish and cl <dig> were significantly enriched in the chiped dna.
therefore, cr-radish and cl <dig> are associated with racenh <dig> figure  <dig> sequences associated with rscenh <dig>  relative fold enrichments of repeats obtained by chip with the anti-brcenh <dig> antibody are shown for radish genomes.
cl <dig> serves as a negative control; cr-radish and cl <dig> were associated with cenh <dig>
discussion
karyotype of radish
up to now, studies on the radish genome were still limited and few cytogenetic and genomic studies were carried out  <cit> .
comparative analysis of rdna and rfk <dig> gene distribution in chromosomes of brassica species and radish were carried through using fish  <cit> .
however, to our knowledge, a complete karyotype analysis that reliably distinguishes each chromosome of radish has not been reported.
chromosome identification is critical for cytological analyses, as well as subsequent studies in genomics, taxonomy, and the evolution of polyploidy, enabling an understanding of the relationship between visible landmarks and genetic or physical map features  <cit> .
the somatic metaphase chromosomes of radish are small and lack feasible markers, which make adequate identification of radish chromosome pairs difficult.
in this study, we used repeatexplorer to conduct genome-wide analysis of repetitive sequences and obtained two useful cytogenetic markers .
together with rdnas, one or two signals were detected on each chromosome .
we are now able to easily identify all  <dig> somatic metaphase chromosomes by the position and intensity of fish signals.
in addition, an integrated metaphase chromosome karyotype was established .
our study provides a valuable basis for future genomic studies.
dynamic nature of radish genome
repetitive sequences contribute significantly to extraordinary genome size variation in higher plants  <cit> .
generally speaking, ltr-retrotransposons are the most abundant element of the genome, especially in big genome species, such as maize  <cit> , wheat  <cit> , and coix  <cit> .
however, the majority of repetitive sequences are satellites, which make up  <dig> % of the radish genome in our study.
a similar high proportion of satellites were found in c. rubella and cucumber, in which more than 20% of the genome sequences are satellite repeats  <cit> .
ordinarily, several to dozens of types of satellite repeats are detected from a number of species  <cit> .
in our study, only three satellite repeats were found in radish, including centromeric repeats, subtelomeric repeats and telomeric repeats .
this is a typical pattern where the satellite dna sequences are appear predominantly in the centromeric, pericentromeric and telomeric regions  <cit> .
the dynamic evolutionary processes of satellite dna may generate changes in its chromosomal location and distribution.
some satellite dna families were found to be species-specific  <cit> , while others were more conserved, and similar sequences may be isolated in closely related species  <cit> .
in our study, we detected  <dig> pairs of 45s rdna signals in early generations of the radish, the same result obtained by koo  <cit> , while only  <dig> pairs of 45s rdna signals were detected in later generations .
it suggests that rdna also have a rapid evolution in the genome.
furthermore radish inbred lines from different areas might contain varied ratio of repetitive sequences.
<dig> % of the 0713d genome is repetitive dna in our study, while repetitive sequences occupied  <dig> % of the japanese radish ‘aokubi’ genome  <cit> .compositions of each type of repetitive elements are also different between these two radishes.
overall, these results demonstrate the highly dynamic nature of radish genome.
rapid evolution of centromere sequence
the centromeres of higher eukaryotes are rich in repetitive dna sequences which include large arrays of satellite repeats and/or retrotransposon-related repetitive sequences  <cit> .
it has been shown that one single major satellite repeat is the dominating sequence in all centromeres in most diploid species  <cit> .
in our study, the similar pattern of one type of centromeric satellite repeat  was detected by immunostaining and the chip test.
however, it has been reported that some plant and animal species contain multiple satellite repeats associated with centromeres, such as in the common bean  <cit> , potato  <cit> , and chicken  <cit> .
centromeric satellite repeats diverge rapidly across species and often do not share any sequence similarity  <cit> .
several centromeric repeats were identified in potato and its closely related wide species s. verrucosum, respectively.
nevertheless, only one single homoeologous centromeric sequence was detected between these two species.
this means centromeric regions of solanum species show rapid evolution.
taxonomic studies and rdna gene space sequence analysis demonstrated that genus brassica is a close relative of the genus raphanus  <cit> .
our results also proved this.
in this study, a new satellite cl <dig> was detected, which is distributed in radish and all tested brassica species and located at the subtelomeric region of all tested species .
even in closely related species, centromeric satellites go through rapid evolution.
cl <dig>  the centromeric satellite repeat, shares high similarities with centbr <dig> and centbr <dig> sequences.
these centbr sequences appeared in the a and c genomes of brassica species, while the corresponding centromeric repeats have not yet been identified in the b genome.
even in the same species, centbr <dig> and centbr <dig> have different distribution patterns on chromosomes  <cit> .
these results indicate that centromeric satellite repeats of cruciferae species evolve rapidly.
CONCLUSIONS
in this study, we used low-coverage sequencing on raphanus sativus l.  to analyze repeat elements.
we revealed the genome structure of radish and found that satellite repeats are most dominating elements, which is differ from most reported species, in which ltr-retrotransposons are the most abundant element of the genome.
the fine karyotyping analysis using satellites and rdnas as markers allow us to easily identify each individual somatic metaphase chromosome.
only one putative cenh <dig>  gene was characterized and cloned from radish.
its deduced amino acid sequence shares high similarities to those of the cenh3s in brassica species.
in addition, immunofluorescence- and chip-based assays demonstrated the functional significance of satellite and centromere-specific retrotransposon at centromeres.
our study provides a valuable basis for future genomic studies in radish.
availability of supporting data
the data sets supporting the results of this article are available in the ncbi sra archive .

 repetitive dnas, including transposable elements and tandem repeats, are the major components of the genomic sequence and the most important factors in genome size variations in higher eukaryotes. based on the mechanism of transposition, transposable elements can be divided into two classes, transposons and retrotransposons. the majority of these elements in plant genome are long terminal repeat retrotransposons and most of them are dispersed throughout all chromosomes. tandem repeats consist of large number of repeat units and are usually found in centromeres, pericentromeres or telomeres. tandem repeats are good cytogenetic markers for chromosome identification and molecular karyotyping. centromeres are specialized regions on chromosomes where centromeric protein and spindle microtubules attach via the kinetochore and typically contain large arrays of satellite repeats and/or retrotransposon-related repetitive sequences in eukaryotes. they are essential for proper chromosome segregation during mitosis and meiosis. although the function of centromeres is conserved in organisms, centromeric repeats appear to evolve rapidly. satellite repeats go through rapid evolution and significant variation between closely related species or even among different chromosomes of the same species. centromeric regions are comprised of repetitive sequences in most species, suggesting that those sequences play important roles in centromere function. centromeres are universally marked by the presence of a centromere-specific histone h, that replaces canonical histone h in centromeric nucleosomes to form functional centromeres. cenh is a good marker to identify the core centromeric sequences by chromatin immunoprecipitation with an anti-cenh antibody. radish, belonging to the family cruciferae, is an important vegetable crop especially in eastern asia. radish root contains various nutritions which play a part in strengthening immunity. radish is a healthy vegetable and is popular in many dishes. although radish is a significant vegetable crop, it still lacks cytogenetic analysis. location of 5s rdna loci and 45s rdna loci were confirmed via fish mapping. these two sequences are located at the pericentromeric heterochromatin regions. a few studies of the radish repetitive dnas were previously reported. first an alphoid-like satellite repeat in radish was found in. it was a big step to get the draft sequences of the japanese radish ‘aokubi’, with a long and thick root, for the study of repetitive elements. it has been estimated that the genome size of the radish is 530 mb and about % of the genome is made of various dna repeats. the transposons and retrotransposons were characterized. nevertheless, up to now, understanding of the repetitive sequences of radish is still not sufficient, especially for the tandem repeats. in this study, 5gb of sequence data was used to analyze the repetitive elements of radish. we found three types of tandem repeats in the radish genome. an integrated metaphase chromosome karyotype was established using tandem repeats, along with rdnas as probes. the coding sequence of cenh of radish was identified. immunostaining and chromatin immunoprecipitation tests demonstrated that both cr-radish and cl are associated with rscenh proteins in radish.  composition of the repetitive sequences in the radish genome 5gb sequencing data, which amounts to × coverage of the radish genome, was obtained from the hiseq platform. repeatexplorer, a graph-based clustering and characterization of repetitive sequence utilities was used for analyzing repetitive elements of the genome. clusters were generated with cluster size threshold of %, and clusters which were annotated putative mitochondrial and plastid contaminations were removed. finally, clusters were used for calculating genome proportions. the genome proportions of each type of repetitive dna are shown in table  about % of the genome is repetitive dnas. according to our results, it has different repetitive dna types: retrotransposons, transposons, rdna and satellites. satellite repeats, which occupy % of the genome, make up the most dominant part of the repetitive dnas in radish. the majority of retrotransposons are ty1/copia and ty3/gypsy retrotransposons, with genome proportions of % and %, respectively. the genome proportion of transposons is only % and the most abundant transposon is hat, with a % genome proportion. estimation of rdna elements abundance showed that they comprise % of the genome.table repeat elements and their proportions in radish elements gp retrotransposon   copia   gypsy   line/sine   unclassified ltr transposon   hat   mutator   dna/cmc-enspm   tc1-mariner rdna satellite unclassified total identification of subtelomeric repeats and centromeric repeats in radish in addition to 5s rdna and 45s rdna, three tandemly organized repeats were identified by bioinformatics analysis of the sequencing data. the cl cl and cl repeats were estimated to make up %, %, and % of the genome, respectively. cl is a telomeric repeat, consisting of a 7 bp monomer, located at both ends of chromosomes. pcr of cl and cl resulted in a ladder like pattern for tandemly organized repetitive units. to ascertain the size of the monomers of cl and cl specific primers were designed for amplifying these two repeats and then sequenced. according to sequencing results, cl consists of ~177 bp monomers, which is almost exactly the same as the size of the alphoid-like satellite repeat reported by grellet. searching genbank and plantsat databases revealed high similarities to centromeric tandem repeats centbr and centbr from brassica species and satellite sequences from sinapis alba. the cl repeat is characterized by a ~348 bp monomer unit and is a newly found satellite. similar to cl the cl sequences shared high similarities to brassica species. in addition, a small part of the cl sequence present in the c. elegans.figure consensus sequence of cl and cl repeats. the black rectangle outlines the positions in the sequence that share similarities to c. elegans. fish result showed that cl is located at the main primary constrictions and cl appears at the subtelomeric regions. on account of cl sharing a high similarity to brassica species, we speculated that cl should have a specific distribution pattern in these species. fish mapping of cl repeats was performed on metaphase chromosomes of several brassica species, including b. rapa, b. nigra, b. oleracea, and b. napus, which are close relatives to radish. overall, cl appeared at subtelomeric regions for all the detected species, although different species have various numbers and varied intensities of signals. intensities of signals are relatively weak in b. rapa, and strong in b. oleracea. a different distribution pattern was detected in b. nigra with strong signals on pairs of chromosomes and weak signals on pairs of chromosomes. therefore the cl repeat is an ancient repeat which appeared before the differentiation of tribe brassiceae and radish. it has maintained its subtelomeric positions in all detected cruciferae species.figure fish mapping of cl and cl repeats. fish mapping of cl in radish; fish mapping of cl25; radish; b. rapa; b. nigra; b. oleracea; b. napus. bars = 5 μm. karyotyping analyses of radish given the lack of dna markers for fish analysis, detailed molecular karyotype analyses of radish have not yet been conducted. repeats identified in this study provide good markers for karyotyping analysis. sequential fish using repetitive dna sequences as probes were performed to identify radish chromosomes. the cl signals appeared at the middle of all of the chromosomes with varied intensities. the cl signals were located at one arm of chromosomes and and both arms of chromosome with one pairs of signals. signals were large and strong on chromosomes and but weak on chromosomes and two pairs of 5s rdna signals were detected, and one pair of strong signals were located at the peri-centromeric region of the short arm of chromosome and the other pair of weak signals appeared at the peri-centromeric region of the short arm of chromosome interestingly, we detected pairs of 45s rdna signals in early generations, which is the same as koo’s results; however, only pairs of the signals were detected generations later. seeds from the new generation were used for karyotyping analysis. in our study, the signals of 45s rdna were located at the long arms of chromosomes and using satellite repeats combined with rdnas as fish probes, we distinctly identified individual somatic chromosome by the position and intensity of their signals. an integrated ideogram of radish metaphase chromosomes is shown in figure 3e.figure the karyotype and ideograph for radish mitotic metaphase chromosomes. the mitotic metaphase chromosomes were counterstained with dapi and pseudocolored in red; fish with the probe of cl and 5s rdna;. the same spread was reprobed with the probe of cl and 45s rdna; individual chromosomes were separated from figure and listed according to their order; ideogram showing the position and intensity of cl and 5s rdna, cl and 45s rdna. bars = 5 μm. cloning of cenh3 to identity cenh in radish, we searched ncbi using the blastn program with the brcenh complementary cdna sequence as the query. two radish cenp-a gene sequences were identified. these two putative cenh open reading frames share high similarity with a small gap and some snps. based on these two sequences, specific primers were designed to isolate the complete rscenh coding region from radish plants. according to cdna sequencing results, three transcripts were detected: a 635 bp length of transcript, a 513 bp length of transcript, and the majority 537 bp length of transcript. to analyze the intron/exon structure of rscenh the full length of genomic dna sequence of rscenh was amplified using the same primers. on the basis of genomic dna results, only one type of dna sequence was found, which has a total length of 1415 bp. this sequence shares 100% identity to the ab and is comprised of nine exons and eight introns. by comparison with the full length genomic dna sequence, a 635 bp length of transcript transformed from the third intron into an exon, a 513 bp length of the transcript has a deletion from part of the forth exon, and the major transcript is 537 bp. considering the translation, alignment to other plant cenh3s, and the proportion of these transcripts, we deemed that the small number of transcripts were produced by mis-splicing from the same loci and the cenh comprises an open reading frame of length 537 bp encoding a predicted 178-amino acid protein. multiple sequence alignment revealed that rscenh shares high identities with cenh from brassica species, 77% identity with brcenh 64% with bncenh and 74% with bocenh several prominent features of the deduced rscenh in comparison with those cenh3s and canonical histone h are as follows. a longer and more divergent n-terminal tail is present in the deduced rscenh sequence that is not alignable to brh. each of the predicted proteins encoded a histone fold domain with similarities to histone h the loop region in the histone fold domain is longer than that of canonical histone h3s. all of these findings demonstrate that the sequence identified is an authentic cenh homolog in radish.figure a multiple alignment of cenh sequences. a multiple alignment of radish, brassica rapa, brassica oleracea, brassica nigra homologs and brassica rapa h. a black rectangle indicates the position of loop region. the right side of the vertical bar is the n-tail region and left side is the histone fold domain. dna sequences associated with rscenh3 a b. rapa -derived cenh antibody was previously used to confirm cenh3-associated centromeric sequences in different brassica species. based on the similarities of cenh3’s sequence between radish and brassica species, we speculated b. rapa -derived cenh antibody should recognize the rscenh protein at core centromeres in radish. to confirm whether it recognizes the rscenh protein, we applied an immunofluorescence assay on somatic cells of radish with the anti-brcenh antibody. signals appeared at the centromeric regions of all metaphase chromosomes. in interphase cells, rscenh signals were located at the edge of the dapi intensively stained heterochromatic regions. it showed that the antibody also could recognize rscenh figure anti-brcenh antibody staining in mitotic cells of radish. rscenh localization on somatic interphase cell and metaphase chromosomes. bars = 5 μm. it has been reported that crb is a core centromeric sequences of brassica species. we also detected a crb-like retrotransposon cl which represents % of the genome and was named cr-radish in radish. to verify if the centromere-specific retrotransposon cr-radish and the 177-bp satellite repeat cl were associated with rscenh protein in radish, we performed an immunofluorescence assay followed by fish on the same set of cells to detect the co-localization of brcenh and centromeric dna repeats. the size of rscenh immuno-signals were relatively uniform among kinetochores while the size of cl signals were uneven among different chromosomes. cl signals were overlapped with the rscenh immuno-signals, although they were significantly larger than the rscenh immuno-signals. these results suggest that only a limited part of the cl sequences are associated with the kinetochore complex. we also conducted anti-brcenh immunostaining followed by fish of the cr-radish retrotransposon. different from that of cl cr-radish signals were smeared and weak. as expected, the fish signals overlapped with most of the immuno-signals. therefore, we propose that the rscenh protein is also associated with cr-radish. dual-color fish showed most signals of cr-radish and cl were co-localized, while the signals of cl were more concentrated than cr-radish signals.figure sequential localization of the anti-brcenh antibody and centromeric repeats on radish. rscenh localization at mitotic metaphase chromosomes of radish; the same cell was hybridized with cl1; merged fluorescence signals from a and b; merged fluorescence signals from c and chromosomes; rscenh localization at mitotic metaphase chromosomes of radish; the same cell was hybridized with cr-radish; merged fluorescence signals from d and e; merged fluorescence signals from g and chromosomes; cr-radish localization at mitotic metaphase chromosomes of radish; the same cell was probed with cl1; merged fluorescence signals from g and h; merged fluorescence signals from h and chromosomes. bars = 5 μm. to further confirm our immunostaining results, chip tests with the anti-brcenh antibody were conducted to assess the association of cl and cr-radish with rscenh fish using the chiped dna as a probe showed high enhanced signals in the centromere regions of all radish chromosomes. in contrast, using mocked dna as a probe showed no obvious signal which indicates that the centromere sequences were specifically pulled down by the anti-brcenh antibody in chip. the chip-qpcr was performed to verify the enrichment of putative centromeric repeats. two specific primers, designed from different regions of cl cl and cr-radish, were used to detect each fragment. the chip-qpcr was repeated three times using cl as extra-centromeric control. rfe value for cl25- was set at and the rfe value of each sequence was normalized using the cl25- as a reference. the rfe of the non-centromeric control cl25- 5s rdna, and 45s rdna were low and similar to each other at  ± ,  ± , and  ± , respectively. in contrast, the rfe of the cr-radish fragments were as high as  ±  and  ± , respectively. similarly, the rfe of the cl fragments were  ±  and  ± , respectively. these results indicate that cr-radish and cl were significantly enriched in the chiped dna. therefore, cr-radish and cl are associated with racenh figure sequences associated with rscenh relative fold enrichments of repeats obtained by chip with the anti-brcenh antibody are shown for radish genomes. cl serves as a negative control; cr-radish and cl were associated with cenh discussion karyotype of radish up to now, studies on the radish genome were still limited and few cytogenetic and genomic studies were carried out. comparative analysis of rdna and rfk gene distribution in chromosomes of brassica species and radish were carried through using fish. however, to our knowledge, a complete karyotype analysis that reliably distinguishes each chromosome of radish has not been reported. chromosome identification is critical for cytological analyses, as well as subsequent studies in genomics, taxonomy, and the evolution of polyploidy, enabling an understanding of the relationship between visible landmarks and genetic or physical map features. the somatic metaphase chromosomes of radish are small and lack feasible markers, which make adequate identification of radish chromosome pairs difficult. in this study, we used repeatexplorer to conduct genome-wide analysis of repetitive sequences and obtained two useful cytogenetic markers. together with rdnas, one or two signals were detected on each chromosome. we are now able to easily identify all somatic metaphase chromosomes by the position and intensity of fish signals. in addition, an integrated metaphase chromosome karyotype was established. our study provides a valuable basis for future genomic studies. dynamic nature of radish genome repetitive sequences contribute significantly to extraordinary genome size variation in higher plants. generally speaking, ltr-retrotransposons are the most abundant element of the genome, especially in big genome species, such as maize, wheat, and coix. however, the majority of repetitive sequences are satellites, which make up % of the radish genome in our study. a similar high proportion of satellites were found in c. rubella and cucumber, in which more than 20% of the genome sequences are satellite repeats. ordinarily, several to dozens of types of satellite repeats are detected from a number of species. in our study, only three satellite repeats were found in radish, including centromeric repeats, subtelomeric repeats and telomeric repeats. this is a typical pattern where the satellite dna sequences are appear predominantly in the centromeric, pericentromeric and telomeric regions. the dynamic evolutionary processes of satellite dna may generate changes in its chromosomal location and distribution. some satellite dna families were found to be species-specific, while others were more conserved, and similar sequences may be isolated in closely related species. in our study, we detected pairs of 45s rdna signals in early generations of the radish, the same result obtained by koo, while only pairs of 45s rdna signals were detected in later generations. it suggests that rdna also have a rapid evolution in the genome. furthermore radish inbred lines from different areas might contain varied ratio of repetitive sequences. % of the 0713d genome is repetitive dna in our study, while repetitive sequences occupied % of the japanese radish ‘aokubi’ genome.compositions of each type of repetitive elements are also different between these two radishes. overall, these results demonstrate the highly dynamic nature of radish genome. rapid evolution of centromere sequence the centromeres of higher eukaryotes are rich in repetitive dna sequences which include large arrays of satellite repeats and/or retrotransposon-related repetitive sequences. it has been shown that one single major satellite repeat is the dominating sequence in all centromeres in most diploid species. in our study, the similar pattern of one type of centromeric satellite repeat was detected by immunostaining and the chip test. however, it has been reported that some plant and animal species contain multiple satellite repeats associated with centromeres, such as in the common bean, potato, and chicken. centromeric satellite repeats diverge rapidly across species and often do not share any sequence similarity. several centromeric repeats were identified in potato and its closely related wide species s. verrucosum, respectively. nevertheless, only one single homoeologous centromeric sequence was detected between these two species. this means centromeric regions of solanum species show rapid evolution. taxonomic studies and rdna gene space sequence analysis demonstrated that genus brassica is a close relative of the genus raphanus. our results also proved this. in this study, a new satellite cl was detected, which is distributed in radish and all tested brassica species and located at the subtelomeric region of all tested species. even in closely related species, centromeric satellites go through rapid evolution. cl the centromeric satellite repeat, shares high similarities with centbr and centbr sequences. these centbr sequences appeared in the a and c genomes of brassica species, while the corresponding centromeric repeats have not yet been identified in the b genome. even in the same species, centbr and centbr have different distribution patterns on chromosomes. these results indicate that centromeric satellite repeats of cruciferae species evolve rapidly.  in this study, we used low-coverage sequencing on raphanus sativus l. to analyze repeat elements. we revealed the genome structure of radish and found that satellite repeats are most dominating elements, which is differ from most reported species, in which ltr-retrotransposons are the most abundant element of the genome. the fine karyotyping analysis using satellites and rdnas as markers allow us to easily identify each individual somatic metaphase chromosome. only one putative cenh gene was characterized and cloned from radish. its deduced amino acid sequence shares high similarities to those of the cenh3s in brassica species. in addition, immunofluorescence- and chip-based assays demonstrated the functional significance of satellite and centromere-specific retrotransposon at centromeres. our study provides a valuable basis for future genomic studies in radish. availability of supporting data the data sets supporting the results of this article are available in the ncbi sra archive. 
immunostaining and chromatin immunoprecipitation tests with anti-brcenh antibody demonstrated that both the centromere-specific retrotransposon and satellite repeat are directly associated with rscenh in radish. immunofluorescence- and chip-based assays demonstrated the functional significance of satellite and centromere-specific retrotransposon at centromeres. radish is a major root vegetable crop especially in eastern asia. radish root contains various nutritions which play an important role in strengthening immunity. repetitive elements are primary components of the genomic sequence and the most important factors in genome size variations in higher eukaryotes. to date, studies about repetitive elements of radish are still limited. to better understand genome structure of radish, we undertook a study to evaluate the proportion of repetitive elements and their distribution in radish. we conducted genome-wide characterization of repetitive elements in radish with low coverage genome sequencing followed by similarity-based cluster analysis. proportions of repetitive elements in radish were estimated and satellite repeats were the most dominating elements. results showed that about 31% of the genome was composed of repetitive sequences. our study provides a valuable basis for future genomic studies in radish. fish signals of two satellite repeats, cl and cl together with 5s rdna and 45s rdna, provide useful cytogenetic markers to identify each individual somatic metaphase chromosome. the distribution pattern of three satellite repeat sequences on radish chromosomes was characterized using fluorescence in situ hybridization. cl was predominantly located at the centromeric region of all chromosomes, cl located at the subtelomeric region, and cl was a telomeric satellite. its deduced amino acid sequence shares high similarities to those of the cenh3s in brassica species. satellite repeats were the most dominating elements of the genome. fine karyotyping analysis was established which allow us to easily identify each individual somatic metaphase chromosome.
 radish is a major root vegetable crop especially in eastern asia. radish root contains various nutritions which play an important role in strengthening immunity. repetitive elements are primary components of the genomic sequence and the most important factors in genome size variations in higher eukaryotes. to date, studies about repetitive elements of radish are still limited. to better understand genome structure of radish, we undertook a study to evaluate the proportion of repetitive elements and their distribution in radish.  we conducted genome-wide characterization of repetitive elements in radish with low coverage genome sequencing followed by similarity-based cluster analysis. results showed that about 31% of the genome was composed of repetitive sequences. satellite repeats were the most dominating elements of the genome. the distribution pattern of three satellite repeat sequences on radish chromosomes was characterized using fluorescence in situ hybridization. cl was predominantly located at the centromeric region of all chromosomes, cl located at the subtelomeric region, and cl was a telomeric satellite. fish signals of two satellite repeats, cl and cl together with 5s rdna and 45s rdna, provide useful cytogenetic markers to identify each individual somatic metaphase chromosome. the centromere-specific histone h has been used as a marker to identify centromere dna sequences. one putative cenh was characterized and cloned from radish. its deduced amino acid sequence shares high similarities to those of the cenh3s in brassica species. an antibody against b. rapa cenh specifically stained radish centromeres. immunostaining and chromatin immunoprecipitation tests with anti-brcenh antibody demonstrated that both the centromere-specific retrotransposon and satellite repeat are directly associated with rscenh in radish.  proportions of repetitive elements in radish were estimated and satellite repeats were the most dominating elements. fine karyotyping analysis was established which allow us to easily identify each individual somatic metaphase chromosome. immunofluorescence- and chip-based assays demonstrated the functional significance of satellite and centromere-specific retrotransposon at centromeres. our study provides a valuable basis for future genomic studies in radish. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords radishrepetitive dnasatellitekaryotypingcenh3centromereissue-copyright-statement© the author 2015 