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(American Journal of Botany. 2008;95:793-804.) doi: 10.3732/ajb.0700007 © 2008 Botanical Society of America, Inc. |
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Genetics |
2 Department of Horticulture, University of Wisconsin-Madison, 1575 Linden Drive, Madison, Wisconsin 53706 USA 3 Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples "Federico II", Via Università 100, 80055 Portici, Italy 4 Department of Genetics, Plant Breeding and Seed Science, Agricultural University of Krakow, Al. 29 Listopada 54, 31-425 Krakow, Poland 5 USDA-ARS, Vegetable Crops Research Unit, Department of Horticulture, University of Wisconsin-Madison, 1575 Linden Drive, Madison, Wisconsin 53706 USA
Received for publication 22 December 2007. Accepted for publication 22 April 2008.
ABSTRACT
Karyotype analysis provides insights into genome organization at the chromosome level and into chromosome evolution. Chromosomes were marked for comparative karyotype analysis using FISH localization of rDNA genes for the first time in Apioideae species including taxa of economic importance and several wild Daucus relatives. Interestingly, Daucus species did not vary in number of rDNA loci despite variation in chromosome number (2n = 18, 20, 22, and 44) and previous publications suggesting multiple loci. All had single loci for both 5S and 18S-25S (nucleolar organizing region) rDNA, located on two different chromosome pairs. The 5S rDNA was on the short arm of a metacentric chromosome pair in D. crinitus (2n = 22) and D. glochidiatus (2n = 44) and on the long arm of a metacentric pair in other Daucus species, suggesting possible rearrangement of this chromosome. For other Apiaceae, from two (Apium graveolens), to three (Orlaya grandiflora), to four (Cuminum cyminum) chromosomes had 18S-25S rDNA sites. Variability for number and position of the 5S rDNA was also observed. FISH signals enabled us to identify 20–40% of the chromosome complement among species examined. Comparative karyotype analysis provides insights into the fundamental aspects of chromosome evolution in Daucus.
Key Words: Apiaceae • Daucus • rDNA genes • fluorescence in situ hybridization • karyotypes • karyotype symmetry
A karyotype describes the phenotypic aspects of the chromosome complement of a species in terms of number, size, arm ratio (or centromere position), and other landmark features of its chromosomes (Levin, 2002
). Karyotypes are dynamic structures evolving through numerical and structural changes (for a comprehensive overview on chromosomal changes in plants, see Levin, 2002). Indeed, the number, size, and arm ratio of chromosome complements may differ even between closely related taxa. For several decades, karyotype diversity has been a crux of plant evolution studies for two main reasons (Levin, 2002). First, chromosome rearrangements often result in partial or complete barriers to interspecific gene flow. Second, karyotypes may provide insights into the relationship between species. However, a serious weakness of karyotype analysis is the paucity of chromosome markers, which has limited the identification of the chromosomal changes responsible for the extant karyotypes. To circumvent this shortcoming, two other main strategies have been used to track the evolutionary events that led to the present karyotypes, namely, comparative genetic mapping and cross-species or comparative chromosome painting. Both approaches are based on the comparison of chromosome colinearity shared between extant species. Cross-species chromosome painting has been widely applied in mammals (Chowdhary et al., 1998) but technical issues have limited its application in plant to a few groups of species (Lysak et al., 2006). Comparative studies in Brassicaceae (Lysak et al., 2006), Graminae (Gale and Devos, 1998), and Solanaceae (Tanksley et al., 1992) have demonstrated that syntenic chromosomal segments between different species have been altered by multiple translocations, inversions, and duplications (and/or deletions), but otherwise they are well conserved among groups of related species.
Without the development of high-density maps, inferences about chromosome evolution rely upon cytological evaluations, using a combination of standard chromosome staining with fluorescence in situ hybridization (FISH). Several repetitive DNA probes have been used to generate FISH-based karyotypes in closely related species belonging to Aegilops, Triticum, Nicotiana, and Pinus (for a review, see Schwarzacher, 2003; Jiang and Gill, 2006). Comparisons of the FISH karyotypes between those closely related species have provided chromosomal evidence of their evolutionary relationship. Among the repetitive DNA probes used for FISH, the gene units encoding ribosomal RNA, both 5S and 18S-25S rDNA, have been widely applied in plants for reliable identifying chromosomes, karyotyping, and supporting the evolutionary relationships among closely related species (Jiang and Gill, 1994; Murata et al., 1997; Hizume et al., 2002; Barto
et al., 2005). The 18S-25S rDNA and 5S rDNA genes are major multigene families in all eukaryotes, representing 8% (Heslop-Harrison, 2000) and 0.7% (Campell et al., 1992) of Arabidopsis genome, respectively. Thus, FISH localization of these gene clusters may also provide valuable information on the genomic organization of a species at the chromosome level (Chung et al., 2008).
The Apiaceae family comprises approximately 250 genera and 2800 species of widely distributed plants. Most Apiaceae of economic relevance, including carrot, celery, coriander, and parsley, belong to the Apioideae subfamily. Besides Daucus carota var. sativus L. (carrot, 2n = 18), approximately other 19 species are ascribed to the Daucus genus. These species are classified in five sections and mainly are 2n = 20 and 22. The genomes of carrot and of celery (2n = 22) are the most studied within the family (Rubatzky et al., 1999). The increasing interest in carrot as a nutriceutical (orange carrots provide up to 30% of the provitamin A consumed in the USA; Simon, 2000) is triggering the development of genomic resources for this crop. In the last two decades, several carrot linkage maps have been developed, based on several types of markers (Just et al., 2007; Simon et al., 2008), and a bacterial artificial chromosome (BAC) clone library representing 22-fold coverage of the carrot genome has been synthesized and is being characterized (Cavagnaro et al., 2008). In contrast to this progress, genome studies at the chromosome level in both Daucus and its family have been based primarily on chromosome counts and morphology. Pimenov et al. (2003) recently collected and reviewed the karyotype formulae of some 300 Apioideae species. Several karyotypes of leading Apioideae crops such as carrot, celery, and coriander, have been published (carrot: Sharma and Gosh, 1954; Sharma and Bhattacharyya, 1959; Hore, 1974; Subramanian, 1986; Schrader et al., 2003; celery: Murata and Orton, 1984; Paul and Datta, 2003; coriander: Das and Mallick, 1989; Poggio et al., 1994). However, the karyological data of the wild relatives of these crop species (including wild Daucus species) are scarce or nonexistent.
The karyotypes in the previously published studies of Apiaceae were made on mitotic chromosomes from plants growing in various environments. The results were collected from different varieties and/or accessions using different nomenclatures and criteria for chromosome classification. Therefore, previously published karyotypes cannot be compared with each other. In addition, within each species, variable numbers of secondary constrictions have been reported. Generally, the cytological significance of these constrictions has not been specified, and as suggested for celery chromosomes by Murata and Orton (1984), they are likely to be unstained regions among chromomeres rather than bona fide nucleolar organizer regions (NOR, that is, the chromosome region harboring the 18S-25S rDNA, often associated with the nucleolus). Fluorescence in situ hybridization is the most reliable approach to assess the number of NORs. However, as far as we know, only two Apioideae species, namely, Daucus carota L. (Schrader et al., 2003) and Thapsia garganica L. (Rasmussen and Avato, 1998), have been studied using FISH with rDNA probes.
This study was undertaken to investigate the number and the distribution of rDNA loci in several cultivated and wild Apioideae species using FISH, with the aim to clarify the number of NORs and to contribute to the genomic characterization of these species using a standard chromosome classification. Based on chromosome measurement and FISH-based chromosome landmarks, karyotypes of Daucus species are compared and grouped using numerical taxonomy approaches. The potential of comparative karyotype analysis to understand the chromosome changes that led to karyotype diversity in Daucus is discussed.
MATERIALS AND METHODS
Plant material and chromosome preparation
Twenty-one species belonging to 11 Apioideae genera and comprising 33 accessions were analyzed in this study. The 10 Daucus species (14 accessions) included belong to section Anisactis DC. [D. glochidiatus (Labill.) Fisher et al.], sect. Daucus L. (D. capillifolius Gilli, D. carota L., D. crinitus Desf., D. guttatus Sm., D. hispidifolius Clos., and D. pusillus Michx.), and sect. Platyspermum DC. (D. broteri Ten. [D. bicolor Sm.], D. littoralis Sm., and D. muricatus L.) as recognized by Sáenz Laín (1981). Information on the origins of the plant material is given in Table 1. Seeds were received from plant breeding stations, research centers, and commercial sources. Surface-sterilized seeds were germinated on Murashige–Skoog medium (Murashige and Skoog, 1962) supplemented with 2% sucrose and 0.22% phytagel at room temperature in the dark. Seeds were also sown in pots in the greenhouse to confirm taxonomical identification. Roots about 2 cm long were treated with 2 mM 8-hydroxyquinoline for 3 h at room temperature in the dark, fixed in freshly prepared methanol:glacial acetic acid (3:1 solution) for at least 24 h, and finally stored in the same solution at –20°C until use. Slides were prepared according to Dong et al. (2000). Slides with good metaphase spreads were selected using a phase-contrast microscope (Axiovert 135, Carl Zeiss MicroImaging, Thornwood, New York, USA) and then stored at room temperature in dry conditions until needed.
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; Gerlach and Dyer, 1980) were used for FISH. Probe pTa71 contains the 18S-25S rDNA genes and the spacer regions. Probe pTa794 consists of a 400-bp BamHI fragment including a 120-bp coding sequence for the 5S rDNA gene. The 18S-25S rDNA probe was labeled with digoxigenin-11-dUTP using a nick translation kit (Roche Diagnostics, Indianapolis, Indiana, USA) according to the manufacturer’s instructions. The 5S rDNA probe was labeled with biotin-16-dUTP (Roche Diagnostics) by PCR using M13 universal primers. FISH was conducted as previously described (Dong et al., 2000). After overnight hybridization at 37°C in a wet chamber and posthybridization washes, 5S and 18S-25S rDNA probes were detected using 1% fluorescein isothiocyanate (FITC)-conjugated antibiotin antibody and 1% rhodamine-conjugated antidigoxygenin antibody (Roche Diagnostics), respectively. Chromosomes were counterstained with 4',6-diamidino-2-phenylindole (DAPI) in ProLong Gold antifade reagent (Invitrogen, Carlsbad, California, USA). Slides were examined with a Zeiss Axiovert 135 fluorescence microscope equipped with separate excitation filter sets for DAPI, FITC, and rhodamine (Carl Zeiss MicroImaging). Images were captured digitally using an AxioCam MRm and MRGrab Software (Carl Zeiss MicroImaging). Grayscale images were acquired for each color channel, merged, and adjusted with Adobe (San Jose, California, USA) Photoshop, version 6.0.1, for contrast and background optimization.
Chromosome measurements and cluster analysis
The 17 species/accessions karyotyped are indicated with an asterisk in Table 1. Chromosomes were measured with MICROMEASURE, a freeware program from Colorado State University (http://www.colostate.edu/Depts/Biology/MicroMeasure). For each species, 5–7 metaphase spreads were measured. The chromosome arm ratio (r = length of the long arm/length of the short arm) was used to classify the centromeric location as recognized by Levan et al. (1964): median region (m), r = 1.00–1.69; submedian (sm), r = 1.7–2.99; subterminal (st), r = 3.00–7.00; and terminal (t) r > 7.00. Mean lengths of the karyotype, of the shortest and of the longest chromosome of the complement, and of the chromosome pair(s) bearing the rDNA sites were calculated. Satellites and nucleolar organizer regions (NOR) were not measured because chromatin decondensation at the secondary constrictions was highly variable (and often considerable), thus greatly affecting the arm ratio. The karyotype symmetry was calculated according to Stebbins (1971). Total form percentage of short arms (TF%) was calculated as follows: TF% = (Sum of the short arm length/Total complement length) x 100.
Hierarchical cluster analysis of the karyotype data was carried out to evaluate karyotype similarity among Daucus species. Orlaya grandiflora was used as an outgroup (Downie et al., 2001). The following karyotype features were considered: chromosome number and mean karyotype length, percentage of metacentric, submetacentric, and subtelocentric chromosomes, karyotype symmetry, relative length (% of the total karyotype length) and arm ratio of the chromosomes with the 5S and the 18S-25S rDNA sites, position (long vs short arm) and relative distance of the 5S rDNA locus from the centromere. The NCSS 97 statistical software, version 2004, was used to standardize a data matrix of the 12 variables, to calculate the average taxonomic distance, and to generate a phenogram (Barto et al., 2005). The unweighted pair-group method (UPGMA) was applied for clustering with the Euclidean distance method and standard deviation used for scaling.
RESULTS
Table 1 summarizes the chromosome numbers (2n) and FISH results for all the accessions of the 21 species included in this study. Species marked by an asterisk in Table 1 were subjected to karyotype analysis, and the results are shown in Table 2.
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). Exceptions were D. pusillus and D. crinitus, which were classified for karyotype symmetry as 2B and 3B, respectively (Table 2). Indeed, both species had a trend toward a bimodal karyotype for the relatively wider variation in chromosome size (ratio between the longest and the shortest chromosome >2:1). Moreover, for D. crinitus, over half of chromosomes had arm ratio >2:1. Nevertheless, the overall predominance of metacentric and submetacentric chromosomes of similar size did not allow the unequivocal identification of the homologous chromosomes, which was possible only for those carrying FISH signals (discussed later). Consequently, for each Daucus species, chromosomes could be roughly divided into three groups according to arm ratio without any further identification possible beyond the FISH signals (Fig. 1).
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Karyotypes of Apiaceae species distantly related to carrot
Chromosome number of Apiaceae species considered outside Daucus varied from 2n = 14 (one species, Cuminum cyminum) to 2n = 20 (three species), 2n = 22 (six species), and 2n = 40 (one species, Pimpinella saxifraga). Orlaya grandiflora (2n = 20) had the shortest karyotype (average length of 77 µm), with chromosome length ranging from 2 to 5 µm. Petroselinum crispum (2n = 22) had the longest karyotype (188 µm), with a range in chromosome length of 6–10 µm (Table 2). The predominance of metacentric and submetacentric chromosomes and the relative size homogeneity were common among these species as observed in Daucus. Indeed, the ratio between the longest and the shortest chromosome usually varied between 1.4:1 and 1.8:1, and several species were classified as 2A for karyotype symmetry. Exceptions were Apium graveolens (Fig. 3A), Coriandrum sativum (Fig. 3C), and Cuminum cyminum (Fig. 3D), which had a greater number of subtelocentric–telocentric chromosomes in their complement and karyotype formulae of 2 m + 18 st + 2 t, 2 sm + 14 st + 6 t and 6 sm + 8 st, respectively. Orlaya grandiflora represented another exception, having chromosomes relatively more heterogeneous in length. Nevertheless, based on chromosome measurement and FISH landmarks (described later), a karyogram was achieved only for Cuminum cyminum (Fig. 3D). In this species, chromosome 1 is the longest submetacentric; chromosomes 2 and 3 both carry the 18S-25S rDNA site on the short arm, but they are morphologically distinguishable, with chromosome 2 submetacentric and 3 subtelocentric. Chromosome 4 is the shortest submetacentric, and chromosomes 5 and 7 are both subtelocentric with no rDNA sites; however, they are morphologically distinguishable, with chromosome 7 nearly telocentric and the shortest chromosome of the complement. Chromosome 6 is subtelocentric and carries the 5S rDNA site on the long arm. As for the other species, chromosomes were grouped according to arm ratio, and the identification of homologous pairs was restricted to those carrying the FISH signals (Fig. 3).
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One chromosome pair carrying 5S rDNA was detected in all the species, except for Pimpinella saxifraga for which four loci (eight chromosomes) were observed (Fig. 2E). Variation was also found in the position of the 5S rDNA. For Anethum graveolens (Fig. 2G), Foeniculum vulgare (Fig. 3E), Pastinaca sativa (Fig. 3G), and Petoselimum crispum (Fig. 3H), the site was detected interstitially on the long arm of a metacentric pair. In Carum carvi (Fig. 3B) and Coriandrum sativum (Fig. 3C), the 5S rDNA probe hybridized to the long arm of one pair of subtelomeric chromosomes in an intercalary position. In Apium graveolens (Fig. 3A) and Orlaya grandiflora (Fig. 3F), it was located within the subterminal region of a metacentric pair (short arm in A. graveolens and long arm in O. grandiflora) and on a subtelocentric pair in Cuminum cyminum. In Pimpinella saxifraga and P. anisum, the 5S rDNA genes were localized on the short arm of eight and two subtelocentric chromosomes, respectively (Fig. 2E, 2F). The chromosome pair bearing the 5S rDNA genes was the longest one in Orlaya grandiflora, among the longest of the complement in Coriandrum sativum, and of medium length in the other species (Table 2).
Karyotype similarities among Daucus species
Hierarchical cluster analysis using 12 karyotype features as variables separated the Daucus species into four groups (Fig. 4). Orlaya grandiflora (used as outgroup to Daucus) separated to another cluster. The first cluster comprised all the 20-chromosome species, both from sect. Platyspermum (D. muricatus, D. littoralis, and D. broteri) and from sect. Daucus (D. guttatus). Beside the same chromosome number and similarities for the chromosomes bearing the 5S and 18S-25S rDNA, these species are similar for the predominance of metacentric chromosomes. Daucus carota and D. capillifolius, which are both from sect. Daucus (Sáenz Laín, 1981) and can be intercrossed (McCollum, 1975), formed another group. These two species were both 2n = 18 and had a similar range in chromosome size and karyotype length. Their karyotype formulae differed only for an additional pair of subtelocentric chromosomes in D. carota compared to the only one of D. capillifolius. Daucus pusillus and D. crinitus (both 2n = 22 and from sect. Daucus) were the only members of other two clusters. These two species were the most asymmetrical among the Daucus analyzed.
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Cytological studies within Apiaceae have been limited, and often little more than the chromosome number is known for most umbellifer vegetables (Rubatzky et al., 1999). The majority of the karyological data refers to the carrot subfamily (Pimenov et al., 2003), that comprises both diploid and polyploid species, with chromosome numbers varying from 2n = 12 to 2n = 154 (Moore, 1971; Pimenov et al., 2003). Cultivated Apioideae are diploid with basic chromosome numbers (x) varying between 7, 9, 10 and 11.
Our chromosome counts are in agreement with those previously reported for the cultivated species. However, the chromosome numbers of several wild Daucus and Pimpinella species are controversial. For example, 10 pairs of chromosomes have been observed in our accession of D. muricatus, in contrast with the 11 pairs previously recorded for this species (Bell and Constance, 1960; Vogt and Oberprieler, 1994). The occurrence of different cytotypes has been reported in at least 14 species of Pimpinella (Castro and Rosselló, 2007), including P. anisum (2n = 18, 20), P. major (2n = 18, 20) and P. saxifraga L. (2n = 18, 20; 2n = 36, 40) (Jurtseva, 1988). Our accessions of P. anisum and P. saxifraga have 20 and 40 chromosomes, respectively. Intraspecific karyotype variants including the variable presence of supernumerary chromosomes have been documented in several plant species (Ebert et al., 1996; Murray and Young, 2001). On the other hand, differences might also be due to species misidentification and technical issues (cytological procedures, abnormal cells or tissues).
Besides chromosome numbers, karyotypes have been reported for several Apioideae crops including carrot (Schrader et al., 2003), celery (Murata and Orton, 1984), and coriander (Poggio et al., 1994). However, to our knowledge, karyotypes of wild Daucus species and Orlaya grandiflora have not been published before. So far, and with only few exceptions (Murata and Orton, 1984; Schrader et al., 2003), karyotype analysis has been done with classical staining that does not allow the identification of individual chromosomes. In addition, most published karyotype formulae are not readily comparable because chromosomes are not classified according to Levan et al. (1964). Nevertheless, common trends can be found among previous cytological investigations and the current study. In particular, the karyotype of our carrot inbred line B493 resembles those described by Sharma and Gosh (1954) and Sharma and Bhattacharyya (1959) for several varieties, which also consisted of medium to short chromosomes with a predominance of median and submedian constrictions. In contrast, our observation differs from that reported by Schrader et al. (2003), who described a much more asymmetrical karyotype for the carrot variety "Lange rote Stumpfe." Discrepancies might be due to the differences in growing conditions, staining technique, and mitotic stage used. Indeed, the carrot karyotype described by Schrader et al. (2003) is based on Giemsa-C banding of long prometaphase chromosomes with a mean karyotype length of 74 µm, which is about 30% longer than the karyotype described in this study. Differences could also reflect karyotype diversity between the carrot varieties analyzed in the two studies. In addition, because Schrader et al. (2003) did not specify the chromosome classification used, direct comparisons between our studies are difficult to make.
Our observations on other Apiaceae generally were similar to previous reports (Apium graveolens: Murata and Orton, 1984; Coriandrum sativum: Poggio et al., 1994; Cuminum cyminum and Foeniculum vulgare: Paul and Datta, 2003; Pastinaca sativa and Petroselinum crispum: Sharma and Bhattacharyya, 1959). However, there is a general disagreement among reports on the number of chromosomes bearing secondary constrictions. Pioneer cytological studies in Apiaceae reported various numbers of chromosomes bearing secondary constrictions (see Pimenov et al., 2003). Hamal et al. (1986) determined the number of interphase nucleoli and the number of the chromosomes with satellites in 35 species representing the three Apiaceae subfamilies. On the basis of their observations, these authors concluded that each species (including Carum carvi and Cuminum cyminum) had a single active NOR per haploid complement. Our results only partially agree with those reported by Hamal et al. (1986). Indeed, we did observe variation among species in the number of the 18S-25S rDNA, which varied from one (two chromosomes) to two loci (four chromosomes). Our observations confirm a single pair of NOR in Daucus carota as already reported by Hamal et al. (1986) and Schrader et al. (2003).
Our study represents the first FISH-based survey in Apioideae on the number of rDNA loci. One interesting finding is the lack of variation in number and position of the 18S-25S rDNA site across the Daucus genus, although the species analyzed have different base chromosome numbers (x = 9, 10, and 11). A single 18S-25S rDNA locus was also observed in the putative disomic tetraploid species D. glochidiatus (2n = 44). Uniformity in the number of chromosomes carrying the NOR have been reported in 11 Quercus species (Zoldos et al., 1999) and in Musa species ascribed to sections Eumusa and Australimusa (Barto et al., 2005).
In contrast, variation in the number of 18S-25S rDNA sites has been extensively reported in plants, both between closely related species and at intraspecific level (Hajdera et al., 2003; Ali et al., 2005; Hasterok et al., 2006; Pedrosa-Harand et al., 2006). On the other hand, the number of chromosomes bearing the rDNA sites in disomic polyploids is not always the sum of the loci present in their putative ancestors. Loss of the rDNA sites during polyploid evolution has been suggested for several plants including species of Glycine (Krishnan et al., 2001) and Trifolium (Ansari et al., 1999). Our results suggest a similar evolutionary change in Daucus.
The number of the 18S-25S rDNA sites varied in the other Apioideae genera included here. Interestingly, Orlaya grandiflora had three 18S-25S rDNA hybridization sites, two of which were clearly NOR. The third weaker signal was in a hemizygous condition, with no obvious association with NOR activity. Orlaya grandiflora is a predominantly outcrossing species, thus the hemizygous signal might indicate the occurrence of individuals with different numbers (two and four) of 18S-25S rDNA hybridization sites in Orlaya populations. Evaluation of more individuals is necessary to confirm this hypothesis. Odd numbers of 18S-25S rDNA sites have been reported in Phaseolus vulgaris (Pedrosa-Harand et al., 2006) and in Brassica carinata where individuals with 8, 9, and 10 sites have been observed (Hasterok et al., 2006). The occurrence of hemizygous sites might also be due to changes in rDNA-related sequences that are known to evolve rapidly (Hwang and Kim, 1999) as demonstrated in Solanum bulbocastanum (Stupar et al., 2002). Unfortunately, chromosome study in Apiales relatives (Araliaceae and Pittosporaceae) are limited to chromosome counts (Yi et al., 2004), thus reports on the number of rDNA loci in families related to Apiaceae are not available for comparison.
It is notable that the number of 5S rDNA sites was constant across the Daucus genus, with one locus in each species analyzed. However, we observed variability in its position—on the long arm of a metacentric pair in most Daucus species and on the short arm in D. crinitus and D. glochidiatus. It is tempting to speculate that a pericentric inversion caused the shift of the 5S rDNA locus in these two species. A pericentric inversion might also determine a shift of the centromere. Nevertheless, if breaks occurred equidistantly from the centromere, the rearrangement would not alter the chromosome arm ratio. Additional cytogenetic markers and/or comparative genetic mapping are needed to confirm this hypothesis.
A wide range of variability was found for the position of the 5S rDNA in different genera of the Apiaceae. Variability in the number of sites was observed in the genus Pimpinella; P. anisum (2n = 20) had one locus (two chromosomes), and P. saxifraga (2n = 40) had four loci (eight chromosomes). The latter species also had two 18S-25S rDNA loci, supporting its polyploidy origin with interesting disparity in reduction of the rDNA loci.
The chromosomal distribution of rDNA has been studied in a large number of plant species to provide insights on the evolution of closely related taxa (Jiang and Gill, 1994; Moscone et al., 1999; Seijo et al., 2004). However, numerical taxonomy approaches have not been widely used to treat karyological data in plants. As far as we know, rare examples are the studies conducted in Solanum (Bernardello et al., 1994), Lathyrus (Seijo and Fernández, 2003), and Musa (Barto et al., 2005). In this study, hierarchical cluster analysis was used to group several Daucus species from sect. Daucus and sect. Platyspermum based on karyotype features. Interestingly, karyotype diversity was mainly found in sect. Daucus. Species under this section differed for chromosome number, karyotype formulae, symmetry, and the location of the 5S rDNA locus. Therefore, they were distributed in four clusters. Carrot and D. capillifolius (sect. Daucus) were grouped together, not only sharing the same chromosome number but also having similar karyotypes overall. As far as it is known, D. capillifolius is the only species crossable with D. carota (McCollum, 1975). On the contrary, all species from sect. Platyspermum (D. broteri, D. littoralis, and D. muricatus) had similar symmetrical karyotypes, with 10 pairs of mainly metacentric chromosomes. These species were placed in the same cluster along with D. guttatus, another 20-chromosome species from sect. Daucus. Systematics of the Daucus species is still fragmentary making it difficult to draw an evolutionary trend without the support of a phylogenetic framework. However, it seems clear that our karyological data do not match the molecular data available.
Molecular analysis of several Daucus species using RFLP of the chloroplast genome (Vivek and Simon, 1999) largely agreed with the morphological classification by Sáenz Laín (1981) and placed the species of sect. Daucus (D. carota, D. capillifolius, D. pusillus, and D. guttatus) in a single group. Daucus littoralis and D. broteri (sect. Platyspermum) formed another group, whereas D. muricatus (also from sect. Platyspermum) was the only member of another cluster. Daucus crinutus was not included in the study. In another phylogenetic study based on ITS sequence data, Lee and Downie (1999) showed that the Daucus genus is split in two groups. One group comprised species of the sections Daucus (D. carota and D. crinitus), Chrysodaucus, Meoides (both sections not included in our study), and Platyspermum (D. muricatus). The other group comprised other species from sections Daucus (D. pusillus) and Platyspermum (D. broteri), and species from sect. Anisactis (D. durieua and D. montanus) not included in this study.
It has been hypothesized that the basic chromosome number in Apiaceae is x = 11 and that a descending dysploid series (including x = 10 and 9) could be derived from it (Moore, 1971; Pimenov et al., 2003). This assumption is made because the wide spread occurrence of x = 11 throughout this subfamily. Indeed, because of the fragmentary knowledge of the phylogeny of the genus, there is no indication either for a potential Daucus ancestor or a hypothetical ancestral karyotype. Nevertheless, it is interesting to note that the reduction in chromosome number from 2n = 22 to 2n = 20 is associated with an overall increase of metacentric pairs and the karyotype symmetry. On the other hand, a further reduction to 2n = 18 is coupled with a large decrease in both the karyotype length and the genome size. Recently, to highlight the karyotype evolution in Arabidopsis thaliana and its relatives, comparative chromosome painting has been applied to the pachytene chromosomes of A. thaliana (2n = 10) (Lysak et al., 2006). The study revealed that the extant A. thaliana karyotype arose through a complex pattern of chromosome rearrangements, involving pericentric inversions, reciprocal translocation, and loss of the minichromosomes in addition to the formation of "fusion chromosomes." Altogether, these events led to both chromosome number reduction (from x = 8 to x = 5) and increased karyotype symmetry (Lysak et al., 2006). It could be speculated that karyotype diversification in Daucus followed similar routes. However, this idea must be tested with more Daucus species and more markers.
Our recent development of a BAC library for D. carota has opened new perspectives to the cytogenetic studies in Daucus (Cavagnaro et al., 2008). To generate additional chromosome landmarks, we are using map-anchored carrot BAC clones as FISH probes on the chromosomes of several Daucus species. This work will create the basis for the integration of genetic and physical maps in D. carota and will possibly shed light on the karyotype evolution in the Daucus genus.
FOOTNOTES
1 The authors thank Dr. R. M. Stupar for assistance with FISH, Dr. D. Grzebelus for assistance with probe preparation, Dr. R. Bara
ski for advice on the statistical analysis, and Dr. D. Senalik for proficient technical assistance. This is contribution no. 167 from DiSSPA. 
6 Author for correspondence (e-mail: psimon{at}wisc.edu)
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