Abstract
Rudbeckia L. are valuable nursery crops that offer broad adaptability and exceptional ornamental merit. However, there is little information on interspecific and interploid crossability and ploidy levels of specific cultivars. The objectives of this study were to determine the ploidy levels and relative DNA contents (genome sizes) of selected species and cultivars, to evaluate self-compatibility and crossability among species and ploidy levels, and to explore reproductive pathways in triploid R. hirta L. with the goal of facilitating future breeding endeavors and development of new hybrids. Reciprocal interspecific crosses were performed between R. hirta cultivars and R. fulgida Ait., R. missouriensis Engelm. ex C.L. Boynton & Beadle, and R. subtomentosa Pursh. as well as reciprocal interploid crosses among four R. hirta cultivars. A combination of relative DNA content analysis and chromosome counts was used to test for hybridity and to determine ploidy levels for selected species, cultivars, and interploid R. hirta F1 hybrids. Of the specific clones tested, R. subtomentosa and R. missouriensis were diploid, R. fuligida varieties were tetraploid, and R. hirta include both diploid and tetraploid cultivars. Mean 1Cx DNA content varied over 320% among species. The interploid R. hirta crosses produced triploids as well as pentaploids and hexaploids. Seedlings from open-pollinated triploid R. hirta appeared, based on diverse phenotypes and DNA contents, to be aneuploids resulting from sexual fertilization, not apomixis. Of the 844 seedlings from interspecific F1 crosses, only one individual, R. subtomentosa ×R. hirta, had a DNA content intermediate between its parents and was confirmed as the only interspecific hybrid. Although most taxa had low self-fertility, seedlings (with genomic sizes similar to their maternal parent) resulted after interspecific crosspollination, indicating that pseudogamy is one reproductive pathway in Rudbeckia species.
The genus Rudbeckia contains ≈30 species of annuals, biennials, and perennials known for their colorful, golden ray corollas and prominent disk-shaped receptacles that can bloom from midsummer through October. Rudbeckia are popular crops and are valued for their ornamental diversity, low-maintenance requirements, and heat and drought resistance.
Rudbeckia include two genetically and morphologically distinct subgenera: Macrocline and Rudbeckia (Urbatsch et al., 2000). Species in the subgenus Rudbeckia include many desirable ornamentals and range from opportunistic annuals to persistent perennials. These species generally flourish in full sun and well-drained soils and are commonly found in recently disturbed habitats such as roadsides or old fields and thrive during primary and secondary succession. The annual species R. hirta includes cultivars with a diverse range of flower colors and forms, including bicoloration and double inflorescences (greater number of ray florets). Petal colors range from a lemon yellow and gold exhibited by ‘Prairie Sun’ to the deep red and mahogany expressed by ‘Cherokee Sunset’. However, R. hirta is short-lived and susceptible to diseases, including rhizoctonia blight (Rhizoctonia sp.) and cercospora leaf spot (Cercospora sp.) (Fulcher et al., 2003; Harkess and Lyons, 1994). Other Rudbeckia species, including R. fulgida, R. missouriensis, and R. subtomentosa, are reliable perennials with superior disease resistance and good performance in southern climates (Armitage, 1997). Interspecific hybridization between durable perennial species and showier annual species could lead to development of valuable new cultivars.
There has been little published on the genetics and breeding of Rudbeckia. Species in Rudbeckia subgenus Rudbeckia have a base chromosome number of x = 19 and polyploids have been reported to exist in R. hirta, R. fulgida var. speciosa, and R. triloba L. (McCrea, 1981; Urbatsch et al., 2000). However, there is little information available on ploidy levels of specific cultivars. Pseudogamy, a form of apomixis, has also been implicated as the sole means of reproduction in triploid R. triloba (McCrea, 1981). A greater understanding of reproductive pathways and occurrence of apomixis would provide valuable information for both breeding and commercial propagation of Rudbeckia.
The objectives of this research were to determine ploidy levels and relative DNA contents of selected species and cultivars, to evaluate self-compatibility and crossability between species, to determine interploid crossability among R. hirta cultivars, and to assess reproductive pathways in triploid R. hirta to better facilitate future Rudbeckia breeding projects.
Materials and Methods
Cytology.
Chromosome counts were performed on R. hirta ‘Toto Gold’, R. hirta ‘Indian Summer’, R. missouriensis, R. subtomentosa, and R. fulgida var. sullivantii ‘Goldsturm’ to calibrate relative DNA content with ploidy level for each species. Root tips were collected and placed in 2 mm 8-hydroxyquioline for 3 to 5 h at 12 °C. Roots were then rinsed with 4 °C distilled water and placed in a 6:3:1 solution of 95% ethanol:chloroform:glacial acetic acid fixative for 24 h at 20 °C. Samples were rinsed and stored in 70% ethanol solution at 4 °C. The next week, samples were removed from storage and transferred to a 30% aqueous ethanol solution for 12 min followed by two 15-min rinses in distilled water. Roots were then hydrolyzed for 1 h at 20 °C in 1 N HCl, followed by a quick rinse in distilled water, and were placed in Feulgen stain for 2 h at room temperature. Root tips were excised and placed on a glass microscope slide with a drop of 1% aceto-carmine stain, squashed with a coverslip, and viewed at ×1500.
Flow cytometry.
Holoploid, 2C DNA contents (i.e., DNA content of the entire nonreplicated, chromosome complement regardless of ploidy level) were determined through flow cytometry for all the parental taxa and progeny (Doležel et al., 1998). Approximately 1.5 cm2 of leaf or petal tissue was chopped with a razor blade in a petri dish containing 400 μL of extraction buffer (CyStain ultraviolet Precise P; Partec, Münster, Germany). The suspension was filtered through 50 μm nylon mesh and nuclei were stained using 1.6 mL staining buffer containing 4′, 6-diamidino-2-phenylindole (DAPI) (CyStain ultraviolet Precise P; Partec). The suspension was analyzed using a flow cytometer with fluorescence excitation provided by a mercury arc lamp (PA-I Ploidy Analyzer; Partec). The mean fluorescence of each sample was compared with an internal standard of known genome size (Pisum sativum L. ‘Ctirad’, 2C = 8.76 ρg; Greihuber et al., 2007). Base 1Cx monoploid DNA content (i.e., DNA content of the nonreplicated base set of chromosomes with x = 19) was calculated for each species as 2C DNA content/ploidy level. DNA content data were subjected to analysis of variance and means were separated using Tukey's honestly significant difference procedure.
Breeding and pollinations.
Taxa selected for breeding included 10 R. hirta cultivars, three R. fulgida varieties, R. missouriensis, and R. subtomentosa (Table 1). Plants were grown in a glass-covered greenhouse in 2006, 2007, and 2008 using standard horticultural practices. Forty-three interspecific crosses were completed in a greenhouse with at least four pollinated inflorescences per cross. The greenhouse was screened to exclude insect pollinators. All pollinations were performed daily by hand and each individual plant was involved in only one reciprocal cross. Pollinations included reciprocal crosses between R. hirta cultivars and the remaining species. Two reciprocal interploid crosses were also performed between R. hirta ‘Cherokee Sunset’ × R. hirta ‘Toto Rustic’ and R. hirta ‘Prairie Sun’ × R. hirta ‘Goldilocks’. Self-pollinations were also performed on separate inflorescences of selected representative taxa: R. hirta ‘Goldilocks’, R. hirta ‘Toto Gold’, R. hirta ‘Toto Rustic’, R. hirta ‘Cherokee Sunset’, R. fulgida var. fulgida, R. fulgida var. speciosa, R. fulgida var. sullvantii ‘Goldsturm’, and R. subtomentosa. Inflorescences were pollinated daily until all disk florets passed anthesis. Achenes were collected after flower senescence and sown. Triploid, F1 interploid progeny were selected and subjected to open-pollination. Achenes from five inflorescences per clone were collected and sown. The reproductive pathway (i.e., apomixis versus sexual) of this population was determined based on relative DNA content and phenotypic variation.
Relative DNA content, ploidy level, and chromosome number of selected Rudbeckia taxa from subgenus Rudbeckia.
Results and Discussion
Chromosome counts documented that R. hirta ‘Toto Gold’ was a diploid (2n = 2x = 38) and R. hirta ‘Indian Summer’ was a tetraploid (2n = 4x = 76) with an average 1Cx value of 3.8 ρg (Table 1). Seedlings of R. missouriensis and R. subtomentosa were confirmed to be diploids with 1Cx values of 8.8 ρg and 11.0 ρg, respectively. Rudbeckia fulgida var. sullivantii ‘Goldsturm’ was found to be tetraploid with a 1Cx value of 8.9 ρg. Based on these standards, ploidy levels of the remaining cultivars were then estimated for each species (Table 1). Although we did not find any diploid R. fulgida, McCrea (1981) reported finding diploid populations of R. fulgida, particularly in the southern portion of its range.
Mean 1Cx DNA content was similar among cultivars of R. hirta regardless of ploidy level (i.e., there was no apparent genome downsizing at higher ploidy levels). However, 1Cx DNA content varied over 320% among species. This considerable variation in 1Cx DNA content among closely related species suggests that recent and substantial changes in genome size have occurred in this subgenus. Accumulation of transposable elements, particularly long terminal repeat retrotransposons, can rapidly accumulate over short evolutionary timeframes (Hawkins et al., 2008) and may have occurred in some species of Rudbeckia. This large variation in DNA content also emphasizes the need to calibrate DNA content with ploidy level separately for each species within this subgenus and provides a convenient way to confirm hybridity in seedlings of interspecific crosses.
Self-compatibility was generally found to be low for all taxa selected for self-pollinations. Rudbeckia hirta ‘Goldilocks’, ‘Toto Rustic’, ‘Cherokee Sunset’, and R. fulgida var. speciosa produced no viable seedlings after selfing. Rudbeckia hirta ‘Toto Gold’ produced 1.2 seedlings per inflorescence, whereas R. fulgida var. fulgida and R. fulgida var. sullvantii ‘Goldsturm’ both produced 0.3 seedlings per inflorescence (Table 2) after selfing. It could not be determined if the limited number of seedlings produced after self-pollinations resulted from low levels of self-compatibility or apomixis.
Seedlings per inflorescence and reproductive pathways of self-pollinated and interspecific crosses varying in ploidy level.
Crosses between species yielded a mean of zero to 23 seedlings per inflorescence with a total of 844 seedlings (Table 2). However, 2C DNA contents of all but one of these seedlings were similar to the maternal parent, suggesting these plants arose through either self-fertilization or apomixis. In some crosses (e.g., R. hirta ‘Goldilocks × R. subtomentosa, R. hirta ‘Cherokee Sunset’ × R. missouriensis), numerous seedlings, with DNA contents similar to the maternal parent, were recovered, although these taxa had very low fertility when selfed. Pseudogamy, a process whereby crosspollination from a different genotype stimulates apomixis, may be involved and has been recorded previously in R. triloba L. (3x) and R. fulgida (4x) (McCrea, 1981). Overall, interspecific crossability among these species was found to be extremely low with production of only one successful hybrid: R. subtomentosa (2C = 21.9 ρg) × R. hirta ‘Toto Gold’ (2C = 7.2 ρg), which yielded a seedling with an intermediate 2C DNA content of 14.7 ρg supporting hybridity (Table 2). Cytology also documented two sets of 19 chromosomes of disparate size, consistent with both parents (Fig. 1). McCrea (1981) also found poor crossability among species in this subgenus and reported that among interspecific crosses attempted with six species, the only successful hybrids were between R. missouriensis and R. fulgida.
Photomicrograph of Rudbeckia subtomentosa (larger chromosomes) × R. hirta ‘Toto Gold’ (smaller chromosomes) H2007-062-001 nucleus with 2n = 2x = 38.
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.44
Photomicrograph of Rudbeckia subtomentosa (larger chromosomes) × R. hirta ‘Toto Gold’ (smaller chromosomes) H2007-062-001 nucleus with 2n = 2x = 38.
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.44
Photomicrograph of Rudbeckia subtomentosa (larger chromosomes) × R. hirta ‘Toto Gold’ (smaller chromosomes) H2007-062-001 nucleus with 2n = 2x = 38.
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.44
Interploid crosses among R. hirta cultivars resulted in 98 viable seedlings, 53 of which were triploids (Table 3). Rudbeckia hirta ‘Cherokee Sunset’ (4x) × R. hirta ‘Toto Rustic’ (2x) produced two triploid, 39 tetraploid (most likely from pseudogamy), two pentaploid, and one hexaploid offspring. Rudbeckia hirta ‘Toto Rustic’ (2x) × R. hirta ‘Cherokee Sunset’ (4x) produced 18 triploids and three tetraploids. The larger than expected ploidy in some individuals may be the result of unreduced gametes in one or both parents. Rudbeckia hirta ‘Prairie Sun’ (4x) × R. hirta ‘Goldilocks’ (2x) resulted in 17 triploids, and the reciprocal cross generated 16 triploids. Interploid cross progeny exhibited intermediate traits.
Ploidy levels of progeny resulting from interploid crosses.
Open-pollination of five inflorescences from each of 13 triploid selections resulted in recovery of 51 seedlings with a broad range of DNA contents (Fig. 2). One population of progeny ranged in ploidy from 2x to 2x + 5 (≈5 extra chromosomes based on DNA content) and most likely resulted from sexual reproduction of triploids crossing with diploid R. hirta plants in the vicinity. A second population of progeny ranged from 3x to 3x + 5 and most likely resulted from either apomixis or sexual reproduction among triploids resulting in aneuploids. There were also four progeny with ploidy levels of 4x or higher and most likely resulted from the union of unreduced gametes (from one or both parents, including outcrosses with diploids or tetraploid R. hirta in the vicinity). Progeny from triploid parents exhibited a broad range of unique phenotypic characteristics, distinct from their respective maternal female parents, further suggesting these plants arose primarily from sexual reproduction and not apomixis.
Frequency distribution of ploidy levels of progeny derived from open-pollinated, triploid Rudbeckia hirta. Ploidy level was estimated based on relative DNA content with each chromosome averaging 0.19 ρg.
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.44
Frequency distribution of ploidy levels of progeny derived from open-pollinated, triploid Rudbeckia hirta. Ploidy level was estimated based on relative DNA content with each chromosome averaging 0.19 ρg.
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.44
Frequency distribution of ploidy levels of progeny derived from open-pollinated, triploid Rudbeckia hirta. Ploidy level was estimated based on relative DNA content with each chromosome averaging 0.19 ρg.
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.44
Results from the cytology and cytometry component of this research documented ploidy levels and relative DNA contents of selected species and cultivars of Rudbeckia. Breeding and crossability components determined a high level of self-incompatibility among these taxa and found that apomixis (pseudogamy) appears to be more prevalent after interspecific pollination in some taxa. Interploid crosses between 2x and 4x R. hirta produced 3x progeny regardless of cross direction. Triploid plants maintained limited fertility, including sexual reproduction through the formation of both unreduced and aneuploid gametes. Although successful interspecific hybridization was rare and difficult to achieve, one diploid interspecific hybrid of R. subtomentosa × R. hirta was verified. Additional efforts to produce interspecific hybrids may lead to development of improved cultivars.
Literature Cited
Armitage, A.M. 1997 Herbaceous perennial plants: A treatise on their identification, culture, and garden attributes 2nd Ed Stipes Champaign, IL
Doležel, J., Greilhuber, J., Lucretti, S., Meister, A., Lysák, M.A., Nardi, L. & Obermayer, R. 1998 Plant genome size estimation by flow cytometry: Inter-laboratory comparison Ann. Bot. (Lond.) 82 17 26
Fulcher, A., Dunwell, W.C. & Wolfe, D. 2003 Rudbeckia taxa evaluation Proc. Southern Nursery Assn. Res. Conf., 48th Annu. Rpt 510 512
Greihuber, J., Temsch, E.M. & Loureiro, J.M. Doležel J., Greilhuber J. & Suda J. 2007 Nuclear DNA content measurement Flow cytometry with plant cells: Analysis of genes, chromosomes and genomes Wiley-VCH Weinheim, Germany 67 101
Harkess, R.L. & Lyons, R.E. 1994 Rudbeckia hirta L.: A versatile North American wildflower HortScience 29 134 227
Hawkins, J.S., Grover, C.E. & Wendel, J.F. 2008 Repeated big bangs and the expanding universe: Directionality in plant genome size evolution Plant Sci. 174 557 562
McCrea, K.D. 1981 Ultraviolet floral patterning, reproductive isolation and character displacement in the genus Rudbeckia (Compositae) PhD Diss Purdue Univ West Lafayette, IN
Urbatsch, L.E., Baldwin, B.G. & Donoghue, M.J. 2000 Phylogeny of the coneflowers and relatives (Heliantheae: Asteraceae) based on nuclear rDNA internal transcribed spacer (ITS) sequences and chloroplast DNA restriction site data Syst. Bot. 25 539 565
