By comparative multicolor FISH, we have physically mapped small chromosome fragments in the sugar beet addition lines PRO1 and PAT2 and analyzed the distribution of repetitive DNA families in species of the section
The characterization of the genome architecture of higher plants is an important scientific task. One of the most unequivocal approaches to reach this aim is to visualize distinctive chromosomal domains directly by fluorescent-in situ-hybridization (FISH). This method is of supreme efficiency to reveal the physical organization of DNA on plant chromosomes at high resolution. It allows the detection and precise localization of repetitive or single-copy sequences on interphase nuclei, chromosomes in mitosis and meiosis or chromatin fibers. After the first application in wheat [
A large portion of plant genomes accounts for repetitive DNA [
Centromeres are essential functional domains of plant chromosomes. They are detectable as primary constrictions or heterochromatic blocks and are responsible for the segregation of the sister chromatids during cell division. The centromere composition was analyzed to different extent in yeast,
The proteins interacting with plant centromere have also attracted attention [
Eukaryotic chromosomes are terminated by specific nucleoprotein complexes—the telomeres. They are important domains responsible for the maintaining of genome stability. Telomeres permit cells to distinguish chromosome ends from double-strand breaks, thus preventing chromosome degradation and fusion [
In this paper, we analyzed the physical organization of the small wild beet chromosome fragments in the two sugar beet mutant lines, PRO1 and PAT2, by multicolor FISH. Pachytene-FISH on meiotic chromosomes was applied to resolve the structure of the wild beet centromeres. Modification of the proteins in the active kinetochore on
Plants were grown under greenhouse conditions. The following wild beet species were included in this study:
Mitotic chromosomes were prepared from the meristem of young plants according to Schwarzacher and Heslop-Harrison [
Meiotic chromosomes were prepared from anthers by a squashing method [
A set of six repetitive DNA probes representing
The clone pLt11 consisting of TTTAGGG repeats was used as a telomeric probe [
Cloned probes shorter than 3 kb were labeled with biotin-16-dUTP or digoxigenin-11-dUTP by PCR using universal primers. The rDNA probe and the telomeric probe pLt11 were labeled with DIG- or BIO-Nick Translation kits (Roche) following the manufacturer’s instructions.
Fluorescent in situ hybridization (FISH) was performed according to Heslop-Harrison et al
Immunostaining was performed according to Houben et al. [
Examination of slides was carried out with a Zeiss Axioplan2 fluorescence microscope equipped with Filter 01 (DAPI), Filter 02 (Cy3), Filter 09 (FITC), and Filter 25 (DAPI/Cy3/FITC). Photographs were taken on Fujicolor SUPERIA 400 colour print film and negative films were digitized on a Nikon LS-1000 scanner. Alternatively, the images were acquired directly with the Applied Spectral Imaging v. 3.3 coupled with the high-resolution CCD camera ASI BV300-20A.
Immunostaining images were acquired directly with a cooled CCD camera. After three-dimensional deconvolution, the resulting data subsets were merged through the
The images were contrast optimized using only functions affecting the whole image equally and printed using Adobe Photoshop 7.0 software.
To analyze the physical organization of the chromosome fragments in the addition lines PRO1 and PAT2, multicolor FISH with
Repetitive probes used for the characterization of the fragment addition lines PRO1 and PAT2.
Probe | Origin | Length, bp | Sequence type | Accession | Reference |
---|---|---|---|---|---|
Satellite | |||||
pTS4.1 | 312 | Z50808 | Schmidt et al. 1990 [ | ||
pTS5 | 153–160 | Z50809 | Schmidt and Heslop-Harrison 1996 [ | ||
pRp34 | 352–358 | AM076755 | Dechyeva and Schmidt 2006 [ | ||
Dispersed | |||||
pAp4 | 1353-1354 | AJ414552 | Dechyeva et al. 2003 [ | ||
Telomere | |||||
pLT11 | not tested | telomeric repeat | not entered | Vershinin et al. 1995 [ | |
Ribosomal genes | |||||
pTa71 | 4642 | 25S-18S gene fragment with spacer | X07841 | Barker et al. 1988 [ |
The in situ hybridization of
Blue fluorescence in each panel shows the chromosomes stained with DAPI. The scale bar in (G) for the panels A-G and in (J) for the panels I and J represents 10
In PRO1, both satellites are detectable only on the chromosomal fragment (Figure
The two centromeric satellites pTS5 and pTS4.1 were hybridized simultaneously to the tetraploid
In PAT2, the
To achieve a higher resolution of the physical organization of the two centromeric satellites, a double-target in situ hybridization with pTS4.1 and pTS5 was performed on meiotic chromosomes of
The hybridization of a
Blue fluorescence in each panel shows the chromosomes stained with DAPI. The scale bar for left and central panels in (Q) represents 10
On the PRO1 metaphase spread, the telomeric DNA was detectable on all sugar beet chromosomes as well as on both ends of the chromosome fragment (Figure
The telomeric probe pLT11 labeled the ends of all
As expected, all PAT2 chromosomes demonstrated telomeric signals (Figure
The localization of the telomeric sequences on the chromosome fragments was complemented by FISH with the subtelomeric satellite pRp34. The probe labeled all but two chromosomal ends of the wild beet
The subtelomeric probe pRp34 was detected on one or both arms of all except two
The
The dispersed repeat pAp4 was scattered over all
To get an insight into the structure and function of the kinetochore, the proteins characteristic for active centromeres were observed on metaphase preparations of
Mitotic beet cells were immunostained with polyclonal antibody against serine 10-phosphorylated histone H3 raised in rabbit [
Proteins in immunostaining probes should preserve their structure resulting in a three-dimensional shape of the nuclei. Such preparations cannot be successfully analyzed by conventional epifluorescence microscopy. Consequently, computational deconvolution microscopy was applied. The immunostaining with the antibody against histone H3 phosphorylated at serine 10 demonstrated that the centromeric histones of
Sugar beet is an important agricultural crop, and the results of genome research in this species might be important to the practical implementation in green biotechnology. Currently, a fine-resolution physical map is under construction and a genome-sequencing project is carried out in the framework GABI–Genome Analysis in Biological System Plant (
Hybridization of
Ribosomal DNA genes in eukaryotes are tandemly arranged in thousands of copies. They reside at the chromosomal loci known as nucleolus organizer regions (NORs) [
The addition line PRO1 [
Previous analysis of the long-range organization of centromeres in the wild beet
To reveal the possible origin of the PRO1 and PAT2 chromosomal fragments, the organization of the centromere-specific satellites pTS4.1 and pTS5 was studied in detail. Hybridization of pTS5 and pTS4.1 on chromosomes of
In PRO1, pTS5 labels one end of the acrocentric fragment, bordered by adjacent pTS4.1 array from one side only (Figure
The telomeres protect the chromosome ends from degradation. Both fragment addition lines PRO1 and PAT2 arose spontaneously in the offspring of
Thus, it was important to find out whether PRO1 and PAT2 chromosome fragments indeed possess telomeres ensuring their stability. Therefore, the next probes tested on the sugar beet hybrids PRO1 and PAT2 were those located at the chromosome ends: the
Additional signals of the subtelomeric satellite pRp34-179 on sugar beet chromosomes (Figures
The presence of the dispersed repetitive family pAp4 specific for the
The allocation of repetitive probes on PRO1 and PAT2 by FISH enabled to propose origins and to develop physical models of the chromosome fragments (Figure
Structural model of the PRO1 and PAT2 chromosomal fragments. Both chromosome fragments are represented according to the distribution patterns of the repetitive DNA sequences mapped by FISH.
The experiments performed in this study demonstrated that the PRO1 fragment is acrocentric. The size of the centromeric pTS5 satellite array has been estimated by fiber FISH to be 115 kb [
There is no conserved DNA sequence responsible for the centromeric function in higher plants [
An important step during the formation of a functional kinetochore is the phosphorylation of the pericentromeric histone H3 [
The fluorescent immunostaining of centromere-associated proteins in the fragment addition line PRO1 allowed the comparison of the histone H3 phosphorylation patterns of
The immunostaining experiment with the antibodies against serine 10-phosphorylated histone H3 and
The authors are grateful to Andreas Houben for the opportunity to perform immunostaining and to Cordula John for assisting with the part of the FISH experiments. This work was funded by BMBF (