The pachytene karyotype
Pollen mother cells at late pachytene clearly displayed eight fully paired bivalents with lengths varying from 29 to 68 µm and a total complement length of 406 µm. Chromo somes were submetacentric or metacentric, with centromere indexes between 27 and 47% (Figure 1a; Table 1). They were numbered according to their corresponding linkage maps, as decided during the Second Medicago truncatula Workshop (Amsterdam, The Netherlands, July 22–23 1999), with their two arms denoted as S (short) and L (long), respectively. DAPI staining of pachytene chromosomes demonstrated striking differences in chromatin density. Brightly fluorescing heterochromatic blocks were detected in the pericentromeric regions of all chromosomes, although their lengths were different for each chromosome (Figure 1a; Table 1). The centromere itself appeared as a primary constriction in which the chromatin fluoresces far more weakly than the flanking heterochromatic regions (Figure 1a). Distal regions of the chromosome arms generally consist of weakly fluorescing euchromatin. In addition to the pericentromeric heterochromatin, smaller heterochromatic knobs were observed on the short arms of chromosomes 3, 4 and 7, and both arms of chromosome 6, but their number was variable and could be observed in few cells only. The total length of all heterochromatic areas is 59.3 µm, about 15% of the length of the complement.
Figure 1. Pachytene chromosomes of Medicago truncatula Jemalong A17.
(a) The complement of pachytene chromosomes. DAPI-stained chromo somes have brightly fluorescent heterochromatin around centromeres (pericentromeric heterochromatin). Chromosomes are numbered according to corresponding linkage groups and indicated by arrowheads at centromere position.
(b) Three chromosomes (3, 5, 6) give FISH signals with a 5S rDNA probe (red). Centromeres of the chromosomes containing a 5S rDNA region are indicated by arrowheads. Chromosome 5, carrying the major 5S rDNA cluster, also contains the 45 rDNA region (green).
(c) Hybridization with 5S rDNA (red) and MtR1 (green) allows identification of all eight pachytene bivalents (see a). Centromeres are indicated by arrowheads.
(d) Three 5S rDNA-carrying chromosomes are dissected from the complement.
Bar = 5 µm in all figures.
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Table 1. Absolute and relative lengths of individual chromosomes and chromosome regions, positions of 5S rDNA, NOR and MtR1 on chromosomes
|Average lengthb||60.3 ± 6.2||49.3 ± 4.1||68.1 ± 5.9||66.0 ± 4.3||49.5 ± 5.2||29.2 ± 3.8||50.4 ± 5.1||33.4 ± 5.4||406.2|
|Total cell complementc||14.9||12.1||16.8||16.2||12.2||7.2||12.4||8.2||100%|
|Centromere indexd||36.4||46.2||27.1||30.2||47.4||46.3||30.0||41.2|| |
|5S rDNA||–||L||–||–||S||S||–||–|| |
|45S rDNA||–||–||–||–||L||–||–||–|| |
|MtR1||L||L||–||S||–||–||L + S||L|| |
Chromosome identification was based on their length, centromere position, size of pericentromeric heterochromatin, and the presence of diagnostic heterochromatic knobs. Eight cells were selected in which all eight bivalents could be discerned and fully traced along their length. Based on this morphometric characterization, the chromosomes could be distinguished as follows (Table 1).
Group of three longest chromosomes (1, 3, 4). These chromosomes measure 60–68 µm and have submedian centromere positions with centromere index (CI) values of 36, 27 and 30%, respectively. Based on centromere position, chromosome 1 can be distinguished from chromosomes 3 and 4. Chromosomes 3 and 4 have similar symmetrical heterochromatic regions, and differ only slightly in arm lengths and centromere positions. Distinction between them without additional diagnostic markers was therefore doubtful.
Group of three medium-sized chromosomes (2, 5, 7). These chromosomes measure approximately 50 µm. The former two have median centromere positions (centromere indexes of 47 and 46%, respectively), whereas chromosome 7 has a submedian centromere (CI = 30%). Furthermore, chromosome 5 has a characteristic pattern of four conspicuous pericentromeric heterochromatic knobs. In contrast, chromosomes 2 and 7 have only one knob on each arm. Furthermore, chromosome 5 contains the secondary constriction (nucleolar organizer region). This weakly fluorescing region is located on the long arm, close to the centromere, and is often clumped together with heterochromatic blocks of other chromosomes.
Group of two smallest chromosomes (6, 8). These chromosomes are 29 and 33 µm long, respectively, and can easily be distinguished by differences in chromatic patterns. Chromosome 6 has several heterochromatic chromomeres on both arms, whereas chromosome 8 has two larger heterochromatic blocks on either side of the centromere.
The total length of the pachytene chromosomes is 406 µm, which is about 20 times longer than that of the mitotic metaphase chromosomes (data not shown). The morphological features of the eight bivalents have been used to construct an ideogram (Figure 2a). We selected the 5S rDNA, 45S rDNA and the MtR1 tandem repeats as additional diagnostic markers to facilitate the identification of the individual chromosomes in the cell complement. FISH hybridization revealed that the 5S rDNA loci are located on chromosomes 2, 5 and 6 (Figure 1b,d). A major 5S rDNA region occurs on the distal part of the pericentromeric heterochromatin of chromosome 5, on the arm containing a single heterochromatic knob. A second, smaller 5S rDNA region is located on the long arm of chromosome 2, close to the border of the pericentromeric heterochromatin. A third 5S rDNA site is present on chromosome 6, 17% of the arm length distally from the centromere. FISH with the 45S rDNA probe demonstrated a bright spot on the secondary constriction of chromosome 5, between two proximal heterochromatic knobs (Figure 1b,d). The same number of 5S and 45S rDNA loci were observed by FISH studies on metaphase chromosomes (Gerbah et al., 1999). The MtR 1 tandem repeat has a 166 bp motif, was identified in two randomly isolated BAC clones, BAC75N01 and BAC53F10 (Table 2), and will be described elsewhere in more detail (O. Kulikova, T. Huguet, H. de Jong and T. Bisseling, unpublished results). This repeat is located in the pericentromeric regions of the chromosome arms 1-L, 2-L, 4-S, 7-S and 7-L, and 8-L (Figure 1c). The MtR1 signal on 8-L is weaker than that of the other MtR1 signals. Thus MtR1 is a good marker to distinguish chromosomes 3 and 4.
Figure 2. Correlation between chromosomes and linkage groups.
(a) Ideogram of pachytene chromosomes and genetic linkage maps of Medicago truncatula Jemalong A17. BAC clones are positioned on the ideogram according to their relative positions in relation to centromeres.
(b) Assignment of linkage group to pachytene chromosomes by FISH with linkage group-specific BAC clones. Chromosomes are digitally sorted out of pachytene complements after hybridization with BAC clones, indicated in bold on the ideogram. For identification of chromosomes, pachytene preparations were reprobed with 5S rDNA (red) and MtR1 (green). Centromeres are indicated by arrowheads.
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Table 2. Characteristics of BAC clones and DNA markers
|BAC clone||Linkage group||Genetic marker||Marker template accession||Marker type and homology of marker template hspa||Distanceb from centromere ± SD|
|75N01|| || ||AQ841077||BESTc; pericentromeric repeat MtR1|| |
|53F10|| || ||AQ841071||BEST ‘–’|| |
|53F10|| || ||AQ841072||BEST ‘–’|| |
|72H13||1||DK049R||AQ841103||BEST; putative beta-fructofuranosidase||18.3 ± 1.2|
|19N23||1||ENOD8||n/a||BEST; BAC contains ENOD8 gene||52.0 ± 2.5|
|69K12||2||DK020R||AQ841084||BEST; similar to putative Arabidopsis proteinase||45.0 ± 2.2|
|51J12||2||DK045R||AQ841099||BEST; no known homology for marker template||90.1 ± 1.4|
|54F15||3||DK123R||AQ841744||BEST; homology to Arabidopsis hypothetical protein Z97335. BAC 54F15 survey sequencing reveals homology to multiple genesd||57.2 ± 1.1|
|33E24||3||DK417L||AQ917383||BEST; similar to NBS-LRR disease resistance protein (AB019186) RPR1 of Oryza sativa||16.2 ± 0.1|
|10F20||4||DK043R||AQ841087||BEST; no known homology for marker template. BAC 10F20 survey sequencing reveals homology to multiple genes||26.3 ± 1.0|
|01P05||4||DK264L||AQ917083||BEST; no known homology for marker template. BAC 01P05 contains putative MYB family transcription factor||43.7 ± 1.2|
|64B21||5||EIL2-1||n/a||BEST; BAC 64B21 is contiguous with BAC 42H09 from which survey sequencing reveals homology to multiple genes, including Arabidopsis ein3-like family||81.0 ± 1.0|
|23A06||5||ENOD40||n/a||PCR marker 3′′ of ENOD40 coding region. BAC 23A06 contains ENOD40 gene and putative receptor protein kinase||40.1 ± 1.6|
|35O12||5||DK139L||AQ841733||BEST; no known homology for marker template. BAC 35012 survey sequencing reveals homology to multiple genes||64.2 ± 1.1|
|58F01||5||DK006R||AQ841074||BEST; no known homology for marker template|| |
|45I09||5||DK039R||AQ841114||BEST; no known homology for marker template. BAC45I09 survey sequencing reveals homology to Mt ESTs|| |
|19L20||6||DK125R||AQ841732||BEST; similar to beta-transducin. BAC 19L20 contains Medicago truncatula cycloartenol synthase gene (Y15366.1)||100.0 ± 0.0|
|73B09||6||DK229L||AQ917196||BEST; homology to tomato callus EST AW029689||83.1 ± 1.0|
|79P21||6||79P21R||n/a||BEST; BAC contains homology to LBS-LRR-TIR family of resistance genes||89.3 ± 2.1|
|37M01||7||DK427R||AQ917398||BEST; no known homology for marker template||57.3 ± 1.1|
|03L06||7||DK274L||AQ917096||BEST; no known homology for marker template||44.8 ± 1.1|
|05K15||8||DK505R||AQ917527||BEST; similar to peptide transporter. BAC contains homology to Arabidopsis genomic DNA by tblastX||37.3 ± 0.1|
|41H08||8||DK455L||AQ917442||BEST; similar to Arabidopsis hypothetical protein. BAC 41H08 contains Medicago truncatula EST AW775698||53.1 ± 0.1|
Together, as shown in Figure 2a and Table 1, the karyotype analysis of the pachytene chromosome morphology and FISH patterns of the 5S rDNA, 45S rDNA, and MtR1 repeats allow identification of all eight chromosomes.
Integration of cytogenetic map and linkage groups
The numbering convention for the eight genetically identified linkage groups of M. truncatula was adopted from M. sativa (Kalóet al., 2000) as determined by comparative map analysis (D.-J. Kim and D.R. Cook, unpublished results). For assigning individual linkage groups to the chromosomes, we selected BACs of each linkage group and used these as probes for FISH mapping to the pachytene chromosomes (Table 2). Concurrent hybridizations with the 5S rDNA and MtR1 repeats were used to assist chromosome identification. Only FISH signals that occurred in at least 90% of the cell complements were considered for quantitative analysis. Examples of representative FISH patterns are shown in Figure 2(b). We measured the distance of the FISH signal in a relative scale from centromere to telomere in seven to ten cells (Table 2). Their distance values were averaged for drawing their positions on the cytogenetic map (Figure 2a).
To provide a frame of reference to the genetic map of M. truncatula, five or more sequence-characterized genetic markers are indicated for each linkage group (Figure 2a). These markers were developed on the basis of BAC end-sequence information (see Experimental procedures). Two to five BAC clones were used for mapping on pachytene chromosomes. Figure 2a shows their genetic map position along with their corresponding position on the pachytene FISH map. Detailed information on individual markers is given in Table 2 and at http://chrysie.tamu.edu/perl-bin/mt_marker_query.2.pl.
As the distal parts of most chromosomes are euchromatic, it is expected that the genetic and FISH map distances would be directly correlated. Table 3 gives a comparison of genetic map distances between six marker pairs and their corresponding microscopic distances on the pachytene FISH map. The ratio of genetic and cytogenetic distance values ranges from 0.9 to 1.9 cm µm−1 for most marker pairs. However, this value is markedly higher (3.0–3.1 cM µm−1) for the pairs located on the small chromosomes 6 and 8.
Table 3. Comparison of genetic and cytogenetic distances between neighbouring BAC clone pairs
|Chromosome||BAC clones||Genetic distance (cM)||Microscopic distance (µm)||cM µm−1|
Resolution of FISH mapping
The BAC clones 58F01 and 59K07 from a BAC contig of chromosome 5 (G. Gualtieri, R. Geurts and T. Bisseling, unpublished results) were selected for estimating the resolution of FISH mapping in a euchromatic region of a pachytene chromosome 5 (Figure 2a). The BACs were hybridized and detected with digoxigenin-FITC (green signal) and biotin-Texas Red (red signal), respectively (Figure 3a,b). A small band of yellow fluorescence in between the red and green spots represents the region of signal overlap. The mid-points of the sequences covered by these BACs are separated by 150 kb. This corresponds to a microscopic distance of approximately 0.5 µm between the centres of the red and green spots on the pachytene chromosome, implying a chromatin density of 300 kb µm−1 for that euchromatin region. With the spatial resolution limit of 0.2 µm for the fluorescence microscope, mapping resolution in this chromosome segment can be estimated at about 60 kb. This is confirmed by a comparable FISH experiment with the BAC clones 45I09 and 63C24 located in the same region of chromosome 5 (Figure 2a). These BACs are about the same size, approximately 40 kb, and are separated by about 55 kb (Figure 3c,d). Fluorescence microscopic observation revealed a prominent yellow spot flanked by small green and red regions, confirming that mapping resolution of adjacent targets in this region is about 60 kb.
Figure 3. Chromatin condensation degree in euchromatic region of chromosome 5.
(a,c)Two-colour FISH with two pairs of BAC clones, BAC59K07 (red) and BAC58F01 (green); BAC63C24 (red) and BAC45I09 (green).
(b,d)Tenfold magnified images (insets in a,d). Yellow fluorescence indicates co-localization of green and red signals. White dashed line indicates distance between BAC pairs in their physical contigs.
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