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ASHS 2024 Annual Conference

 

Use of Flow Cytometry to Assess Total Genomic Content for Asclepias spp.

Authors:
Mary Lewis Department of Horticulture and Institute of Plant Breeding, Genetics and Genomics, University of Georgia, 1111 Miller Plant Science Building, Athens, GA 30602, USA

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John M. Ruter Department of Horticulture and Institute of Plant Breeding, Genetics and Genomics, University of Georgia, 1111 Miller Plant Science Building, Athens, GA 30602, USA

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Abstract

Interest in plant species that support pollinator health has been increasing in recent years. As a result, research into these historically overlooked species is increasing. One such taxon is milkweed (Asclepias spp.), a genus primarily native to North America that serves as an oviposition and food source for various pollinators, especially the monarch butterfly (Danaus plexippus L.). Although exhaustive research has been conducted on Asclepias flower morphology, seed production, and pollinator impact, little cytological work has been published. Knowing the genome size of species can predict their ability to hybridize and the potential of genetic variability within a genus. Our study used 15 different Asclepias species and four interspecific Asclepias hybrids, and the total genomic content was calculated using propidium iodide. We found the 2C genome size ranged from 0.65 to 1.24 picograms. To our knowledge, our research presents data on eight species with previously unknown genomic content and is the first to report 2C values for interspecific Asclepias hybrids.

Asclepias is a member of the Apocynaceae family, having 130 species native to North America, six to South America, and up to 250 species in Africa. (Blackwell 1964; Fishbein 1996, 2008; Goyder 2001; Stevens 1983; Woodson 1954; Wyatt and Broyles 1994). Known for its ecological importance to many migratory North American butterfly species (Brower 1969; Brower et al. 1972; Hutchings 1923; Malcolm 1991), the family possesses one of the most complex reproductive structures aside from orchids in the plant kingdom (Wyatt 1976). The family’s unique reproductive strategy has evolved to promote outcrossing in nature, yet interspecific hybridization has been reported in the past (Bookman 1984; Weitemier et al. 2015). Phylogenetic studies in Asclepias currently divide the genus into 17 clades (Agrawal and Fishbein 2006, 2008; Agrawal et al. 2008; Fishbein 1996; Fishbein et al. 2011). These previously published studies indicate a division across the family into broadly geographic clades that could imply a high degree of relatedness among species within these clades. However, not all species within clades share the same affinity toward interspecific hybridization, raising questions as to what other factors are limiting interspecific compatibility (Lewis et al. 2021). Although phylogenic relationships are tentative indicators of how genetically similar Asclepias spp. are to one another, flow cytometry (FCM) may offer additional insights into genetic similarities and fertility barriers among species used in conjunction with other species’ genomic comparison methods.

Flow cytometry is generally used to estimate genome size and general ploidy (Doležel et al. 2007). FCM research has been used in the Asclepiadoideae family (a subfamily of Apocynaceae) and Asclepias spp. to determine ploidy and total genomic content for several species. These species include Asclepias curassavica L., Asclepias fascicularis Decne., Asclepias incarnata L., Asclepias latifolia Torr., Asclepias salicifolia Lodd., Asclepias speciosa Torr., Asclepias syriaca L., Asclepias verticillata L., and Asclepias tuberosa L.; all being diploid (Darlington and Wylie 1956; Moyer 1936). In addition, 22 Asclepias species have a base chromosome count of 11 (2n = 2x = 22) (Chromosome Counts Database 2021; Darlington and Wylie 1956; Gadella et al. 1969; Heiser and Whitaker 1948). A 2001 study by Albers and Meve determined that in an analysis of more than 650 species within the Apocynaceae family, only 3% deviated from the x = 11 chromosome count, and polyploidy was not observed in any Asclepias spp. sampled. With only a small number of species in the family documented, there is still work to be done to ensure that ploidy levels are consistent across the species.

Total genomic content, measured ordinarily using propidium iodide (PI), is usually characterized by C-value. C-value is the number of base pairs (picograms) of DNA in a whole chromosome complement (Greilhuber 2005; Swift 1950). PI stain is then used, which binds to AT and GC regions, providing estimations of total DNA content and genome size with greater accuracy (Doležel et al. 2007).

Published reports documenting total genomic content for Asclepias have been minimal. Those species having total genomic content registered include A. curassavica L., A. incarnata, A. syriaca, A. tuberosa, and A. verticillata (Bai et al. 2012). The relatively small genome size of Asclepias is intriguing based on the available literature. In angiosperms, 1.4 to 3.5 pg (2C-value) is considered a very small to small genome (Soltis et al. 2003). Asclepias genome size (2C-values) ranges from 0.7 to 1.1 pg, with A. verticillata recorded as the smallest and A. tuberosa as the largest (Bai et al. 2012). Given that total DNA content among species with published genome sizes can vary from 40% to 70% (Verloove et al. 2017), it is essential to understand the range of sizes using a more extensive complement of species. However, the known differences in DNA content may help explain why there have been few documented naturally occurring interspecific hybrids, given the number of species present in North America (Kephart et al. 1988; Zonneveld 2019).

The objective of this study was to determine the total genomic content of 15 Asclepias species native to North America and four synthetic hybrids between the maternal parent A. tuberosa and pollen parents, including Asclepias hirtella, Asclepias purpurascens, A. speciosa, and A. syriaca. Results will increase the number of reported and documented species and could potentially be used to aid and identify hybridization opportunities between species in the future.

Materials and Methods

Plant material.

The species used and measured for this study were Asclepias amplexicaulis Sm., Asclepias angustifolia Schweigg, Asclepias asperula Decne., A. curassavica, A. fascicularis, A. hirtella Woodson, A. incarnata, A. latifolia, Asclepias linaria Cav., A. purpurascens L., A. speciosa, Asclepias subverticillata Gray, A. syriaca, A. tuberosa, Asclepias variegata L., and Asclepias viridis Walter. Four hybrids were also analyzed: A. tuberosa × A. hirtella, A. tuberosa × A. purpurascens, A. tuberosa × A. speciosa, and A. tuberosa × A. syriaca (Table 1). Seeds were obtained from various sources (Table 1) and subsequently cold-moist stratified for 30 d at 3 to 4 °C in a washed builder’s sand substrate. Upon removal from stratification, seeds were germinated in Jan 2020 at the University of Georgia Athens campus, College Station Greenhouse Complex (lat. 33.9480°N, long. 83.3773°W). Seeds were placed at surface level in 804 inserts (T.O. Plastics, Minneapolis, MN) containing 100% perlite (Carolina Perlite Co. Inc., Gold Hill, NC) to a depth of 5.2 cm, with a topdressing of vermiculite (TX401; BWI, Greer, SC) of 0.635 cm depth. Supplemental light was provided by light-emitting diode arrays (Fluence Spyder with PhysioSpec; Fluence Technologies Inc., Austin, TX), providing a photosynthetic photon flux density of 250 μmol·m−2·s−1 at the substrate surface and 14-h daylength (Albrecht and Lehmann 1991). After planting, seed trays were placed on a mist bench with a misting cycle applying municipal water (pH 6.2, alkalinity of 11 ppm) set at 6 s every 10 min. Greenhouse temperatures were maintained at 25 °C days and 18 °C at night. Upon germination and expansion of the first set of true leaves, seedlings were transplanted into 804 inserts (T.O. Plastics) held by 1020 greenhouse trays (T.O. Plastics) filled with 80% milled peat (Sungro Peat Moss Grower Grade Orange, Agwam, MA) and 20% perlite (Carolina Perlite Co. Inc.). Once established, seedlings were transplanted into 3.97 L (#1) containers (Classic 400; Nursery Supply, Agwam, MA) containing an 80% bark (3/8-inch particle size) and 20% milled peat (Foothills Compost, Gainesville, GA). Seedlings were irrigated with municipal water and fertilized twice a week with Peter’s 20N–4.4P–16.6K liquid soluble fertilizer (Scotts Co., Marysville, OH) at 100 ppm.

Table 1.

Asclepias species used and the seed source’s nursery locations. More than one seed source was used for this experiment to ensure the germination of at least one plant per species.

Table 1.

DNA C-values and internal standard.

Immature leaf tissue was used in the determination of DNA content and ploidy in this study. Samples were harvested from actively growing stems with newly expanded leaves 4 to 6 weeks after germination. Collection time varied slightly because of differences in leaf size among species. For consistent results, only one plant was used per species from which all samples were taken. Fresh leaf material was harvested and processed for each species immediately after collection. The internal standard used was Pisum sativum L. ‘Ctirad’. This genotype has a published C-value closest to the estimated C-value of Asclepias spp. (0.1–3.0 pg) (Bai et al. 2012). P. sativum ‘Ctirad’ was germinated and grown alongside Asclepias plants in Jan 2020, and newly expanded leaf tissue was harvested for use employing identical techniques.

CyStain PI Absolute P kit (Sysmex America Inc., Lincolnshire, IL) was used to prepare all samples. About 1 cm2 of the species of interest and the internal standard P. sativum ‘Ctirad’ were coarsely chopped with a new razor blade in 500 µL of iced extraction buffer in 100 × 15-mm petri dishes (Thermo Fisher Scientific, Waltham, MA) for 90 s. The solution was then filtered through a 30-μm filter (CellTrics, Sysmex America Inc.) and into a 3.5-mL sample tube (Sysmex America Inc.) to isolate somatic nuclei from other cellular debris. Without centrifuging, 2 mL of staining buffer was added, followed by 12 μL of PI and 6 μL of RNase stock solutions. PI-stained samples were incubated in darkness for at least 30 min at 4 °C (refrigerator temperature). Leaf samples estimating C-values were then run on a CytoFLEX S flow cytometer (Beckman Coulter, Hialeah, FL) operating at 488 nm at the University of Georgia Athens Center for Tropical and Emerging Global Diseases Cytometry Shared Resource Laboratory Coverdell Center. Five replicates of each accession were prepared and analyzed. A minimum of 5000 particles were analyzed for each sample. Sample runs were rejected if the coefficient of variation was greater than 3% (Marie and Brown 1993). Further analysis to determine differences between genome sizes across all species was performed with JMP statistical software (version 13.0; SAS Institute, Cary, NC). Data were analyzed to determine normality and homogeneity, and a one-way analysis of variance test was conducted. With a resulting P value of <0.0001, a separation of treatment means using Tukey’s honestly significant difference was then performed on the results. Only data showing significant differences (P ≤ 0.05) among treatments are reported.

The 2C-value was calculated using the following formula:
(meanofsamplepeak/meanofstandardpeak)×2CDNAamount(pg)ofthestandard.
Reported 2C DNA for P. sativum ‘Ctirad’ was 9.09 pg (Dirihan et al. 2013).

Results and Discussion

The 2C genome size ranged from 0.645 to 1.239 pg among species examined in this study (Table 2, Fig. 1), with A. tuberosa × A. hirtella hybrid having the largest genome size and A. subverticillata having the smallest. Those species classified as having small genome sizes (between 0.645 and 0.659 pg) included A. subverticillata (0.645 pg), A. curassavica (0.652 pg), A. angustifolia (0.66 pg), and A. fascicularis (0.69 pg) (Table 2, Fig. 1). A. linaria, A. purpurascens, and A. speciosa shared slightly larger genome sizes, ranging from 0.781 to 0.819 pg in size (Table 2). A. syriaca, A. variegata, and A. viridis had similar and slightly larger genome sizes, ranging from 0.91 to 0.954 pg (Table 2). Species with 2C levels ranging from 1.038 to 1.070 pg (Table 2) included A. latifolia, A. tuberosa × A. syriaca hybrid, A. tuberosa × A. speciosa hybrid, A. hirtella, and A. tuberosa × A. purpurascens hybrids. A. asperula and A. tuberosa had the largest DNA content of any species observed in this study, being 1.12 and 1.15 pg in size (Table 2, Fig. 1). The largest genome measured in this study was the hybrid between A. tuberosa and A. hirtella, being 1.239 pg in size (Table 2, Fig. 1).

Table 2.

The calculated 2C (pg) and million base pairs (Mbp) with standard deviation (SD) values for all 19 Asclepias species and hybrids.

Table 2.
Fig. 1.
Fig. 1.

The total genome size of all Asclepias species ranked by size in picograms (pg). Error bars indicate standard deviation. Blue-colored bars indicate hybrid species, purple indicates their pollen parents, green indicates the maternal parent (Asclepias tuberosa), silver indicates a pollen parent that failed to hybridize with A. tuberosa, and pink indicates all other species not used in hybridizations but still analyzed.

Citation: HortScience 58, 5; 10.21273/HORTSCI17137-23

Despite slightly varying methods in this study and the published work of Bai et al. (2012) and Kubešová et al. (2010), results were similar to the species analyzed in their studies. Using the same internal standard, Bai et al. (2012) estimated the DNA content of A. incarnata to have a 2C value of 0.7 pg, similar to our finding of 0.734 pg. In the Bai et al. (2012) study, A. tuberosa was 1.1 pg in size, similar to the results of this research at 1.15 pg. Similarly, for A. syriaca, the genome size estimated by Bai et al. (2012) was 0.9 pg, similar to this study’s estimated value of 0.91 pg. Kubešová et al. (2010) used a differing standard, Solanum lycopersicum L. (tomato), but still had a similar 2C value in A. syriaca (0.84 pg) to that measured in this study (0.91 pg). Bainard et al. (2012) used Glycine max (soybean) as an internal standard when estimating the DNA content of A. syriaca, yet still reported similar values (0.86 pg) to those in this study (0.91 pg). This consistency among results from multiple published reports using varying FCM protocols is encouraging. Furthermore, this consistency infers that results obtained on other taxa measured in this study can be presumed (at a minimum) precise.

Regarding ploidy, Kubešová et al. (2010) offer the only report on ploidy in Asclepias. In that study, A. syriaca was determined to be diploid with a genome size of 0.91 pg. Their calculated DNA content mirrored that of A. syriaca in this study and was in the range of all observed species (Table 2). Therefore, it can be assumed that the estimated ploidy of all other species observed in this study are diploid, as there appear to be no significantly larger genome sizes observed. Changes in ploidy (e.g., tetraploidy) can be observed through significantly greater DNA content estimations compared with their diploid counterparts in ornamental species with autopolyploid genotypes (Lattier et al. 2019; Rothleutner et al. 2016). Based on these data, the assumption of diploid was made, and the equation from Dirihan et al. (2013) tentatively confirmed that no polyploidy was observed in this study (Table 2). Further research attempts were made to visually karyotype via root and pollen squashes to verify and validate chromosome counts and estimated ploidy. However, clear images to make definitive chromosome counts were unsuccessful because of the species’ extremely small chromosomes. It did appear from this preliminary work that both A. syriaca and A. tuberosa × A. syriaca hybrids had the same chromosome counts (2n = 22). However, because of imperfect imaging, we do not base any results on those findings (Lewis 2021).

In a study by Lewis et al. (2021), interspecific hybrids were attempted using A. tuberosa as the maternal parent and seven other species as pollen parents. Of seven pollen-donor species, only four successfully hybridized with A. tuberosa: A hirtella, A. purpurascens, A. speciosa, and A. syriaca. A potential reason for the failure of A. curassavica, A. fascicularis, and A. incarnata (as paternal parents) to hybridize with A. tuberosa could be a difference in genome size. Referring to Table 2, with A. tuberosa as the maternal parent, species that failed to make successful hybrids with A. tuberosa (1.15 pg) did have smaller genome sizes (0.65–0.73 pg). In addition, based on the amplified fragment length polymorphism–based phylogeny study by Weitemier et al. (2015) in Asclepias, species unable to hybridize with A. tuberosa were in a more distantly related clade. Of the four species that did hybridize, all were located within about the same clade (K and L), whereas the three that failed to hybridize were significantly removed (clade F). Our results for genome size add an additional layer to predict future successful hybridizations. Knowing genetic and genome size similarities may be valuable when planning hybridization work. Although correlation does not equal causation, genome size may play a role in the success of controlled and natural interspecific hybridization events via a lack of chromosomal pairing.

Typically, successful interspecific hybrids between species have intermediate genome sizes, as seen in Cirsium, Cornus, and Magnolia (Bureš et al. 2004; Parris et al. 2010; Shearer and Ranney 2013). Interestingly, the hybrid A. tuberosa × A. hirtella measured in this study deviated from this trend, having a genome size greater than either parent (Table 2). Further research is needed to assess why the genome was larger, as this deviates from normal hybridization behavior. One possible avenue for future research would be to examine karyotypes and gene copy numbers from genome sequencing. In doing so, it would be possible to confirm if there is or is not polyploidization and to delve deeper into the specific reasons for the increase in genomic content. Genome sizes calculated during this project should be helpful for plant breeders in predicting which species of Asclepias might have enhanced potential for success when attempting interspecific hybridization. Our research serves as a reference and starting point and reveals that Asclepias species have a small reference genome size that varies among species.

This study documents the total genomic content of 15 Asclepias species and four hybrids. Generally, the range of Asclepias species tends to be between 0.65 pg and 1.24 pg. Based on previous research and results discovered in this study, it is also concluded that the estimated ploidy of all observed Asclepias species are diploids. Thus, there appear to be barriers to hybridization, whereby genome size is a good indicator of the potential success or failure of a natural or controlled hybridization event. In the future, breeders may be able to use genome size as a guide to identifying likely parents for successful crosses. These findings provide a starting point for future breeding efforts and expand the general knowledge surrounding Asclepias species. In doing so, impacts will benefit the commercial market and pollinator relationships with Asclepias. Improving the variability of morphological features, the range at which the species can be found, and vigor would positively affect the monarch butterfly and other pollinators.

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  • Fig. 1.

    The total genome size of all Asclepias species ranked by size in picograms (pg). Error bars indicate standard deviation. Blue-colored bars indicate hybrid species, purple indicates their pollen parents, green indicates the maternal parent (Asclepias tuberosa), silver indicates a pollen parent that failed to hybridize with A. tuberosa, and pink indicates all other species not used in hybridizations but still analyzed.

  • Agrawal AA & Fishbein M. 2006 Plant defense syndromes Ecology. 87 S132S149

  • Agrawal AA & Fishbein M. 2008 Phylogenetic escalation and decline of plant defense strategies Proc Natl Acad Sci USA. 105 1005710060

  • Agrawal AA, Lejeunesse MJ & Fishbein M. 2008 Evolution of latex and its constituent defensive chemistry in milkweeds (Asclepias) Entomol Exp Appl. 128 126138

    • Search Google Scholar
    • Export Citation
  • Albers F & Meve U. 2001 A karyological survey of Asclepiadoideae, Periplocoideae, and Secamonoideae, and evolutionary considerations within Apocynaceae sl. Ann Missouri Bot Garden. 88 4 624656 https://doi.org/10.2307/3298637

    • Search Google Scholar
    • Export Citation
  • Albrecht ML & Lehmann JT. 1991 Daylength, cold storage, and plant-production method influence growth and flowering of Asclepias tuberosa HortScience. 26 120121 https://doi.org/10.21273/HORTSCI.26.2.120

    • Search Google Scholar
    • Export Citation
  • Bai C, Alverson WS, Follansbee A & Waller DM. 2012 New reports of nuclear DNA content for 407 vascular plant taxa from the United States Ann Bot. 110 16231629 https://doi.org/10.1093/aob/mcs222

    • Search Google Scholar
    • Export Citation
  • Bainard JD, Bainard LD, Henry TA, Fazekas AJ & Newmaster SG. 2012 A multivariate analysis of variation in genome size and endoreduplication in angiosperms reveals strong phylogenetic signal and association with phenotypic traits New Phytol. 196 12401250 https://doi.org/10.1111/j.1469-8137.2012.04370.x

    • Search Google Scholar
    • Export Citation
  • Blackwell WH. 1964 Synopsis of the 23 species of Asclepias (Asclepiadacea) in Tamaulipas and Nuevo Leon including two new species, Asclepias bifida and Asclepias prostrata Southwest Nat. 9 171180

    • Search Google Scholar
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Mary Lewis Department of Horticulture and Institute of Plant Breeding, Genetics and Genomics, University of Georgia, 1111 Miller Plant Science Building, Athens, GA 30602, USA

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John M. Ruter Department of Horticulture and Institute of Plant Breeding, Genetics and Genomics, University of Georgia, 1111 Miller Plant Science Building, Athens, GA 30602, USA

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Contributor Notes

We thank Julie Nelson, the lab manager at the University of Georgia Coverdell Center, for her guidance and support for this project. In addition, we appreciate the contributions of Alana Edwards and Rebekah Maynard for their assistance in machinery training and sample preparation techniques required for data collection.

J.M.R. is the corresponding author. E-mail: ruter@uga.edu.

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