Cytogenetic Characterization and Nuclear DNA Content of Diploid and Tetraploid Forms of Stokes Aster

in HortScience
View More View Less
  • 1 Department of Horticultural Science, North Carolina State University, Campus Box 7609, Raleigh, NC 27695-7609
  • 2 Department of Horticultural Science, North Carolina State University, Campus Box 7522, Raleigh, NC 27695-7522
  • 3 Crop Science Department, North Carolina State University, Greenhouse Unit 3, Campus Box 7629, Raleigh, NC 27695-7629

Stokesia laevis (J. Hill) Greene is a herbaceous perennial native to the southeastern United States. Most cultivars of Stokesia are diploid (2n = 2x = 14) except for ‘Omega Skyrocket’, a tetraploid (2n = 4x = 28) form selected from a natural population. A comparative study of the karyotypes and meiotic behavior of diploid cultivars, seed-derived accessions of ‘Omega Skyrocket’, synthetically derived autotetraploids, and triploid progeny from these taxa strongly suggest that ‘Omega Skyrocket’ is an autotetraploid form of Stokesia. Total karyotype length, 161 μm and 293 μm, and average chromosome length, 11.5 μm and 10.5 μm, of the diploid cultivars and tetraploid accessions of ‘Omega Skyrocket’, respectively, were determined. The karyotype of the diploid cultivars consisted of eight metacentric (m) and six submetacentric (sm) chromosomes with average arm ratio values ranging from 1.12 to 2.06. The karyotype of ‘Omega Skyrocket’ consisted of 23 m chromosomes and 5 sm chromosomes with average arm ratio values ranging from 1.22 to 2.02. Meiotic pairing in the diploids was normal. No meiotic irregularities such as laggards or bridges were observed and disjunction was balanced (7:7). Accessions of ‘Omega Skyrocket’ demonstrated a high frequency (60%) of quadrivalent formation; however, later stages of meiosis were regular with balanced disjunction (14:14) occurring in 95% of the cells. Meiotic configurations in synthetically derived autotetraploids and triploid hybrids from crosses of diploid cultivars × ‘Omega Skyrocket’ consisted of univalents, bivalents, trivalents, quadrivalents, and pentavalents. Abnormalities, including laggards, unequal and/or premature disjunction, chromosome bridges, and chromosome stickiness were observed. Average nuclear 2C DNA content was 20.3 pg for the diploid cultivars and 39.9 pg for the newly synthesized autotetraploids. Average nuclear 2C DNA content for ‘Omega Skyrocket’ was 37.3 pg, which was 8.2% less than twice the average 2C DNA content of the diploid accessions and 6.4% less than the newly synthesized autotetraploids, suggesting that genomic downsizing in ‘Omega Skyrocket’ has occurred. Similarity of the karyotypes of the diploids and ‘Omega Skyrocket’ and the slight reduction in nuclear DNA content suggest that ‘Omega Skyrocket’ has diverged little from its original diploid progenitor.

Abstract

Stokesia laevis (J. Hill) Greene is a herbaceous perennial native to the southeastern United States. Most cultivars of Stokesia are diploid (2n = 2x = 14) except for ‘Omega Skyrocket’, a tetraploid (2n = 4x = 28) form selected from a natural population. A comparative study of the karyotypes and meiotic behavior of diploid cultivars, seed-derived accessions of ‘Omega Skyrocket’, synthetically derived autotetraploids, and triploid progeny from these taxa strongly suggest that ‘Omega Skyrocket’ is an autotetraploid form of Stokesia. Total karyotype length, 161 μm and 293 μm, and average chromosome length, 11.5 μm and 10.5 μm, of the diploid cultivars and tetraploid accessions of ‘Omega Skyrocket’, respectively, were determined. The karyotype of the diploid cultivars consisted of eight metacentric (m) and six submetacentric (sm) chromosomes with average arm ratio values ranging from 1.12 to 2.06. The karyotype of ‘Omega Skyrocket’ consisted of 23 m chromosomes and 5 sm chromosomes with average arm ratio values ranging from 1.22 to 2.02. Meiotic pairing in the diploids was normal. No meiotic irregularities such as laggards or bridges were observed and disjunction was balanced (7:7). Accessions of ‘Omega Skyrocket’ demonstrated a high frequency (60%) of quadrivalent formation; however, later stages of meiosis were regular with balanced disjunction (14:14) occurring in 95% of the cells. Meiotic configurations in synthetically derived autotetraploids and triploid hybrids from crosses of diploid cultivars × ‘Omega Skyrocket’ consisted of univalents, bivalents, trivalents, quadrivalents, and pentavalents. Abnormalities, including laggards, unequal and/or premature disjunction, chromosome bridges, and chromosome stickiness were observed. Average nuclear 2C DNA content was 20.3 pg for the diploid cultivars and 39.9 pg for the newly synthesized autotetraploids. Average nuclear 2C DNA content for ‘Omega Skyrocket’ was 37.3 pg, which was 8.2% less than twice the average 2C DNA content of the diploid accessions and 6.4% less than the newly synthesized autotetraploids, suggesting that genomic downsizing in ‘Omega Skyrocket’ has occurred. Similarity of the karyotypes of the diploids and ‘Omega Skyrocket’ and the slight reduction in nuclear DNA content suggest that ‘Omega Skyrocket’ has diverged little from its original diploid progenitor.

Stokesia (J. Hill) Greene is a monotypic genus comprised of a single species (S. laevis) that is native to the coastal plain of the southeastern United States (i.e., Mississippi, Louisiana, Alabama, Georgia, northern Florida, and South Carolina) (Bailey, 1949; Gunn and White, 1974). Stokesia is a member of the Vernonieae tribe (Asteraceae) that includes 121 genera and ≈1500 species with global distribution (Robinson, 1999a, 1999b, 2007). There are 21 species of Vernonieae representing four genera (i.e., Vernonia Schreb., Stokesia, Elephantopus L., Pseudelephantopus Rohr) found in the southeastern United States with 19 of these being indigenous (Gunn and White, 1974; Jones, 1982). Stokesia is the only genus, however, that is restricted to the United States (Gettys and Werner, 2002; Jones, 1982).

Members of the Vernonieae tribe are extremely diverse in form, habitat, and ecology, indicating that this tribe has evolved and radiated over time. Examples of this diversity include the woody tree, Vernonia arborea Ham., which is the tallest species (greater than 30 m) in the Asteraceae family and the small aquatic plant, Pacourina edulis Aubl., which has edible leaves. Additionally, there are a number of small acaulescent perennial taxa (e.g., Vernonia guineensis Benth., V. acrocephala Klatt, V. chthonocephala O. Hoffm.) that thrive in fire-maintained savannahs in Africa (Jones, 1977, 1982). In general, members of the Vernonieae tribe are perennial, herbaceous plants, shrubs, small trees, or vines with alternate leaves showing pinnate venation. Flowers characteristically form large homogamous discoid heads with one to many perfect flowers that are deep purplish red to blue or sometimes white (Jones, 1982). Base chromosome number (x) is highly variable in the Vernonieae tribe. New World taxa have a basic chromosome number of x = 8, 10–19 and often include many polyploid forms (e.g., n = 20, 26–30, 33–39, 51, 68) (Jones, 1977; Ruas et al., 1991). In contrast, most Old World taxa have a lower base number of 9 or 10 (Jones, 1977). Despite their overall diversity, all members of this tribe have uniquely shaped “Vernonioid” styles that are semicylindrical with acutely branched tips that present the hirsute surface of the stigma along the interior (Jones, 1982).

Stokesia has many of the essential features (e.g., perennial life cycle, alternate leaves, and “Vernonioid” style) that are typical of the Vernonieae tribe; however, floral and cytological characteristics of Stokesia are distinct. Specifically, Stokesia has zygomorphic flowers with ligulate ray florets that are deeply five-lobed and a reduced deciduous pappus, which are more characteristic of the Mutiseae and Lactuceae tribes (Jansen et al., 1991; Jones, 1977, 1982; Robinson, 1999c). Additionally, Stokesia has a low chromosome base number (x = 7) and large chromosomes, which are atypical of most Vernonieae (Gaus et al., 2005; Gunn and White, 1974; Jones, 1974). Despite these differences, Stokesia is consistently placed in the Vernonieae tribe (Jansen et al., 1991; Keeley et al., 2007; Keeley and Jansen, 1994; Kim et al., 1998; Kim and Jansen, 1995).

Stokesia is an attractive genus with large aster-like flowers that are characteristically pale blue or lavender in color with some cultivars having violet, pale yellow, pale pink, or albescent-colored flowers. Cultivars of Stokesia are 30 to 60 cm tall with many, moderately (three to five) branched flower scapes. Exceptions include ‘Omega Skyrocket’ [minimally (one to three) branched flower scapes greater than 1 m] and ‘Peachies Pick’ [highly (five to seven) branched flower scapes 70 to 80 cm] (Gettys and Werner, 2002).

‘Omega Skyrocket’ is an atypical example of stokes aster that was derived from a wild population discovered in Colquitt County, GA, near the town of Omega by R. Determann, S. Determann, and O. Johnson of the Atlanta Botanical Garden (Gettys and Werner, 2002). This natural population was uniform and consisted of plants with tall (greater than 1 m), upright flower scapes. ‘Omega Skyrocket’ is primarily sold by seed; however, some clonal accessions are also available.

Preliminary work demonstrated that cultivars of Stokesia studied thus far are diploids (2n = 2x = 14) except for ‘Omega Skyrocket’, which is a tetraploid form (2n = 4x = 28) (Gaus et al., 2005). The cytological characteristics of mitosis and meiosis as well as an absolute genome size estimate for this species have not been described. The objectives of this work were to: 1) prepare a karyotype; 2) characterize the meiotic behavior of diploid, triploid, and tetraploid accessions of Stokesia laevis, including ‘Omega Skyrocket’; and 3) determine the absolute nuclear 2C DNA content of diploid and tetraploid plants using flow cytometry.

Materials and Methods

Plant material.

Diploid accessions of Stokesia (‘Alba’, ‘Colorwheel’, ‘Honeysong Purple’, ‘Mary Gregory’, ‘Peach Melba’, ‘Peachies Pick’, ‘Purple Parasols’) were acquired and maintained as asexually propagated clones with the exception of ‘Alba’, which was grown from true-to-type seed. Accessions of the naturally occurring tetraploid, ‘Omega Skyrocket’, were grown from seed acquired from Jelitto Seed Co. (Schwarmstedt, Germany). Triploid plants were produced by crossing ‘Alba’, ‘Peach Melba’, ‘Peachies Pick’, and ‘Purple Parasols’ with four different accessions of ‘Omega Skyrocket’. Synthetic autotetraploid forms of diploid cultivars were produced using colchicine [N-(5,6,7,9-tetrahydro-1,2,3,10-tetra-methoxy-9-oxobenzo(a)heptalen-7-yl)acetamide] or oryzalin (3,5-dinitro-N4N4-dipropylsulfanilamide). Small (2.5 cm diameter) asexually reproduced plantlets were fully submerged for 24 to 48 h in a saturated solution of 0.2% oryzalin, prepared by diluting 1 mL Surflan® A.S. (United Phosphorus Inc., Trenton, NJ) (40.4% oryzalin) in 200 mL distilled water, or one to two drops (≈250 μL) of 2.0% colchicine were applied twice daily (0800 hr, 1600 hr) directly to the meristematic whorl of each plantlet for 3 d. Plants used for cytological examination were grown in sand under ambient light conditions in the greenhouses at North Carolina State University, Raleigh. Daytime temperatures in the greenhouse ranged from 25 to 35 °C. Plants were fertilized daily with 100 mg·L−1 nitrogen of Peters 20N–8.7P–16.6K General Purpose or Peters EXCEL Cal-Mag 15N–2.2P–12.5K fertilizer (The Scotts Co., Marysville, OH).

Cytological techniques.

Roots were collected between 1000 hr and 1200 hr on warm, sunny days. Roots were pretreated in a solution of 2 mm 8-hydroxyquinoline plus colchicine (0.5 g·L−1) for 2 to 3 h at 4 °C (Dhesi and Stalker, 1994), fixed in freshly prepared Carnoy's solution (1 part glacial acetic acid:3 parts chloroform:6 parts 95% ethanol) for 48 h at room temperature (RT), and stored at 4 °C in 70% ethanol. Roots were hydrolyzed in 1 N HCl at 60 °C for 8 min, washed in distilled water, and stained in Schiff's reagent for a minimum of 30 min in the dark. Root tips were removed and squashed in 1% acetocarmine. Cells were observed using a light microscope (Carl Zeiss Photomicroscope 3; Carl Zeiss MicroImaging, Inc., Thornwood, NY) under ×630 and ×1000 magnification. Digital photographs of well-spread metaphase plates were taken using a Sony Cybershot F717 camera (Tokyo, Japan). Total karyotype length (TKL), the total length of individual chromosomes (TCL), and the length of the short (SA) and long (LA) arm of each chromosome were measured from digital photographs using MicroMeasure version 3.3 software (Reeves, 2001). Average chromosome length (ACL) and arm ratio (AR = LA/SA) were calculated from these measurements. Ideograms were constructed based on chromosome length and arm ratios. Chromosome nomenclature followed Levan et al. (1964) (m = metacentric, sm = submetacentric, st = subtelocentric). Intrachromosome asymmetry (A1) was calculated following the formula A1 = 1 – [Σ(si/li)/n], where si is the average length of the short arms in every chromosome, li is the average length of the long arm of every chromosome, and n is the number of chromosomes. This asymmetry index was adapted from the formula presented in Romero Zarco (1986), which calculated A1 using measurements of homologous chromosome pairs, not individual chromosomes. Interchromosome asymmetry (A2) was calculated following the formula A2 = sd/x, where sd is the standard deviation and x is the mean of the chromosome lengths (Romero Zarco, 1986). The index of karyotype symmetry (TF% = 100 ΣS ΣL−1) was also calculated (Huziwara, 1962).

Meiotic chromosomes were observed in developing pollen mother cells (PMCs). Immature buds (1 to 1.5 cm in diameter) were collected on bright sunny days between 0900 hr and 1000 hr and the sepals were removed. Buds were fixed in freshly prepared Carnoy's solution for 48 h at RT and stored in 70% ethanol at 4 °C. Flower buds were dissected under a dissecting microscope and three to six anthers were removed and squashed in 1% acetocarmine. Cells were observed at ×630 and ×1000 magnification.

Nuclear DNA content determination.

Absolute nuclear 2C DNA content was determined using a Becton-Dickinson FASCan flow cytometer (San Jose, CA) equipped with a 488-nm argon laser. Pisum sativum L. ‘Citrad’, with a known 2C DNA content of 8.76 pg (Greilhuber et al., 2007), was used as an internal standard. Nuclei of the internal standard and sample were extracted and stained simultaneously according to protocols provided with the Partec CyStain PI Absolut P kit (Partec GmbH, Munster, Germany). Cold (0 °C) extraction buffer (500 μL) was added to ≈2 cm2 of newly expanded leaf tissue of the internal standard and sample and finely chopped for 30 s and incubated for 30 s at RT. The solution was filtered using Partec CellTrics™ disposable filters (Munster, Germany) with a pore size of 50 μm. Cold (0 °C) staining buffer (1 mL staining buffer, 12 μL of PI stock, 6 μL RNase stock) was added and samples were incubated for 2 h at 4 °C protected from light before they were analyzed. Replications (four to five) over 2 d were analyzed for each plant with 5,000 to 10,000 nuclei analyzed per replication. Peaks used for analysis had coefficient of variation values of 6% or less. Conversion of mass values into number of base pairs (bps) was calculated using the factor 1 pg = 978 Mbp (Doležel et al., 2003).

Statistical analysis.

Data were subjected to analysis of variance using the PROC GLM procedure and means were separated by least significant difference (P ≤ 0.05) using SAS 9.1 software (SAS Institute, Cary, NC).

Results

Karyotype.

Somatic chromosomes from seven diploid (2n = 2x = 14) accessions (‘Alba’, ‘Colorwheel’, ‘Honeysong Purple’, ‘Mary Gregory’, ‘Peach Melba’, ‘Peachies Pick’, ‘Purple Parasols’) and three seed-derived accessions of ‘Omega Skyrocket’ (OSR) (2n = 4x = 28) were studied (Fig. 1). In all cases, chromosome counts were consistent with previous reports (Gaus et al., 2005; Gunn and White, 1974). There were few differences among the karyotypes of the diploid cultivars; thus, a single representative karyotype was constructed based on 33 well-spread metaphase plates (14, ‘Honeysong Purple’; 14, ‘Peachies Pick’; 5, ‘Alba’) (Fig. 2A). Similarly, there were few differences among the accessions of ‘Omega Skyrocket’, so one karyotype was constructed from 35 well-spread metaphase plates (14, OSR100; 15, OSR182; 6, OSR200) (Fig. 2B). Because chromosome morphology was very similar, individual chromosomes could not be definitively identified in all cases, thus preventing the direct comparison of chromosomes from the diploid accessions to the chromosomes of ‘Omega Skyrocket’.

Fig. 1.
Fig. 1.

Somatic chromosomes of Stokesia laevis (A) ‘Peachies Pick’ (2n = 2x = 14) and (B) ‘Omega Skyrocket’ (2n = 4x = 28). Scale bar = 10 μm.

Citation: HortScience horts 43, 7; 10.21273/HORTSCI.43.7.2005

Fig. 2.
Fig. 2.

Karyotype ideograms of Stokesia laevis. (A) Average karyotype of ‘Honeysong Purple’, ‘Alba’, ‘Peachies Pick’ (2n = 2x = 14). (B) ‘Omega Skyrocket’ (2n = 4x = 28). Metacentric chromosomes (AR < 1.7) in gray. Submetacentric chromosomes (AR ≥ 1.7) in black. Scale bar = 2 μm.

Citation: HortScience horts 43, 7; 10.21273/HORTSCI.43.7.2005

Average TKL for the four diploid accessions was 161 ± 29 μm with an ACL of 11.5 ± 2.0 μm. Chromosomes ranged in length from 8.3 μm to 16.1 μm (Table 1). The average karyotype of the diploids consisted of eight metacentric (m) chromosomes with average ARs ranging from 1.12 to 1.58 and six submetacentric (sm) chromosomes with average ARs ranging from 1.72 to 2.06 (Table 1; Fig. 2). The average TKL was 293 ± 53 μm and ACL was 10.5 ± 1.9 μm for the three tetraploid accessions of ‘Omega Skyrocket’. Chromosomes ranged in size from 7.3 μm to 15.4 μm. The average karyotype of ‘Omega Skyrocket’ consisted of 23 m chromosomes with average ARs ranging from 1.22 to 1.68 and five sm chromosomes with average ARs ranging from 1.71 to 2.02 (Table 1; Fig. 2). The TKL (293 μm) for ‘Omega Skyrocket’ was ≈9% less than two times the average TKL (161 × 2 = 322 μm) of the diploids. The overall symmetry of the karyotypes of ‘Omega Skyrocket’ and the diploid accessions were very similar. Karyotype symmetry (TF%) was 66.68% and 67.93% for the diploids and ‘Omega Skyrocket’, respectively. Intrachromosome asymmetry (A1) and interchromosome asymmetry (A2) indices were 0.341 and 0.277 for the diploids and 0.328 and 0.289 for ‘Omega Skyrocket’, respectively.

Table 1.

Karyotypic parameters of Stokesia laevis.

Table 1.

Meiotic behavior.

Meiosis was observed in PMCs of diploids, triploids, tetraploid accessions of ‘Omega Skyrocket’, and synthetic autotetraploids (Tables 2 and 3; Figs. 38). Chromosome pairing in the diploids was normal with each bivalent, usually possessing two chiasmata (Fig. 3). No meiotic irregularities such as laggards or bridges were observed and disjunction was equal (7:7) at later stages of meiosis. Average chromosome configuration of triploid taxa was 4.9I + 5.2II + 1.9III (Table 2). The most frequent configurations were 2III + 5II + 5I, 3III + 4II + 4I, and 1III + 6II + 6I, which accounted for 25%, 19%, and 18% of the cells, respectively (Fig. 4, data not shown). Lagging chromosomes (one to five) were present in either Anaphase I or Anaphase II in 53% of the 34 PMCs observed (Fig. 5). Disjunction at Anaphase I and II in triploid cells without lagging chromosomes produced nearly balanced cells (63%) with chromosome configurations of 10:11 and unbalanced configurations of 9:12 (31%) and 8:13 (6%), respectively (data not shown, Fig. 5). The average chromosome configuration of tetraploid accessions of ‘Omega Skyrocket’ was 0.1I + 5.5II + 0.1III + 4.2IV (Table 2). The most frequent configurations were 5IV + 4II and 4IV + 6II, which accounted for 27% and 24% of the cells, respectively (Fig. 6, data not shown). Equal disjunction (14:14) was observed in 95% of the 84 anaphase cells observed (Fig. 7A). The average chromosome configuration of the eight synthetic autotetraploids was 0.3I + 2.8II + 0.3III + 5.3IV (Table 2). The most common configurations were 7IV, 6IV + 2II, 5IV + 4II, and 4IV + 6II, which accounted for 25%, 22%, 15%, and 10% of the cells, respectively (data not shown). Although laggards were observed in only one cell (Fig. 7B), only 38% of the 40 anaphase cells were equally balanced (14:14) (data not shown).

Table 2.

Chromosome associations in pollen microsporocytes of different cytotypes of Stokesia laevis.

Table 2.
Table 3.

Average nuclear DNA content of different Stokesia laevis as determined by flow cytometric measurements of PI-stained nuclei.

Table 3.
Fig. 3.
Fig. 3.

Meiotic chromosome associations of diploid Stokesia laevis ‘Peachies Pick’, 7 ring bivalents at Metaphase I (2n = 2x = 14). Scale bar = 10 μm.

Citation: HortScience horts 43, 7; 10.21273/HORTSCI.43.7.2005

Fig. 4.
Fig. 4.

Meiotic chromosome associations of triploid Stokesia laevis (2n = 3x = 21). Scale bar = 10 μm. (A) ‘Purple Parasols’ × ‘Omega Skyrocket’; 1 frying pan trivalent, 6 bivalents, 6 univalents at Metaphase I. (B) ‘Peachies Pick’ × ‘Omega Skyrocket’; 3 trivalents (2 frying pans + 1 Y-shape), 4 bivalents, 4 univalents at Metaphase I.

Citation: HortScience horts 43, 7; 10.21273/HORTSCI.43.7.2005

Fig. 5.
Fig. 5.

Meiotic chromosome abnormalities observed in triploid hybrids of Stokesia laevis (2n = 3x = 21). Scale bar = 10 μm. (A) ‘Peachies Pick’ × ‘Omega Skyrocket’ with unbalanced balanced disjunction (12:9) at Anaphase I; arrow indicates possible chromosome bridge. (B) ‘Peachies Pick’ × ‘Omega Skyrocket’ with 10 laggards (circled) at Anaphase I. (C) ‘Purple Parasols’ × ‘Omega Skyrocket’, unbalanced disjunction (10:11) at Anaphase I.

Citation: HortScience horts 43, 7; 10.21273/HORTSCI.43.7.2005

Fig. 6.
Fig. 6.

Meiotic chromosome associations of Stokesia laevis ‘Omega Skyrocket’ (2n = 4x = 28). Scale bar = 10 μm. Metaphase I configurations. (A) Four figure-8 quadrivalents and six bivalents; arrow indicates interlocking bivalents. (B) Four quadrivalents (2 figure-8s, 2 rings) and 6 bivalents. (C) Five quadrivalents (all figure-8s) and 4 bivalents (2 rings + 2 rods).

Citation: HortScience horts 43, 7; 10.21273/HORTSCI.43.7.2005

Fig. 7.
Fig. 7.

Meiotic chromosome associations of tetraploids of Stokesia laevis (2n = 4x = 28). Scale bar = 10 μm. (A) Anaphase I of ‘Omega Skyrocket’; equal disjunction (14:14); dotted line indicates division line. (B) Telophase II of 4x-‘Peach Melba’; lagging chromosomes indicated by arrows.

Citation: HortScience horts 43, 7; 10.21273/HORTSCI.43.7.2005

Fig. 8.
Fig. 8.

Flow cytometry histograms of different cytotypes of Stokesia laevis. Pisum sativum L. ‘Citrad’ is the internal standard. (A) ‘Honeysong Purple’ (2n = 2x = 14). (B) ‘Omega Skyrocket’ accession 100; naturally occurring tetraploid (2n = 4x = 28). (C) 4x-‘Purple Parasols’; synthetic autotetraploid (2n = 4x = 28).

Citation: HortScience horts 43, 7; 10.21273/HORTSCI.43.7.2005

Bivalent frequency was significantly higher and quadrivalent frequency was significantly lower (P < 0.05) in PMCs of ‘Omega Skyrocket’ relative to that observed in PMCs of the newly synthesized tetraploids (Table 2). Triploid taxa produced more (P < 0.05) univalents and trivalents than the other taxa.

Nuclear DNA content estimation.

2C nuclear DNA content was determined for six diploid accessions (‘Alba’, ‘Colorwheel’, ‘Peachies Pick’, ‘Honeysong Purple’, ‘Purple Parasols’, ‘Peach Melba’), five accessions of ‘Omega Skyrocket’, and five synthetic autotetraploids (4x-‘Alba’, 4x-‘Colorwheel’, 4x-‘Peachies Pick’, 4x-‘Purple Parasols’, 4x-‘Peach Melba’) (Table 3; Fig. 8). Chromosome number was confirmed for all of the diploids tested, three of the ‘Omega Skyrocket’ accessions, and all of the synthetic tetraploids except for 4x-‘Peach Melba’ (data not shown). There was no difference (P < 0.001) between measurements taken on different days, so replications were pooled for analysis. Average 2C nuclear DNA content was significantly different across all groups (P < 0.001) (Table 3). Average 2C DNA content of the synthetic autotetraploids (39.9 ± 0.3 pg) was approximately twice (196%) the average 2C nuclear DNA content of the diploid accessions (20.3 ± 0.1 pg). Average 2C DNA content of the natural tetraploid accessions of ‘Omega Skyrocket’ (37.3 ± 0.5 pg) was ≈6.4% less than the synthetic autotetraploids and 8.2% less than twice the average 2C DNA content of the diploid accessions. The basic monoploid genome size (1Cx) for the diploids, ‘Omega Skyrocket’, and the synthetic tetraploids was 10.2 pg, 9.3 pg, and 10.0 pg, respectively. All accessions had an intermediate-sized genome (i.e., 3.5 pg < 1Cx > 14.0 pg) (Leitch et al., 2005).

Discussion

All members of the Vernonieae tribe classified thus far from India and North, South, and Central America, including Stokesia, have chromosomes that are fairly symmetrical and classified mainly as either metacentric or submetacentric (Dematteis, 1998, 2002; Dematteis and Fernández, 1998; De Oliveira et al., 2007; Esteves Mansanares et al., 2007; Mathew and Mathew, 1983; Ruas et al., 1991; Smith and Jones, 1987). The TCLs of Stokesia (i.e., 8 to 16 μm), however, were significantly greater than that observed for any of the other Vernonieae species (i.e., 1 to 4 μm). Stokesia also differs from other species in the tribe because it has an unusually low base chromosome number of x = 7, which is only present in Vernonia appendiculata Less., a species native to Madagascar (Rabakonandrianina and Carr, 1987). Stokesia is a New World species but it shares a low base chromosome number (x) that is more typical of an Old World species of the Vernonieae tribe (i.e., x = 9, 10). This is not surprising, however, because phylogenetic studies using chloroplast DNA restriction site data and sequence data from the chloroplast, ndhF gene, noncoding spacer trnL-F, and the nuclear rRNA ITS region illustrate that New and Old World species of the Vernonieae tribe are not mutually exclusive (Keeley and Jansen, 1994), and Stokesia in particular is often associated with Old World species despite its New World origin (Keeley et al., 2007).

Similarities between the karyotypes of the diploid cultivars and ‘Omega Skyrocket’ and the high frequency (60%) of quadrivalent formation in ‘Omega Skyrocket’ both indicate that ‘Omega Skyrocket’ is an autotetraploid form of Stokesia laevis (Tables 1 and 2; Figs. 2 and 6). In theory, autotetraploids exhibiting a high frequency of quadrivalents are expected to have unequal chromosomal disjunction during later stages of meiosis resulting in a corresponding reduction in fertility. Studies show that selection over several generations for increased seed set in autotetraploid populations is associated with a decrease in quadrivalent frequency and a corresponding increase in mean bivalent frequency (Bremer and Bremer-Reinders, 1954; Hilpert, 1957; Kumar et al., 1993; Swaminathan and Sulbha, 1959). However, in other studies, selection for increased seed set in autotetraploid populations is correlated with an unexpected increase in the quadrivalent frequency and a corresponding decrease in univalent and trivalent frequencies (Crowley and Rees, 1968; Hazarika and Rees, 1967; McCollum, 1958; Narasinga Rao and Pantulu, 1982). In Stokesia, the high frequency (60%) of quadrivalent formation in the autotetraploid accessions of ‘Omega Skyrocket’ did not result in a correspondingly high frequency of unequal disjunction and a reduction in fertility. In fact, later stages of meiosis in these accessions were regular and produced equally balanced gametes 95% of the time. Furthermore, the relatively high pollen viability (74%) of ‘Omega Skyrocket’ (unpublished data) is further evidence that quadrivalent formation does not affect fertility in this taxa.

In the synthetically derived autotetraploids, 76% of the chromosomes were involved in quadrivalent formation, a slight but significant increase relative to ‘Omega Skyrocket’, which is a more established autotetraploid form. This observation was not surprising because quadrivalent frequency in newly synthesized autopolyploids often decreases over subsequent generations after the initial polyploidization event (Gilles and Randolph, 1951; Santos et al., 2003). Later stages of meiosis in the synthetic autotetraploids were irregular and unequal disjunction and chromosome stickiness were observed in a high percentage of cells, unlike ‘Omega Skyrocket’ in which meiosis was normal. The disparity between these two groups of tetraploids may be attributed, in part, to the “types” (e.g., ring, box, zigzag chain, figure-eight) of quadrivalents formed by each group (Figs. 6 and 8). Quadrivalents of ‘Omega Skyrocket’ were primarily figure-eight types or zigzag chains with alternately oriented centromeres (data not shown) that presumably produced balanced 2 × 2 disjunction at Anaphase. In contrast, quadrivalents of the synthetic autotetraploids included zigzag chains and figure-eight types as well as rings with adjacently oriented centromeres and indifferently oriented configurations that presumably resulted in unbalanced disjunction in some of the cells.

Triploids from crosses of diploid cultivars × ‘Omega Skyrocket’ had more (P ≤ 0.05) univalents and trivalents (Table 2), which likely contributed to the formation of a high number of unbalanced gametes. Interestingly, an average chromosome configuration of 1.9III + 5.2II + 4.9I observed for the triploids suggests that chromosomal homology between ‘Omega Skyrocket’ and the diploids was similar enough to support trivalent formation but not similar enough to prevent the formation of a relatively high number of univalents. These results indicate that some genomic rearrangement in ‘Omega Skyrocket’ has probably occurred since the initial doubling event; however, enough chromosomal affinity still remains between ‘Omega Skyrocket’ and the diploid cultivars such that chromosomal pairing still occurs in the triploid hybrids.

The ≈9% reduction in TKL in ‘Omega Skyrocket’ relative to twice the karyotype of the diploids and the corresponding reduction in nuclear DNA content indicate that TKL and nuclear DNA content for this species are closely correlated, a trend frequently observed in other species (Cerbah et al., 1995; Dimitrova and Greilhuber, 2000; Garnatje et al., 2004; Moscone et al., 2003; Srivastava and Lavania, 1991; Torrell and Vallès, 2001). The reduction in nuclear DNA content and corresponding reduction in TKL indicate that differences in TKL between the diploids and ‘Omega Skyrocket’ is likely attributable to actual genomic downsizing and not an artifact of different chromosome condensation. Genomic downsizing in polyploid plants is a widespread phenomenon in which the monoploid genome size (i.e., 1Cx = 2C DNA content divided by ploidy level; Greilhuber et al., 2005) decreases with increasing ploidy level (Leitch and Bennett, 2004). In this study, the monoploid genome size of ‘Omega Skyrocket’ (9.3 pg) was less than the basic genome size of the diploid accessions (10.2 pg). Genomic downsizing of similar magnitude has been identified in other polyploids (Bennett et al., 2000; Emshwiller, 2002; Hörandl and Greilhuber, 2002; Pecinka et al., 2006; Raina et al., 1994).

According to Rees (1984), changes in DNA content (not attributable to aneuploidy or chromosome arm loss) can occur in two ways, whereby change is either proportional to chromosome size (Brandham and Doherty, 1998; Naranjo et al., 1998; Poggio et al., 2007) or change is achieved by the equal addition or subtraction of DNA to each chromosome irrespective of chromosome size (Pringle and Murray, 1993; Raina et al., 1994; Raina and Rees, 1983). Chromosomes of Stokesia are morphologically very similar preventing the definitive identification of individual chromosomes; thus, comparison of homologous chromosome pairs in the diploids to the homologous chromosome pairs in ‘Omega Skyrocket’ was not possible. However, if the chromosomes were grouped into homologous pairs according to TCL only and compared, the reduction in DNA in ‘Omega Skyrocket’ appeared to be proportionally distributed relative to chromosome length such that longer chromosomes lost more DNA than shorter chromosomes (data not shown).

Mechanisms capable of reducing genome size in plants include unequal and illegitimate recombination of retroelement sequences, inefficient double-stranded DNA break repair systems, mutational bias of deletions over insertions, and the targeted elimination of specific DNA sequences (e.g., rDNA) (reviewed in Bennett and Leitch, 2005; Bennetzen, 2002; Bennetzen et al., 2005; Petrov, 2001; Tate et al., 2005; Wendel, 2000; Wendel et al., 2002). Rapid genomic rearrangement (e.g., elimination of repetitive DNA, loss of amplified fragment length polymorphism and random amplified polymorphic DNAbands) in nascent allo- and autopolyploids has been identified in Brassica L. and Aegilops L. hybrids (Ozkan et al., 2001; Shaked et al., 2001; Song et al., 1995) and in autotetraploids of Paspalum L. and Eragrostis Host (Martelotto et al., 2007; Mecchia et al., 2007). These studies demonstrate that genomic rearrangement, and possibly genomic downsizing, in polyploids can occur immediately after an allo- or autopolyploidization event.

Literature Cited

  • Bailey, L.H. 1949 Manual of cultivated plants. Revised ed Macmillian New York, NY

    • Export Citation
  • Bennett, M.D., Bhandol, P. & Leitch, I.J. 2000 Nuclear DNA amounts in angiosperms and their modern uses—807 new estimates Ann. Bot. (Lond.) 86 859 909

    • Search Google Scholar
    • Export Citation
  • Bennett, M.D. & Leitch, I.J. 2005 Genome size evolution in plants 90 151 Gregory T.R. The evolution of the genome Elsevier Amsterdam, The Netherlands

  • Bennetzen, J.L. 2002 Mechanisms and rates of genome expansion and contraction in flowering plants Genetica 115 29 36

  • Bennetzen, J.L., Ma, J. & Devos, K.M. 2005 Mechanisms of recent genome size variation in flowering plants Ann. Bot. (Lond.) 95 127 132

  • Brandham, P.E. & Doherty, M.J. 1998 Genome size variation in the Aloaceae, an angiosperm family displaying karyotypic orthoselection Ann. Bot. Suppl. A 67 73

    • Search Google Scholar
    • Export Citation
  • Bremer, G. & Bremer-Reinders, D.E. 1954 Breeding of tetraploid rye in The Netherlands: Methods and cytological investigations Euphytica 3 49 63

  • Cerbah, M., Coulaud, J., Godelle, B. & Siljak-Yakovlev, S. 1995 Genome size, fluorochrome banding, and karyotype evolution in some Hypochoeris species Genome 38 689 695

    • Search Google Scholar
    • Export Citation
  • Crowley, J.G. & Rees, H. 1968 Fertility and selection in tetraploid Lolium Chromosoma 24 300 308

  • Dematteis, M. 1998 Karyotype analysis in some Vernonia species (Asteraceae) from South America Caryologia 51 279 288

  • Dematteis, M. 2002 Cytotaxonomic analysis of South American species of Vernonia (Vernonieae: Asteraceae) Bot. J. Linn. Soc. 139 401 408

  • Dematteis, M. & Fernández, A. 1998 Karyotypes of seven South American species of Vernonia (Asteraceae) Cytologia (Tokyo) 63 323 328

  • De Oliveira, V.M., Forni-Martins, E.R. & Semir, J. 2007 Cytotaxonomy or species of Vernonia, section Lepidaploa, group Axilliflorae (Asteraceae: Vernonieae) Bot. J. Linn. Soc. 154 99 108

    • Search Google Scholar
    • Export Citation
  • Dhesi, J.S. & Stalker, H.T. 1994 Enhancing techniques for studying mitotic peanut chromosomes Peanut Sci. 21 92 94

  • Dimitrova, D. & Greilhuber, J. 2000 Karyotype and DNA-content evolution in ten species of Crepis (Asteraceae) distributed in Bulgaria Bot. J. Linn. Soc. 132 281 297

    • Search Google Scholar
    • Export Citation
  • Doležel, J., Bartoš, J., Volgmayr, H. & Greilhuber, J. 2003 Nuclear DNA content and genome size of trout and human Cytometry 51 127 128

  • Emshwiller, E. 2002 Ploidy levels among species in the ‘Oxalis turberosa Alliance’ as inferred by flow cytometry Ann. Bot. (Lond.) 89 741 753

    • Search Google Scholar
    • Export Citation
  • Esteves Mansanares, M., Forni-Martins, E.R. & Semir, J. 2007 Cytotaxonomy of Lychnophoriopsis Sch. Bip. and Paralychnophora MacLeish species (Asteraceae: Vernonieae: Lychnophorinae) Bot. J. Linn. Soc. 154 109 114

    • Search Google Scholar
    • Export Citation
  • Garnatje, T., Vallès, J., Garcia, S., Hidalgo, O., Sanz, M., Canela, M.A. & Siljak-Yakovlev, S. 2004 Genome size in Echinops L. and related genera (Asteraceae, Cardueae): Karyological, ecological and phylogenetic implications Biocell 96 117 124

    • Search Google Scholar
    • Export Citation
  • Gaus, J., Werner, D. & Tallury, S. 2005 Polyploidy in stokes aster (Stokesia laevis) HortScience 40 1101 (abstr.).

  • Gettys, L.A. & Werner, D.J. 2002 Stokes aster HortTechnology 12 138 142

  • Gilles, A. & Randolph, L.F. 1951 Reduction in quadrivalent frequency in autotetraploid maize during a period of 10 years Amer. J. Bot. 38 12 17

  • Greilhuber, J., Doležel, J., Lysák, M.A. & Bennett, M.D. 2005 The origin, evolution and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to describe nuclear DNA contents Ann. Bot. (Lond.) 95 255 260

    • Search Google Scholar
    • Export Citation
  • Greilhuber, J., Temsch, E.M. & Loureiro, J.C.M. 2007 Nuclear DNA content measurement Doležel J., Greilhuber J. & Suda J. Flow cytometry with plant cells WILEY-VCH Verlag GmbH & Co KGaA, Weinheim, Germany

    • Search Google Scholar
    • Export Citation
  • Gunn, C.R. & White, G.A. 1974 Stokesia laevis. Taxonomy and economic value Econ. Bot. 28 130 135

  • Hazarika, M.H. & Rees, H. 1967 Genotypic control of chromosome behavior in rye. X. Chromosome pairing and fertility in autotetraploids Heredity 22 317 332

    • Search Google Scholar
    • Export Citation
  • Hilpert, G. 1957 Effect of selection for meiotic behavior in auto-tetraploid rye Hereditas 43 318 322

  • Hörandl, E. & Greilhuber, J. 2002 Diploid and autotetraploid sexuals and their relationships to apomicts in the Ranunculus cassubicus group: Insights from DNA content and isozyme variation Plant Syst. Evol. 234 85 100

    • Search Google Scholar
    • Export Citation
  • Huziwara, Y. 1962 Karyotype analysis in some genera of Compositae. VIII. Further studies on the chromosomes of Aster Amer. J. Bot. 49 116 119

  • Jansen, R.K., Michaels, H.J. & Palmer, J.D. 1991 Phylogeny and character evolution in the Asteraceae based on chloroplast DNA restriction site mapping Syst. Bot. 16 98 115

    • Search Google Scholar
    • Export Citation
  • Jones, S.B. 1974 Vernonieae (Compositae) chromosome numbers Bull. Torrey Bot. Club 101 31 34

  • Jones, S.B. 1977 Vernonieae—Systematic review Heywood V.H., Harborne J.B. & Turner B.L. The biology and chemistry of the Compositae Vol. 1 Academic Press London, UK

    • Search Google Scholar
    • Export Citation
  • Jones, S.B. 1982 The genera of Vernonieae (Compositae) in the southeastern United States J. Arnold Arbor. 63 489 507

  • Keeley, S.C., Forsman, Z.H. & Chan, R. 2007 A phylogeny of the ‘evil tribe’ (Vernonieae: Compositae) reveals Old/New World long distance dispersal: Support from separate and combined congruent datasets (trnL-F, ndhF, ITS) Mol. Phylogenet. Evol. 44 89 103

    • Search Google Scholar
    • Export Citation
  • Keeley, S.C. & Jansen, R.K. 1994 Chloroplast DNA restriction site variation in the Vernonieae (Asteraceae), an initial appraisal of the relationship of New and Old World taxa and the monophyly of Vernonia Plant Syst. Evol. 193 249 265

    • Search Google Scholar
    • Export Citation
  • Kim, H.-G., Keeley, S.C., Vroom, P.S. & Jansen, R.K. 1998 Molecular evidence for an African origin of the Hawaiian endemic Hesperomannia (Asteraceae) Proc. Natl. Acad. Sci. USA 95 15440 15445

    • Search Google Scholar
    • Export Citation
  • Kim, K.-J. & Jansen, R.K. 1995 ndhF sequence evolution and the major clades in the sunflower family Proc. Natl. Acad. Sci. USA 92 10379 10383

  • Kumar, H., Mercykutty, V.C. & Srivastava, C.P. 1993 Fertility improvement in autotetraploids of pea—Selection for seed-set and disjunction index Plant Breeding 110 81 83

    • Search Google Scholar
    • Export Citation
  • Leitch, I.J. & Bennett, M.D. 2004 Genome downsizing in polyploid plants Biol. J. Linnean Soc. 82 651 663

  • Leitch, I.J., Soltis, D.E., Soltis, P.S. & Bennett, M.D. 2005 Evolution of DNA amounts across land plants (Embryophyta) Ann. Bot. (Lond.) 95 207 215

  • Levan, A., Fredga, K. & Sandberg, A.A. 1964 Nomenclature for centromeric position on chromosomes Hereditas 52 201 220

  • Martelotto, L.G., Ortiz, J.P.A., Stein, J., Espinoza, F., Quarin, C.L. & Pessino, S.C. 2007 Genome rearrangements derived from autopolyploidization in Paspalum sp Plant Sci. 172 970 977

    • Search Google Scholar
    • Export Citation
  • Mathew, P.M. & Mathew, A. 1983 Studies on the South Indian Compositae V. Cytotaxonomic consideration of the tribes Vernonieae and Eupatorieae Cytologia (Tokyo) 48 679 690

    • Search Google Scholar
    • Export Citation
  • McCollum, G.D. 1958 Comparative studies of chromosome pairing in natural and induced tetraploid Dactylis Chromosoma 9 571 605

  • Mecchia, M.A., Ochogavía, A., Selva, J.P., Laspina, N., Felitti, S., Martelotto, L.G., Spangenberg, G., Echenique, V. & Pessino, S.C. 2007 Genome polymorphisms and gene differential expression in a ‘back-and-forth’ ploidy-altered series of weeping lovegrass (Eragrostis curvula) J. Plant Physiol. 164 1051 1061

    • Search Google Scholar
    • Export Citation
  • Moscone, E.A., Baranyi, M., Ebert, I., Greilhuber, J., Ehrendorfer, F. & Hunziker, A.T. 2003 Analysis of nuclear DNA content in Capsicum (Solanaceae) by flow cytometry and feulgen densitometry Ann. Bot. (Lond.) 92 21 29

    • Search Google Scholar
    • Export Citation
  • Naranjo, C.A., Ferrari, M.R., Palmero, A.M. & Poggio, L. 1998 Karyotype, DNA content and meiotic behaviour in five South American species of Vicia (Fabaceae) Ann. Bot. (Lond.) 82 757 764

    • Search Google Scholar
    • Export Citation
  • Narasinga Rao, P.S.R.L. & Pantulu, J.V. 1982 Fertility and meiotic chromosome behavior in autotetraploid pearl millet Theor. Appl. Genet. 62 345 351

    • Search Google Scholar
    • Export Citation
  • Ozkan, H., Levy, A. & Feldman, M. 2001 Allopolyploidy-induced rapid genome evolution in the wheat (Aegilops-Triticum) group Trends Genet. 19 141 147

  • Pecinka, A., Suchánková, P., Lysak, M., Trávníček, B. & Doležel, J. 2006 Nuclear DNA variation among several Central European Koeleria taxa Ann. Bot. (Lond.) 98 117 122

    • Search Google Scholar
    • Export Citation
  • Petrov, D. 2001 Evolution of genome size: New approaches to an old problem Trends Genet. 19 141 147

  • Poggio, L., Gonzáles, G. & Naranjo, C.A. 2007 Chromosome studies in Hippeastrum (Amaryllidaceae): Variation in genome size Bot. J. Linn. Soc. 155 171 178

    • Search Google Scholar
    • Export Citation
  • Pringle, G.J. & Murray, B.G. 1993 Karyotypes and C-banding patterns in species of Cyphomandra Mart. ex Sendtner (Solanaceae) Bot. J. Linn. Soc. 111 331 342

    • Search Google Scholar
    • Export Citation
  • Rabakonandrianina, E. & Carr, G.D. 1987 Chromosome numbers of Madagascar plants Ann. Mo. Bot. Gard. 74 123 125

  • Raina, S.N., Parida, A., Koul, K.K., Salimath, S.S., Bisht, M.S., Raja, V. & Khostoo, T.N. 1994 Associated chromosomal DNA changes in polyploids Genome 37 560 564

    • Search Google Scholar
    • Export Citation
  • Raina, S.N. & Rees, H. 1983 DNA variation between and within chromosome complements of Vicia species Heredity 51 335 346

  • Rees, H. 1984 Nuclear DNA variation and the homology of chromosomes Grant W.F. Plant biosystematics Academic Press Toronto, Canada

  • Reeves, A. 2001 MicroMeasure: A new computer program for the collection and analysis of cytogenetic data Genome 44 439 443

  • Robinson, H. 1999a Generic and subtribal classification of American Vernonieae Smithsonian Contrib. Bot. 89 1 16

  • Robinson, H. 1999b Revision of paleotropical Vernonieae (Asteraceae) Proc. Biol. Soc. Washington (USA) 112 220 247

  • Robinson, H. 1999c Two new subtribes, Stokesiinae and Pacourininae, of the Vernonieae (Asteraceae) Proc. Biol. Soc. Wash. 112 216 219

  • Robinson, H. 2007 Vernonieal Kadereit J. & Jeffery C. Asterales. The families and genera of vascular plants Vol. 8 (K. Kubitzki, Series Ed.). Springer Berlin, Germany

    • Search Google Scholar
    • Export Citation
  • Romero Zarco, C. 1986 A new method for estimating karyotype asymmetry Taxon 35 526 530

  • Ruas, P.M., Ruas, C.F., Vieira, A.O.S., Matzenbacher, N.I. & Martins, N.S. 1991 Cytogenetics of genus Vernonia Schreber (Compositae) Cytologia (Tokyo) 56 239 247

    • Search Google Scholar
    • Export Citation
  • Santos, J.L., Alfarao, D., Sanchez-Moran, E., Armstrong, S.J., Franklin, F.C.H. & Jones, G.H. 2003 Partial diploidization of meiosis in autotetraploid Arabidopsis thaliana Genetics 165 1533 1540

    • Search Google Scholar
    • Export Citation
  • Shaked, H., Kashkush, K., Ozkan, H., Feldman, M. & Levy, A.A. 2001 Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome to wide hybridization and allopolyploid wheat Plant Cell 13 1749 1759

    • Search Google Scholar
    • Export Citation
  • Smith, G.L. & Jones S.B. Jr 1987 Cytotaxonomic studies of Piptocarpha subgenus Hypericoides (Compositae: Vernoniea) Rhodora 89 35 40

  • Song, K.M., Lu, P., Tang, K.L. & Osborn, T.C. 1995 Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution Proc. Natl. Acad. Sci. USA 92 7719 7723

    • Search Google Scholar
    • Export Citation
  • Srivastava, S. & Lavania, U.C. 1991 Evolutionary DNA variation in Papaver Genome 34 763 768

  • Swaminathan, M.S. & Sulbha, K. 1959 Multivalent frequency and seed fertility in raw and evolved tetraploids of Brassica campestris var. toria Plant Breeding 90 385 392

    • Search Google Scholar
    • Export Citation
  • Tate, J.A., Soltis, D.E. & Soltis, P.S. 2005 Polyploidy in plants 372 414 Gregory T.R. The evolution of the genome Elsevier Amsterdam, The Netherlands

  • Torrell, M. & Vallès, J. 2001 Genome size in 21 Artemisia L. species (Asteraceae, Anthemideae): Systematic, evolutionary and ecological implications Genome 44 231 238

    • Search Google Scholar
    • Export Citation
  • Wendel, J.F. 2000 Genome evolution in polyploids Plant Mol. Biol. 42 225 249

  • Wendel, J.F., Cronn, R.C., Johnston, J.S. & Price, H.J. 2002 Feast and famine in plant genomes Genetica 115 37 47

If the inline PDF is not rendering correctly, you can download the PDF file here.

Contributor Notes

We gratefully acknowledge Layne Snelling and Janet Dow for their superb technical assistance.

Graduate student. Current address: Center for Applied Genetic Technologies, University of Georgia, 111 Riverbend Road, Athens, GA 30602.

Director, J.C. Raulston Arboretum.

Senior Researcher.

To whom reprint requests should be addressed; e-mail jgbarb@uga.edu

  • View in gallery

    Somatic chromosomes of Stokesia laevis (A) ‘Peachies Pick’ (2n = 2x = 14) and (B) ‘Omega Skyrocket’ (2n = 4x = 28). Scale bar = 10 μm.

  • View in gallery

    Karyotype ideograms of Stokesia laevis. (A) Average karyotype of ‘Honeysong Purple’, ‘Alba’, ‘Peachies Pick’ (2n = 2x = 14). (B) ‘Omega Skyrocket’ (2n = 4x = 28). Metacentric chromosomes (AR < 1.7) in gray. Submetacentric chromosomes (AR ≥ 1.7) in black. Scale bar = 2 μm.

  • View in gallery

    Meiotic chromosome associations of diploid Stokesia laevis ‘Peachies Pick’, 7 ring bivalents at Metaphase I (2n = 2x = 14). Scale bar = 10 μm.

  • View in gallery

    Meiotic chromosome associations of triploid Stokesia laevis (2n = 3x = 21). Scale bar = 10 μm. (A) ‘Purple Parasols’ × ‘Omega Skyrocket’; 1 frying pan trivalent, 6 bivalents, 6 univalents at Metaphase I. (B) ‘Peachies Pick’ × ‘Omega Skyrocket’; 3 trivalents (2 frying pans + 1 Y-shape), 4 bivalents, 4 univalents at Metaphase I.

  • View in gallery

    Meiotic chromosome abnormalities observed in triploid hybrids of Stokesia laevis (2n = 3x = 21). Scale bar = 10 μm. (A) ‘Peachies Pick’ × ‘Omega Skyrocket’ with unbalanced balanced disjunction (12:9) at Anaphase I; arrow indicates possible chromosome bridge. (B) ‘Peachies Pick’ × ‘Omega Skyrocket’ with 10 laggards (circled) at Anaphase I. (C) ‘Purple Parasols’ × ‘Omega Skyrocket’, unbalanced disjunction (10:11) at Anaphase I.

  • View in gallery

    Meiotic chromosome associations of Stokesia laevis ‘Omega Skyrocket’ (2n = 4x = 28). Scale bar = 10 μm. Metaphase I configurations. (A) Four figure-8 quadrivalents and six bivalents; arrow indicates interlocking bivalents. (B) Four quadrivalents (2 figure-8s, 2 rings) and 6 bivalents. (C) Five quadrivalents (all figure-8s) and 4 bivalents (2 rings + 2 rods).

  • View in gallery

    Meiotic chromosome associations of tetraploids of Stokesia laevis (2n = 4x = 28). Scale bar = 10 μm. (A) Anaphase I of ‘Omega Skyrocket’; equal disjunction (14:14); dotted line indicates division line. (B) Telophase II of 4x-‘Peach Melba’; lagging chromosomes indicated by arrows.

  • View in gallery

    Flow cytometry histograms of different cytotypes of Stokesia laevis. Pisum sativum L. ‘Citrad’ is the internal standard. (A) ‘Honeysong Purple’ (2n = 2x = 14). (B) ‘Omega Skyrocket’ accession 100; naturally occurring tetraploid (2n = 4x = 28). (C) 4x-‘Purple Parasols’; synthetic autotetraploid (2n = 4x = 28).

  • Bailey, L.H. 1949 Manual of cultivated plants. Revised ed Macmillian New York, NY

    • Export Citation
  • Bennett, M.D., Bhandol, P. & Leitch, I.J. 2000 Nuclear DNA amounts in angiosperms and their modern uses—807 new estimates Ann. Bot. (Lond.) 86 859 909

    • Search Google Scholar
    • Export Citation
  • Bennett, M.D. & Leitch, I.J. 2005 Genome size evolution in plants 90 151 Gregory T.R. The evolution of the genome Elsevier Amsterdam, The Netherlands

  • Bennetzen, J.L. 2002 Mechanisms and rates of genome expansion and contraction in flowering plants Genetica 115 29 36

  • Bennetzen, J.L., Ma, J. & Devos, K.M. 2005 Mechanisms of recent genome size variation in flowering plants Ann. Bot. (Lond.) 95 127 132

  • Brandham, P.E. & Doherty, M.J. 1998 Genome size variation in the Aloaceae, an angiosperm family displaying karyotypic orthoselection Ann. Bot. Suppl. A 67 73

    • Search Google Scholar
    • Export Citation
  • Bremer, G. & Bremer-Reinders, D.E. 1954 Breeding of tetraploid rye in The Netherlands: Methods and cytological investigations Euphytica 3 49 63

  • Cerbah, M., Coulaud, J., Godelle, B. & Siljak-Yakovlev, S. 1995 Genome size, fluorochrome banding, and karyotype evolution in some Hypochoeris species Genome 38 689 695

    • Search Google Scholar
    • Export Citation
  • Crowley, J.G. & Rees, H. 1968 Fertility and selection in tetraploid Lolium Chromosoma 24 300 308

  • Dematteis, M. 1998 Karyotype analysis in some Vernonia species (Asteraceae) from South America Caryologia 51 279 288

  • Dematteis, M. 2002 Cytotaxonomic analysis of South American species of Vernonia (Vernonieae: Asteraceae) Bot. J. Linn. Soc. 139 401 408

  • Dematteis, M. & Fernández, A. 1998 Karyotypes of seven South American species of Vernonia (Asteraceae) Cytologia (Tokyo) 63 323 328

  • De Oliveira, V.M., Forni-Martins, E.R. & Semir, J. 2007 Cytotaxonomy or species of Vernonia, section Lepidaploa, group Axilliflorae (Asteraceae: Vernonieae) Bot. J. Linn. Soc. 154 99 108

    • Search Google Scholar
    • Export Citation
  • Dhesi, J.S. & Stalker, H.T. 1994 Enhancing techniques for studying mitotic peanut chromosomes Peanut Sci. 21 92 94

  • Dimitrova, D. & Greilhuber, J. 2000 Karyotype and DNA-content evolution in ten species of Crepis (Asteraceae) distributed in Bulgaria Bot. J. Linn. Soc. 132 281 297

    • Search Google Scholar
    • Export Citation
  • Doležel, J., Bartoš, J., Volgmayr, H. & Greilhuber, J. 2003 Nuclear DNA content and genome size of trout and human Cytometry 51 127 128

  • Emshwiller, E. 2002 Ploidy levels among species in the ‘Oxalis turberosa Alliance’ as inferred by flow cytometry Ann. Bot. (Lond.) 89 741 753

    • Search Google Scholar
    • Export Citation
  • Esteves Mansanares, M., Forni-Martins, E.R. & Semir, J. 2007 Cytotaxonomy of Lychnophoriopsis Sch. Bip. and Paralychnophora MacLeish species (Asteraceae: Vernonieae: Lychnophorinae) Bot. J. Linn. Soc. 154 109 114

    • Search Google Scholar
    • Export Citation
  • Garnatje, T., Vallès, J., Garcia, S., Hidalgo, O., Sanz, M., Canela, M.A. & Siljak-Yakovlev, S. 2004 Genome size in Echinops L. and related genera (Asteraceae, Cardueae): Karyological, ecological and phylogenetic implications Biocell 96 117 124

    • Search Google Scholar
    • Export Citation
  • Gaus, J., Werner, D. & Tallury, S. 2005 Polyploidy in stokes aster (Stokesia laevis) HortScience 40 1101 (abstr.).

  • Gettys, L.A. & Werner, D.J. 2002 Stokes aster HortTechnology 12 138 142

  • Gilles, A. & Randolph, L.F. 1951 Reduction in quadrivalent frequency in autotetraploid maize during a period of 10 years Amer. J. Bot. 38 12 17

  • Greilhuber, J., Doležel, J., Lysák, M.A. & Bennett, M.D. 2005 The origin, evolution and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to describe nuclear DNA contents Ann. Bot. (Lond.) 95 255 260

    • Search Google Scholar
    • Export Citation
  • Greilhuber, J., Temsch, E.M. & Loureiro, J.C.M. 2007 Nuclear DNA content measurement Doležel J., Greilhuber J. & Suda J. Flow cytometry with plant cells WILEY-VCH Verlag GmbH & Co KGaA, Weinheim, Germany

    • Search Google Scholar
    • Export Citation
  • Gunn, C.R. & White, G.A. 1974 Stokesia laevis. Taxonomy and economic value Econ. Bot. 28 130 135

  • Hazarika, M.H. & Rees, H. 1967 Genotypic control of chromosome behavior in rye. X. Chromosome pairing and fertility in autotetraploids Heredity 22 317 332

    • Search Google Scholar
    • Export Citation
  • Hilpert, G. 1957 Effect of selection for meiotic behavior in auto-tetraploid rye Hereditas 43 318 322

  • Hörandl, E. & Greilhuber, J. 2002 Diploid and autotetraploid sexuals and their relationships to apomicts in the Ranunculus cassubicus group: Insights from DNA content and isozyme variation Plant Syst. Evol. 234 85 100

    • Search Google Scholar
    • Export Citation
  • Huziwara, Y. 1962 Karyotype analysis in some genera of Compositae. VIII. Further studies on the chromosomes of Aster Amer. J. Bot. 49 116 119

  • Jansen, R.K., Michaels, H.J. & Palmer, J.D. 1991 Phylogeny and character evolution in the Asteraceae based on chloroplast DNA restriction site mapping Syst. Bot. 16 98 115

    • Search Google Scholar
    • Export Citation
  • Jones, S.B. 1974 Vernonieae (Compositae) chromosome numbers Bull. Torrey Bot. Club 101 31 34

  • Jones, S.B. 1977 Vernonieae—Systematic review Heywood V.H., Harborne J.B. & Turner B.L. The biology and chemistry of the Compositae Vol. 1 Academic Press London, UK

    • Search Google Scholar
    • Export Citation
  • Jones, S.B. 1982 The genera of Vernonieae (Compositae) in the southeastern United States J. Arnold Arbor. 63 489 507

  • Keeley, S.C., Forsman, Z.H. & Chan, R. 2007 A phylogeny of the ‘evil tribe’ (Vernonieae: Compositae) reveals Old/New World long distance dispersal: Support from separate and combined congruent datasets (trnL-F, ndhF, ITS) Mol. Phylogenet. Evol. 44 89 103

    • Search Google Scholar
    • Export Citation
  • Keeley, S.C. & Jansen, R.K. 1994 Chloroplast DNA restriction site variation in the Vernonieae (Asteraceae), an initial appraisal of the relationship of New and Old World taxa and the monophyly of Vernonia Plant Syst. Evol. 193 249 265

    • Search Google Scholar
    • Export Citation
  • Kim, H.-G., Keeley, S.C., Vroom, P.S. & Jansen, R.K. 1998 Molecular evidence for an African origin of the Hawaiian endemic Hesperomannia (Asteraceae) Proc. Natl. Acad. Sci. USA 95 15440 15445

    • Search Google Scholar
    • Export Citation
  • Kim, K.-J. & Jansen, R.K. 1995 ndhF sequence evolution and the major clades in the sunflower family Proc. Natl. Acad. Sci. USA 92 10379 10383

  • Kumar, H., Mercykutty, V.C. & Srivastava, C.P. 1993 Fertility improvement in autotetraploids of pea—Selection for seed-set and disjunction index Plant Breeding 110 81 83

    • Search Google Scholar
    • Export Citation
  • Leitch, I.J. & Bennett, M.D. 2004 Genome downsizing in polyploid plants Biol. J. Linnean Soc. 82 651 663

  • Leitch, I.J., Soltis, D.E., Soltis, P.S. & Bennett, M.D. 2005 Evolution of DNA amounts across land plants (Embryophyta) Ann. Bot. (Lond.) 95 207 215

  • Levan, A., Fredga, K. & Sandberg, A.A. 1964 Nomenclature for centromeric position on chromosomes Hereditas 52 201 220

  • Martelotto, L.G., Ortiz, J.P.A., Stein, J., Espinoza, F., Quarin, C.L. & Pessino, S.C. 2007 Genome rearrangements derived from autopolyploidization in Paspalum sp Plant Sci. 172 970 977

    • Search Google Scholar
    • Export Citation
  • Mathew, P.M. & Mathew, A. 1983 Studies on the South Indian Compositae V. Cytotaxonomic consideration of the tribes Vernonieae and Eupatorieae Cytologia (Tokyo) 48 679 690

    • Search Google Scholar
    • Export Citation
  • McCollum, G.D. 1958 Comparative studies of chromosome pairing in natural and induced tetraploid Dactylis Chromosoma 9 571 605

  • Mecchia, M.A., Ochogavía, A., Selva, J.P., Laspina, N., Felitti, S., Martelotto, L.G., Spangenberg, G., Echenique, V. & Pessino, S.C. 2007 Genome polymorphisms and gene differential expression in a ‘back-and-forth’ ploidy-altered series of weeping lovegrass (Eragrostis curvula) J. Plant Physiol. 164 1051 1061

    • Search Google Scholar
    • Export Citation
  • Moscone, E.A., Baranyi, M., Ebert, I., Greilhuber, J., Ehrendorfer, F. & Hunziker, A.T. 2003 Analysis of nuclear DNA content in Capsicum (Solanaceae) by flow cytometry and feulgen densitometry Ann. Bot. (Lond.) 92 21 29

    • Search Google Scholar
    • Export Citation
  • Naranjo, C.A., Ferrari, M.R., Palmero, A.M. & Poggio, L. 1998 Karyotype, DNA content and meiotic behaviour in five South American species of Vicia (Fabaceae) Ann. Bot. (Lond.) 82 757 764

    • Search Google Scholar
    • Export Citation
  • Narasinga Rao, P.S.R.L. & Pantulu, J.V. 1982 Fertility and meiotic chromosome behavior in autotetraploid pearl millet Theor. Appl. Genet. 62 345 351

    • Search Google Scholar
    • Export Citation
  • Ozkan, H., Levy, A. & Feldman, M. 2001 Allopolyploidy-induced rapid genome evolution in the wheat (Aegilops-Triticum) group Trends Genet. 19 141 147

  • Pecinka, A., Suchánková, P., Lysak, M., Trávníček, B. & Doležel, J. 2006 Nuclear DNA variation among several Central European Koeleria taxa Ann. Bot. (Lond.) 98 117 122

    • Search Google Scholar
    • Export Citation
  • Petrov, D. 2001 Evolution of genome size: New approaches to an old problem Trends Genet. 19 141 147

  • Poggio, L., Gonzáles, G. & Naranjo, C.A. 2007 Chromosome studies in Hippeastrum (Amaryllidaceae): Variation in genome size Bot. J. Linn. Soc. 155 171 178

    • Search Google Scholar
    • Export Citation
  • Pringle, G.J. & Murray, B.G. 1993 Karyotypes and C-banding patterns in species of Cyphomandra Mart. ex Sendtner (Solanaceae) Bot. J. Linn. Soc. 111 331 342

    • Search Google Scholar
    • Export Citation
  • Rabakonandrianina, E. & Carr, G.D. 1987 Chromosome numbers of Madagascar plants Ann. Mo. Bot. Gard. 74 123 125

  • Raina, S.N., Parida, A., Koul, K.K., Salimath, S.S., Bisht, M.S., Raja, V. & Khostoo, T.N. 1994 Associated chromosomal DNA changes in polyploids Genome 37 560 564

    • Search Google Scholar
    • Export Citation
  • Raina, S.N. & Rees, H. 1983 DNA variation between and within chromosome complements of Vicia species Heredity 51 335 346

  • Rees, H. 1984 Nuclear DNA variation and the homology of chromosomes Grant W.F. Plant biosystematics Academic Press Toronto, Canada

  • Reeves, A. 2001 MicroMeasure: A new computer program for the collection and analysis of cytogenetic data Genome 44 439 443

  • Robinson, H. 1999a Generic and subtribal classification of American Vernonieae Smithsonian Contrib. Bot. 89 1 16

  • Robinson, H. 1999b Revision of paleotropical Vernonieae (Asteraceae) Proc. Biol. Soc. Washington (USA) 112 220 247

  • Robinson, H. 1999c Two new subtribes, Stokesiinae and Pacourininae, of the Vernonieae (Asteraceae) Proc. Biol. Soc. Wash. 112 216 219

  • Robinson, H. 2007 Vernonieal Kadereit J. & Jeffery C. Asterales. The families and genera of vascular plants Vol. 8 (K. Kubitzki, Series Ed.). Springer Berlin, Germany

    • Search Google Scholar
    • Export Citation
  • Romero Zarco, C. 1986 A new method for estimating karyotype asymmetry Taxon 35 526 530

  • Ruas, P.M., Ruas, C.F., Vieira, A.O.S., Matzenbacher, N.I. & Martins, N.S. 1991 Cytogenetics of genus Vernonia Schreber (Compositae) Cytologia (Tokyo) 56 239 247

    • Search Google Scholar
    • Export Citation
  • Santos, J.L., Alfarao, D., Sanchez-Moran, E., Armstrong, S.J., Franklin, F.C.H. & Jones, G.H. 2003 Partial diploidization of meiosis in autotetraploid Arabidopsis thaliana Genetics 165 1533 1540

    • Search Google Scholar
    • Export Citation
  • Shaked, H., Kashkush, K., Ozkan, H., Feldman, M. & Levy, A.A. 2001 Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome to wide hybridization and allopolyploid wheat Plant Cell 13 1749 1759

    • Search Google Scholar
    • Export Citation
  • Smith, G.L. & Jones S.B. Jr 1987 Cytotaxonomic studies of Piptocarpha subgenus Hypericoides (Compositae: Vernoniea) Rhodora 89 35 40

  • Song, K.M., Lu, P., Tang, K.L. & Osborn, T.C. 1995 Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution Proc. Natl. Acad. Sci. USA 92 7719 7723

    • Search Google Scholar
    • Export Citation
  • Srivastava, S. & Lavania, U.C. 1991 Evolutionary DNA variation in Papaver Genome 34 763 768

  • Swaminathan, M.S. & Sulbha, K. 1959 Multivalent frequency and seed fertility in raw and evolved tetraploids of Brassica campestris var. toria Plant Breeding 90 385 392

    • Search Google Scholar
    • Export Citation
  • Tate, J.A., Soltis, D.E. & Soltis, P.S. 2005 Polyploidy in plants 372 414 Gregory T.R. The evolution of the genome Elsevier Amsterdam, The Netherlands

  • Torrell, M. & Vallès, J. 2001 Genome size in 21 Artemisia L. species (Asteraceae, Anthemideae): Systematic, evolutionary and ecological implications Genome 44 231 238

    • Search Google Scholar
    • Export Citation
  • Wendel, J.F. 2000 Genome evolution in polyploids Plant Mol. Biol. 42 225 249

  • Wendel, J.F., Cronn, R.C., Johnston, J.S. & Price, H.J. 2002 Feast and famine in plant genomes Genetica 115 37 47

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 337 30 3
PDF Downloads 50 19 3