Chromosome characterization in Thinopyrum ponticum ( Triticeae , Poaceae ) using in situ hybridization with different DNA sequences

Thinopyrum ponticum (2n = 10x = 70, JJJJJ) belongs to the Triticeae tribe, and is currently used as a source of pathogen resistance genes in wheat breeding. In order to characterize its chromosomes, the number and position of 45S and 5S rDNA sites, as well as the distribution of the repetitive DNA sequences pAs1 and pSc119.2, were identified by fluorescent in situ hybridization. The number of nucleoli and NORs was also recorded after silver nitrate staining. Seventeen 45S and twenty 5S rDNA sites were observed on the short arms of 17 chromosomes, the 45S rDNA was always located terminally. On three other chromosomes, only the 5S rDNA site was observed. Silver staining revealed a high number of Ag-NORs (14 to 17) on metaphase chromosomes, whereas on interphase nuclei there was a large variation in number of nucleoli (one to 15), most of them (82.8%) ranging between four and nine. The pAs1 probe hybridized to the terminal region of both arms of all 70 chromosomes. In addition, a disperse labeling was observed throughout the chromosomes, except in centromeric and most pericentromeric regions. When the pSc119.2 sequence was used as a probe, terminal labeling was observed on the short arms of 17 chromosomes and on the long arms of five others. The relative position of 45S and 5S rDNA sites, together with the hybridization pattern of pAs1 and pSc119.2 probes, should allow whole chromosomes or chromosome segments of Th. ponticum to be identified in inbred lines of wheat x Th. ponticum.

Due to the importance of Th. ponticum genes in wheat breeding, cytological markers associated with chromosomes or chromosome segments have been used in gene introgression programs.The use of fluorescent in situ hybridization (FISH) has made it possible to locate several specific DNA sequences throughout the chromosomes of various species of Triticeae, allowing alien chromatin to be identified in interspecific hybrids.Probes corresponding to multigenes, such as 45S and 5S rDNA, and highly repetitive DNA sequences, are the most commonly used markers of specific chromosomes or genome regions.For example, in Triticum aestivum L., Mukai et al. (1990;1991) labeled five chromosome pairs with 45S rDNA sites (1AS, 1BS, 6BS, 5DS, 7DL) and six pairs with 5S rDNA sites (1AS, 1BS, 1DS, 5AS, 5BS e 5DS).The pAs1 probe, originally obtained from Aegilops tauschii Coss (D genome), allowed the D genome chromosomes (Rayburn and Gill, 1986) to be identified, along with small sites on chromosomes 1A, 4A, 1B, 3B, 6B and 7B in wheat (Mukai et al., 1993).Similarly, the pSc119.2probe from Secale cereale L. (R genome) distinguished all B genome chromosomes and 4A, 5A, 2D, 3D, 4D e 5D chromosomes.The simultaneous use of these two probes allowed 17 out of 21 chromosome pairs of T. aestivum to be identified (Mukai et al., 1993).More recently, Cuadrado et al. (2000) characterized the seven pairs of the B genome and some chromosomes of the A and D genomes of T. aestivum using two simple sequence repeats (SSR), (AAC) 5 and (AAG) 5 .
Besides the FISH, another procedure used to characterize chromosomes has been silver staining, which was developed to detect nucleoli in interphase cells and nucleolus organizer regions (NORs) in metaphase chromosomes (Fernandez-Gomez et al., 1969;Hizume et al., 1980).The activity of 45S rDNA genes is usually associated with NORs and secondary constrictions, and has been studied at cytological level by recording the number and volume of nucleoli in interphase cells and NORs in metaphase chromosomes by silver staining (Martini and Flavell, 1985;Vieira et al., 1990;Lima-Brito et al., 1998).
In the genus Thinopyrum, the localization of rDNA sites has been reported in some species.In Th. distichum (Thunb.)Á. Löve (2n = 4x = 28, JJ) 1 , two 45S rDNA sites have been located in homeologous groups 5 and 6, and two 5S rDNA sites have been found in homoeologous group 5 and in a non-defined group (Fominaya et al., 1997).In Th. elongatum (Host) D. R. Dewey [= Agropyron elongatum (Host) P. Beauv., Elytrigia elongata (Host) Nevski, Lophopyrum elongatum (Host) Á. Löve] (2n = 2x = 14, E), the 5S rDNA sites were associated with chromosomes 1E and, possibly, 5E (Dvorák et al., 1989).Similarly to related diploid species, the decaploid Th. ponticum has two 45S rDNA loci per monoploid genome, in all a total of 20 sites (Li and Zhang, 2002).Highly repetitive DNA sequences, such as pSc119.2cloned from rye (Lapitan et al., 1987) and pLeUCD2 of Th. elongatum (Zhang and Dvorák, 1990;Kim et al., 1993), have been isolated and used in the characterization of Thinopyrum chromosomes and for the identification of introgressed alien chromatin in wheat x Thinopyrum hybrids.In the present study, FISH was used to identify the number and position of 45S and 5S rDNA sequences, as well as the distribution of the highly repetitive DNA sequences pAs1 and pSc119.2 in the Th.ponticum genome.The number of nucleoli and NORs was also recorded to elucidate whether all 45S rDNA loci were active in this allopolyploid.

Plant material and chromosome preparation
The clone of Th. ponticum used in this study was the PF Ag. el.84001 accession, maintained under cultivation at Embrapa Trigo, Passo Fundo (Rio Grande do Sul, Brazil).
Root tips were pretreated in ice-cold water for 24 h, fixed in ethanol: acetic acid (3:1, v/v) and then stored at -20 °C.The material was digested in an enzyme mixture containing 2% (w/v) cellulase (Onozuka R10) and 20% (v/v) pectinase (Sigma) for 2 to 3 h at 37 °C.Afterward, each root tip was squashed in a drop of 45% acetic acid and frozen in liquid nitrogen.

Fluorescent in situ hybridization (FISH)
FISH was performed essentially as described by Heslop-Harrison et al. (1991) at 85% stringency.No immunocytochemistry was necessary to detect the direct rhodamine labeling.Biotin-labeled probes were detected using avidin-rhodamine conjugate (Vector), while digoxigenin-labeled probes were detected using antidigoxigenin-fluorescein isotiocyanate (FITC) conjugate (Roche).All preparations were counterstained with 2 µg/mL 4', 6-diamidino-2-phenylindole (DAPI) and mounted in Vectashield H-1000 (Vector).Photographs were taken on Fujicolor Super HG or Kodak Ultra 400 print film, and scanned at 300 dpi.The images were optimized for best contrast and brightness with Adobe Photoshop 6.0.

Silver staining
The silver staining technique employed was that described by Vieira et al. (1990).After cold treatment, roots were fixed in 50% ethanol, glacial acetic acid and 37% formaldehyde (18:1:1, v/v/v) for a minimum of 4 h at room temperature and a maximum of three days at 0 °C to 2 °C.Fixed roots were washed several times in distilled water to eliminate any residues of fixative.Once clean, they were immersed overnight in an aqueous solution of 20% AgNO 3 at 60 °C.Residual silver was eliminated by washing with distilled water and the staining developed with 1% hydroquinone and 10% formaldehyde (1:1, w/v) for one to 10 min, depending on the intensity required.After a further wash with distilled water, the root tips were squashed in 45% acetic acid containing a drop of 1% carmine, which aids viewing in normal light microscopy.
The number of nucleoli was recorded in 1,064 interphase nuclei and the number of active NORs was ob-served in 42 metaphases.Best cells were photographed on Kodak Imagelink ASA 25 print film, and scanned at 300 dpi.The images were optimized for best contrast and brightness with Adobe Photoshop 6.0.

In situ hybridization
Both simultaneous and sequential FISH with a twoby-two combination of probes were used to study the metaphase chromosomes of Th. ponticum.The 5S and the 45S rDNA genes were located simultaneously using pTa794 and pTa71 probes, respectively.For both sequences, minimal differences of intensities were detected between loci.Twenty 5S rDNA sites were observed at interstitial position in the short arms of 20 chromosomes.Seventeen 45S rDNA were located at a terminal position in the short arms of 17 out of the 20 chromosomes carrying the 5S rDNA, on a double labeling pattern (Figures 1a, b).Considering the number of rDNA sites, as well as the ploidy level (2n = 10x = 70) and the chromosome morphology of Th. ponticum, double labeling seemed to be restricted to chromosomes of two homoeologous groups, both submetacentrics.
The pAs1 probe produced terminal labels on both arms of all 70 chromosomes.In addition, dispersed labeling was observed throughout the chromosomes, except for centromeric and most pericentromeric regions (Figure 1c).The pSc119.2 probe produced labeling on terminal positions of the short arms of 17 chromosomes.Two of them, with different morphologies, also showed telomeric labels on the long arm.In addition, pSc119.2labels were observed on the long arm of three chromosomes with 45S rDNA sites (Figure 1d).

Silver staining
An attempt was made to analyze the activity of different NORs in cells undergoing interphase to metaphase.Silver staining revealed a high number of Ag-NOR sites (14 to 17).Seventeen were observed in 76% of the 46 metaphases studied (Figure 2a).However, in the 1,064 interphase nuclei studied, the number of nucleoli varied from one to 15, most (82.8%)showing four to nine nucleoli (Figure 2b).

Ribosomal genes
The species of the Triticeae tribe vary greatly in number of 45S ribosomal DNA sites.For example, Ae. comosa Sibth.& Sm.ssp.comosa shows nine pairs of rDNA loci distributed among their seven chromosome pairs, whereas S. cereale has only one pair of these loci (Appels et al., 1980).Nevertheless, the main 45S rDNA loci occur preferentially in the homoeologous groups 1, 5 and 6 (Lacadena et al., 1988;Badaeva et al., 1996).In Th. elongatum (2n = 2x = 14, E) and in Th. distichum (2n = 4x = 28, JJ), the 45S rDNA loci are located on the short arms of two chromosome pairs named as 5 and 6 (Dvorák et al., 1984;Fominaya et al., 1997).In the Th.ponticum analyzed, the 45S rDNA sites were located in a similar position in 17 chromosomes, suggesting that, except in three chromosomes, the ribosomal genes are present in two chromosome pairs of each one of the five genomes (JJJJ s J s ).The occurrence of these sites in 17 instead of 20 chromosomes, as observed by Li and Zhang (2002), most likely indicates a reduction in the number of repeats or a deletion of sites from this accession.A similar reduction has already been described in other polyploids (Linde-Laursen et al., 1992) and it may be more frequent in higher polyploids.In common wheat, the elimination of DNA sequences is one of the main responses to allopolyploidization, occurring after the formation of the polyploid and involving a large fraction of the genome, including 45S rDNA repetitive units (Shaked et al., 2001).For example, in the chromosome 1AS of T. monococcum L., the number of repetitive units of 45S rDNA was largely reduced after its incorporation into the polyploid wheats (Jiang and Gill, 1994).In addition, the terminal position of this locus in Th. ponticum might facilitate its partial or total elimination in some sites (Pestsova et al., 1998).
The number of Ag-NOR sites of Th. ponticum revealed after silver staining was generally similar to that of 45S rDNA labeled after FISH (17).However, the mean number of nucleoli per nucleus was much lower.This indicates that all or nearly all rDNA sites are necessary for nor-mal cell function.At interphase, the Ag-NOR sites tend to coalesce, as suggested by Lacadena et al. (1988).
In Triticeae, the 5S rDNA sites consist of a 120 bp coding sequence alternating with non-transcribed spacers, which are classified into two types according to their length and sequence (Gerlach and Dyer, 1980).In most genomes the sites with short spacers are located in homeologous group 1, whereas those with long spacers are located in group 5 (Reddy and Appels, 1989;Dvorák et al., 1989).In Th. elongatum, the presence of 5S rDNA in the short arms of chromosomes of the homeologous group 1 and probably 5 has been suggested (Scoles et al., 1988;Dvorák et al., 1989).In Th. ponticum, we found two 5S rDNA sites per haploid genome.Considering the distribution of these sites in other species, and the proximity of these sites to the 45S rDNA sites, it is tempting to conclude that these chromosomes belong to homoeologous groups 1 and 5.

pSc119.2 and pAs1 repetitive sequences
The sequence pSc119.2from S. cereale has been widely used in FISH experiments due to its high copy number and polymorphic location in many species of the Triticeae.Lapitan et al. (1987), studying the distribution of this sequence in three species of Thinopyrum, found 10 terminal and only one subterminal site in Th. bessarabicum (Savul & Rayss) Á. Löve (2n = 2x = 14, J genome).In Th. elongatum (2n = 2x = 14, E genome), these authors found 10 terminal sites plus other sites distributed throughout the entire length of all chromosomes, except in the telomeres of one chromosome.On the other hand, in the hexaploid Th. intermedium (Host) Barkworth & D.R. Dewey (JJ s St), the pSc119.2sequence hybridized to a few chromosomes.Similarly, in the decaploid Th. ponticum studied in the present work, a reduction in the number of pSc119.2sites was found, when compared to those of the two diploid species referred to above.
The pAs1 probe has also been widely used in the Triticeae.In T. aestivum, the identification of all chromosomes was possible when this probe was associated with a GAA-rich satellite sequence.The authors observed 48 pAs1 bands (Pedersen and Langridge, 1997) and most large sites were located on the terminal region of D genome chromosomes, as described by Rayburn and Gill (1986).In fact, most large blocks of tandem repetitive sequences are usually located in a similar position in all the chromosomes of a given species.It is possible that these chromosome regions favor or show greater tolerance to the presence of such tandem repetitions (Flavell, 1982).In the present study, Th. ponticum showed terminal bands on both arms of the 70 chromosomes.Another characteristic of this probe in Th. ponticum was the disperse labeling throughout all the chromosomes, except in the centromeric and most of the pericentromeric regions.This diffuse labeling in Th. ponticum, unlike that found in T aestivum, makes this sequence an important tool for chromosome differentiation in T. aestivum x Th. ponticum hybrids.
These data show that the number and position of the sites revealed by the probes pTa 71, pTa794, pAs1 and pSc119.2are valuable markers to characterize and identify the chromosomes of Th. ponticum in inbred lines of wheat x Th. ponticum.

Chromosome characterization in Thinopyrum ponticum 507 Figure 1 -
Figure 1 -Location of specific sites in the chromosomes of Thinopyrum ponticum.a. Complete metaphase with 2n = 70 DAPI stained chromosomes.b.The same cell showing seventeen 45S rDNA sites (red) and 20 sites of 5S rDNA (green).c.Reprobing of the same cell revealing the distribution of the highly repetitive DNA sequence pAs1.d.Partial prometaphase displaying 15 sites of 45S rDNA (red) and the terminal locating of the pSc119.2probe (green).Bars = 10 µm.

Figure 2 -
Figure 2 -Analysis of NOR activity, using the silver-staining procedure.a. Partial metaphase showing the maximum number of Ag-NORs found in a single cell (17 sites).b.Interphase nuclei with six nucleoli.Bar = 10 µm.