Crop Science 42:192-196 (2002)
© 2002 Crop Science Society of America
TURFGRASS SCIENCE
Ploidy Determination in Agrostis Using Flow Cytometry and Morphological Traits
Stacy A. Bonos*,
Karen A. Plumley and
William A. Meyer
Dep. of Plant Science, 59 Dudley Road, Foran Hall, New Brunswick, NJ 08901-8520
* Corresponding author (bonos{at}eden.rutgers.edu)
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ABSTRACT
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The taxonomic classification of the genus Agrostis is one of the most complicated of the grass genera. Classification based upon morphological and anatomical characters is difficult and complicated by the presence of intermediate forms and the misapplication of names. Determining ploidy levels of new germplasm can assist in species determination and is necessary before initiating breeding or genetics studies. The objectives of this study were to (i) evaluate the use of laser flow cytometry as a quick, reliable tool to determine ploidy level and aid in Agrostis species determination, and (ii) identify morphological characters associated with DNA content or ploidy level. The six Agrostis species evaluated were A. canina L. subsp. canina, A. canina L. subsp. montana (Hartm.) Hartm., A. palustris Huds. [= A. stolonifera var. palustris (Huds.) Farw.], A. tenuis Sibth. (= A. capillaris L.), A. castellana Boiss. & Reut., and A. alba L. Ploidy level was determined by flow cytometry and root tip chromosome counts. Plant height, panicle height, flag leaf length, flag leaf width, and highest internode length of mature field-grown spaced plants were measured. Significant differences in 2C DNA content were found between species (P < 0.01) differing in ploidy level. Flow cytometry was effective in differentiating between diploid, tetraploid, and hexaploid species. Chromosome numbers previously reported and those observed in this study were positively correlated with 2C nuclear DNA content (r = 0.98, P < 0.01). Flag leaf length was the only morphological measurement taken that was significantly positively correlated to DNA content (r = 0.98, P < 0.001). The results of this study indicate that laser flow cytometry is a quick, reliable tool to determine ploidy levels and infer certain species of Agrostis. This technique will aid breeders to quickly and accurately determine ploidy levels of new germplasm collections.
Abbreviations: CRBC, chicken red blood cell stock • PBS, phosphate buffered saline
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INTRODUCTION
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THE TAXONOMIC CLASSIFICATION of the genus Agrostis is one of the most complicated of the grass genera. Distinguishing Agrostis species and subspecies was difficult in the past because the species are very similar to one another and often confused in botanical works (Piper, 1918). Even today, with better references of the taxonomic classifications (Hitchcock, 1971), the similarities between species and the known existence of hybrids and natural variation (Bradshaw, 1957; Stuckey and Banfield, 1946) make separating species difficult (Monteith, 1930; Shildrick, 1976). This is especially problematic to turfgrass breeding programs interested in studying several Agrostis species.
The common Agrostis species used for turf range in ploidy level from diploid to hexaploid, and can naturally form interspecific semi-sterile hybrids (Bradshaw, 1957; Wipff and Rose-Fricker, 2000). Keith Jones was the first researcher to extensively study the genus and the hybrids that form between species (Jones, 1956a, b,c). He conducted classic cytogenetic studies and used the hybrids to determine cytological relationships between Agrostis species. He determined that velvet bentgrass (A. canina L. subsp. canina) is a diploid (2n = 2x = 14) that regularly forms seven bivalents, and that brown velvet bentgrass (A. canina L. subsp. montana (Hartm.) Hartm.) is most likely an autotetraploid (2n = 4x = 28), possibly derived from chromosome duplication within or between ecotypes of the diploid subsp. canina (Jones, 1956a). He found that colonial bentgrass (A. tenuis Sibth. = A. capillaris L.) is a segmental allotetraploid (2n = 4x = 28), with one of its ancestors belonging to A. canina L. Creeping bentgrass [A. palustris Huds. = A. stolonifera var. palustris (Huds.) Farw.] is a strict allotetraploid (2n = 4x = 28) with well-differentiated genomes, with one of the ancestral diploid type in common with colonial bentgrass, and the other genome of unknown origin (Jones, 1956b). Warnke et al.'s (1998) work using isozyme analysis of creeping bentgrass supports Jones' observation of the strict allotetraploid behavior of creeping bentgrass. Jones also concluded that the hexaploid redtop (A. alba L.) (2n = 6x = 42) shares homology with both the tetraploids: creeping and colonial bentgrass (Jones, 1956c). Jones determined that hybrids of these species are detected by differences in chromosome number and type of chromosome pairing. Although these techniques can identify hybrids, species, and ploidy levels, cytogenetics is difficult, labor intensive, and requires long hours of microscope evaluations. A quick and dependable way to determine ploidy level in this genus is needed to improve efficiency of species identification.
Morphological traits are also used in species identification (Hitchcock, 1971). However, in many situations morphological traits are highly variable and cannot definitively determine species. One of the biggest taxonomic problems within the Agrostis genus is ‘Highland’ bentgrass. Highland bentgrass, until recently, was treated as a cultivar of colonial bentgrass in the U.K. (Shildrick, 1976), but Highland is on the borderline of the morphological characters described for colonial bentgrass, and is very distinctive in turfgrass performance (Shildrick, 1976). The features of Highland bentgrass are quite distinctive from other cultivars of colonial bentgrass, which include vigorous spread by rhizomes, formation of aerial tillers under high mowing height, a prominent, jagged ligule twice the size of normal colonial bentgrass and similar in size to creeping bentgrass, blue-green leaf color, tall open growth habit, and good winter color (Shildrick, 1976). Scholz (1965) considered Highland bentgrass to be an aberrant form, which may be a distinct species belonging to dryland bentgrass (A. castellana Boiss. & Reut.) based on plant and seed examination. The description of dryland bentgrass, particularly leaf color and heading records, suggest that dryland bentgrass is very similar to Highland bentgrass (Shildrick, 1976). Those who suggest Highland bentgrass is similar to brown velvet bentgrass, however, dispute this classification. Dryland bentgrass is reported as a hexaploid with 2n = 6x = 42 chromosomes (Bjorkman, 1954), while brown velvet bentgrass and colonial bentgrass are reported as tetraploids with 2n = 4x = 28 chromosomes (Jones, 1956b). If flow cytometry can determine ploidy levels quickly, then it may be easy to discern the taxonomic identity of Highland bentgrass as well as other Agrostis species with different ploidy levels.
These taxonomic problems between Highland bentgrass and colonial bentgrass, as well as other Agrostis species, is especially evident under spaced plant nursery situations. Bentgrass breeding programs interested in breeding several Agrostis species collect germplasm and grow individual plants in spaced plant nurseries. Morphological differences in some plants grown in nursery situations make species identification difficult, especially between dryland bentgrass and colonial bentgrass, between colonial bentgrass and creeping bentgrass, and even between colonial and velvet bentgrass in some cases. Some individual aberrant plants have characteristics intermediate of either species, which could lead to misclassification of individual plants. Misclassified plants with possibly different chromosome numbers that become incorporated into crossing blocks lead to sterility and species hybridization. Quick, reliable ploidy identification of plant collections may reduce misclassification of plant material, improve species identification of aberrant plants, and improve efficiency of Agrostis breeding programs.
Stuckey and Banfield (1946) reported on morphological variations in some species of Agrostis in Rhode Island. They identified A. capillaris and A. alba using the standard reference of nomenclature at the time (Manual of Grasses – Hitchcock, 1935). A. capillaris was identified on the basis of a short truncated ligule and more delicate growth habit compared with the pointed ligule and course texture of A. alba. Some plants in their study were A. capillaris in all respects except for one or two characters. For example, a long pointed ligule or dense basal branches on the panicle. Other plants showed a blending of one or more characters. They found chromosome counts of plants to range from 28 to 42 with no correlation between morphological type and chromosome number.
Some morphological measurements such as ligule and leaf texture are quick and easy to identify, while other morphological measurements used for species determination, such as panicle type or heading differences, can be time consuming, labor intensive, and time-specific, and most are not completely definitive, especially if a blending of traits is present. Ploidy determination using flow cytometry reduces the need for many morphological measurements and chromosome counts, and flow cytometry can be used on seedlings or mature plants with rapid, reliable results. Flow cytometry has effectively determined ploidy level in other turfgrass species (Arumuganathan et al., 1999; Huff and Bara, 1993; Huff and Palazzo, 1998; Johnson et al., 1998; Treadway and Huff, 1998; Velguth and White, 1992) and other plant species (Arumugananthan and Earle, 1991; Brummer et al., 1999; Vogel et al., 1999). Huff and Palazzo (1998) showed that flow cytometry effectively distinguished certain fine fescue species. The objective of this study was to (i) evaluate the use of laser flow cytometry as a quick, reliable tool to determine ploidy level and identify certain Agrostis species, and (ii) identify morphological traits, such as those used in Plant Variety Protection applications, that may be associated with ploidy level. Rapid determination of ploidy levels of new collections and morphologically aberrant plants, using flow cytometry, would allow breeding programs to develop productive crossing blocks that contain plants with similar chromosome numbers.
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MATERIALS AND METHODS
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Plant Material
Six Agrostis species, 1 diploid, 3 tetraploids, and 2 hexaploids were evaluated in this study: Velvet bentgrass (2n = 2x = 14), brown velvet bentgrass (2n = 4x = 28), creeping bentgrass (2n = 4x = 28), colonial bentgrass (2n = 4x = 28), dryland bentgrass (2n = 6x = 42), and redtop (2n = 6x = 42). Plant material was acquired from a number of different sources. The redtop and dryland bentgrass seed lots were acquired from the former Agribiotech, Inc. The velvet and colonial bentgrasses were acquired from the USDA-ARS Plant Introduction collection in Pullman, WA. Seeds were germinated under greenhouse conditions with natural daylength in Promix BX potting soil then transplanted to a spaced plant nursery trial established on 1 July 1997 on a Nixon loam (fine-loamy, mixed, mesic Typic Hapludult). Mowing was withheld to allow inflorescence development and anthesis. The nursery received a total of 4.6 g m-2 of N as 10–4.5–8.3 (N–P–K) on 24 Oct 1997. Irrigation was applied to the trial only to avoid severe wilt stress.
Flow Cytometry
Three to ten genotypes of each of the six species were evaluated using flow cytometry. The samples were prepared for flow cytometry using a combination of techniques reported in Arumuganathan and Earle (1991), and Huff and Palazzo, (1998). Fully expanded leaves were collected from the field-grown plants and washed in distilled water. The leaves (200-mg fresh weight) were placed in a plastic petri dish with 2 mL of chopping buffer [per liter: 4.26 g MgCl2, 8.84 g sodium citrate, 4.23 g morpholinepropanesulfonic acid, pH of the buffer was adjusted to 7.0 with 5 M sodium hydroxide, 3.0 mL Triton X-100, 50 µL propidium iodide, and 30 mL chicken red blood cell stock (CRBC) (acquired from Pocono Rabbit Farm, Canadensis, PA)]. Each step was performed on ice. Leaves were chopped with a scalpel to 
0.3 mm. One mL of the chopping buffer containing leaf nuclei was filtered through 30-µm nylon mesh into 5-mL test tubes. Two milliliters of phosphate buffered saline (PBS) [per liter: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 dissolved in 1 L of dH2O, adjusted to pH 7.4 with HCl] was added to each test tube. The test tubes were centrifuged in a desktop centrifuge at 10 000 rpm for 5 min. The supernatant was aspirated and the pellet was resuspended in 2 mL of PBS. The sample was centrifuged a second time under the same conditions. The supernatant was aspirated and the pellet was resuspended in 1 mL of PBS. At this time, 10µL of propidium iodide [1 mg 1 mL-1 dH2O] and 1 µL RNase A (1 mg mL-1 dH2O) were added. Samples were analyzed at 488 nm with an Epics Profile flow cytometer (Coulter Corp., Miami, FL). The plant nuclear 2C DNA content, measured in picograms, was determined using the following ratio:
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At least two replicated flow cytometry measurements of 2C DNA content from separate nuclei extractions were taken for each of the genotypes representing the six Agrostis species within in each flow cytometric run. "Run" refers to the entire set of samples analyzed on the flow cytometer on a specific date. Three complete flow cytometry runs were averaged to obtain the mean 2C DNA content for each species.
Morphological Measurements
The use of morphological traits for species determination in Agrostis is not always definitive. Stuckey and Banfield (1946) evaluated ligule length, panicle type, and texture, and found no correlation between morphological type and chromosome number. The morphological measurements, plant height, panicle length, flag leaf length, flag leaf width, and highest internode length were evaluated in this study to identify traits that may be associated with ploidy level, and subsequently used to aid in species determination. Morphological measurements were taken on 16 June 1998, 10 d after anthesis, based on the parameters specified in the USDA Plant Variety Protection application form for Bentgrass (USDA, Beltsville, MD). One plant height measurement per plant was made from the soil surface to the top of the third tallest panicle. Five panicle length measurements per plant were taken from the flag leaf to the top of the panicle. Three flag leaf blade length measurements per plant were taken from the ligule to the tip of the leaf blade. Three flag leaf blade width measurements per plant were taken at the widest point of the leaf. Flag leaf length and width measurements were chosen randomly from the plants, using the same leaf for length and width measurements. Highest internode length measurements were taken from the base of the plant to the tallest internode.
Cytology
Ploidy determination by chromosome counts was performed on at least 5 genotypes from A. canina (diploid), A. capillaris and A. palustris (tetraploid), and A. castellana and A. alba (hexaploid). Since two tetraploid species were counted, A. canina subsp. Montana was not counted. Chromosome counts were conducted on root tips of newly-germinated seeds. Seeds were germinated under greenhouse conditions with natural daylength in Promix BX potting soil. Seedlings were grown for 3 wk. Root tips were collected hourly to identify the time of day when the maximum number of metaphase cells was observed. The collected root tips were fixed in a 2:1 ethanol:glacial acetic acid solution for at least 2 h. The tips were washed in dH2O and enzyme digested for 1.5 h at 37°C in a solution of 5 mg cellulase, 1.0 mL pectinase, and 5 mg drysilase dissolved in 15 mL of Sodium Acetate, pH 4.8. The tips were washed in dH2O and stored in the fixative solution described above. Samples were analyzed immediately. Chromosomes were treated with acetocarmine stain and viewed under x1000 magnification with phase contrast microscopy.
Statistical Analysis
All data were subjected to analysis of variance for a randomized complete block design. Species means were separated using Tukey's studentized range test at P 
0.05. DNA content (2C) was correlated with reported and observed chromosome numbers. Mean morphological measurements for each species were also correlated with mean DNA content and reported and observed chromosome numbers.
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RESULTS AND DISCUSSION
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Observed chromosome counts were in agreement with those previously reported for the five turfgrass species (Table 1) (Fig. 1)
. Various protocols for root tip chromosome counts suggest collecting root tips in the early morning to maximize the number of cells in metaphase. Results from this study indicate that Agrostis species may differ in the time of day of cell division. For example, A. alba and A. castellana had a high concentration of dividing cells when cells were fixed at 0730 h, A. canina sub. canina and A. capillaris at 1330 h, and A. palustris at 1730 h.
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Table 1. Mean 2C DNA content for genotype samples of six Agrostis species having different 2n chromosome numbers.
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Fig. 1. Microscopic views of grass root tips in metaphase indicating chromosome numbers of (A) velvet bentgrass (2n = 2x = 14), and (B) creeping bentgrass (2n = 4x = 28), magnification at x1000.
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Flow cytometry analysis of intact leaf nuclei demonstrated an accurate ability to measure DNA content and determine ploidy level of Agrostis species. DNA content was significantly positively correlated with reported and observed chromosome numbers (r = 0.98, P < 0.01). Significant differences in 2C DNA content were found between diploid, tetraploid, and hexaploid Agrostis species (Table 1). The relative fluorescence signal intensities reflecting differences in DNA content between Agrostis ploidy levels is illustrated in Fig. 2
. The diploid species had an average nuclear DNA content of 3.42 pg, the tetraploid species had an average of 5.81 pg, and the hexaploid species had an average of 8.45 pg. The nuclear DNA content of the tetraploid species is in agreement with the nuclear DNA content of creeping bentgrass ‘Penncross’ reported by Arumugananthan et al. (1999). Most importantly, flow cytometry was effective in distinguishing between colonial bentgrass, a tetraploid, and dryland bentgrass, a hexaploid, indicating Highland bentgrass can be distinguished from colonial bentgrass using laser flow cytometry.
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Fig. 2. Histogram of propidium iodide fluorescence intensity of leaf tissue from Agrostis species indicating relative differences in signal intensity of Agrostis ploidy levels compared with the internal standard.
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Noticeably, the tetraploid DNA content is less than two times the diploid DNA content, and the hexaploid is less than three times the diploid DNA content; this could be because of genome differences among species, or possibly the deletion of repetitive DNA in the higher ploidy levels.
Although flow cytometry was not as effective in distinguishing between species within the same ploidy level, colonial bentgrass tended to have a higher DNA content than creeping bentgrass (Table 1) (This was significant on two sampling dates – data not shown). To confirm this, flow cytometry studies should be repeated with other genotypes of each species to determine potential genome differences between the two species by studying the GC:AT ratio. Results of this study support the correlation results of other research, indicating that flow cytometry can simply, dependably, and effectively distinguish between ploidy levels and therefore identify certain species of Agrostis that differ in ploidy levels.
Morphological measurements were also correlated to ploidy level. Correlations between morphological measurements and DNA content, and between morphological measurements and chromosome number were similar; therefore, only the correlations to DNA content are reported. Flag leaf length was the only morphological measurement significantly positively correlated to DNA content and chromosome number with an r-value of 0.98 (P 0.001) (Table 2). No other morphological measurements were associated with DNA content or chromosome number (Table 2). This data indicates that flag leaf length of mature spaced plants is a good indicator of ploidy level. The other morphological measurements evaluated in this study, along with those by Stuckey and Banfield (1945), indicate that these morphological measurements are not good indicators of ploidy level or DNA content.
Although there was no correlation between most of the morphological measurements and DNA content, significant (P = 0.05) differences in morphological measurements were found between Agrostis species (Table 3). There were significant differences between colonial bentgrass and dryland bentgrass based on the morphological measurements evaluated in this study. These measurements, however, along with flag leaf length measurements, are taken after anthesis and flowering. These measurements are based on mature plants and are not effective in distinguishing between these two species prior to anthesis when crossing blocks need to be developed. Flow cytometry is used effectively on seedlings and immature plants. The success of flow cytometry in distinguishing between species that differ in ploidy level is important for breeding programs because it can identify the ploidy of morphologically aberrant plants before anthesis so that plants with similar chromosome numbers can be chosen for the development of productive crossing blocks. This research identifies the potential benefits of flow cytometry as a quick and reliable alternative to chromosome counts and morphological measurements for ploidy determination and certain species determination in Agrostis.
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Table 3. Morphological measurements of six Agrostis species taken from spaced plants in a field trial established in July 1998.
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NOTES
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Research was supported by the Rutgers Center for Turfgrass Science, New Jersey Agricultural Experiment Station, and the New Jersey Turfgrass Association. Journal No. D-12264-1-01.
Received for publication January 19, 2001.
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ABSTRACT
INTRODUCTION
