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TURFGRASS SCIENCE

Determination of the Level of Variation in Polyploidy among Kentucky Bluegrass Cultivars by Means of Flow Cytometry

^a Dep. of Entomology, Univ. of Wisconsin-Madison, 1630 Linden Dr., Madison, WI 53706
^b Dep. of Plant Pathology, Univ. of Wisconsin-Madison, 1630 Linden Dr., Madison, WI 53706

^* Corresponding author (jung{at}plantpath.wisc.edu)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Kentucky bluegrass (Poa pratensis L.) is an important cool-season grass species in the turfgrass and forage industries. Understanding the genetics of Kentucky bluegrass is useful in developing improved cultivars and hybrids. However, studying the genetics of Kentucky bluegrass can often be difficult because of the high variation in ploidy level that results from its facultative apomictic reproductive nature. Flow cytometry provides an easy and accurate method for assessing this variation by quantifying DNA content. The purpose of our study was to determine the level of variation in ploidy in Kentucky bluegrass by analyzing its DNA content using flow cytometry. In addition, DNA content was compared with genetic similarity derived from DNA marker data, and was also correlated with chromosome number. Twenty-two cultivars of Kentucky bluegrass were selected for the study by considering the range of variability in morphological traits and genetic distance derived from DNA marker data. We found that the DNA content of Kentucky bluegrass genotypes from the 22 cultivars ranged from 5.39 to 17.69 pg of DNA/2C and that a majority of the genotypes had a DNA content value in the range of 7 to 13 pg. A significant correlation between DNA content and chromosome number was detected. Euploid chromosome numbers (x = 7) with a range from the pentaploid (2n = 5x = 35) to the quindecaploid (2n = 15x = 105) were found along with aneuploid numbers. The results of this research could aid both breeders and researchers in studying the genetics of the species and in improving Kentucky bluegrass cultivars via intra- and interspecific hybridizations.

Abbreviations: RAPD, random amplified polymorphic DNA

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
KENTUCKY BLUEGRASS is a cool-season grass that is used in many different environments. Kentucky bluegrass has a number of characteristics that make it highly valuable to various industries. It is a hardy grass that produces strong, rhizomatous growth, and it is also a very aggressive grass. These characteristics make it ideal for uses in forage areas, lawns, and golf courses. As a forage grass, Kentucky bluegrass is used for grazing on >15 million ha of pasture throughout the northeastern and north central USA and substantial areas of Europe and Canada (Bashaw and Funk, 1987). On golf courses, it is commonly used in fairways and roughs. It is also widely used for home lawns, where it is blended with other cultivars or with other turfgrass species. The importance of Kentucky bluegrass to the various turfgrass industries necessitates extensive research with improvement as the goal. Understanding the genetics of Kentucky bluegrass is the key to improving varieties through the selection of desirable traits. However, research into the genetics of Kentucky bluegrass can be difficult because of a complex genome that can affect many different variables. For instance, genome size has adaptive significance and influences phenotype by the expression of its genic content and by the physical effects of its mass and/or volume (Bennett and Leitch, 1995). In angiosperms, DNA amount has been shown to correlate with a wide range of important characters such as minimum generation time and ecological behavior. Large variations in ploidy level exist within and between cultivars because of the facultative apomictic nature of reproduction in Kentucky bluegrass. Therefore, it is not possible to assign a specific ploidy level to Kentucky bluegrass cultivars. The production of reduced and unreduced eggs accounts for the differences in chromosome number. Research has demonstrated that the chromosome number in Kentucky bluegrass can vary from 28 to 140, which corresponds to euploid chromosome numbers with x = 7 (Löve and Löve, 1975; Barcaccia et al., 1997).

The ability to quickly estimate DNA amount would facilitate Kentucky bluegrass research by allowing the inclusion or exclusion of DNA amount as a variable in an experiment. Estimating DNA amount can also be valuable to plant breeders who are trying to detect true hybrids from crosses. One tool that has proven useful in estimating DNA amount in Kentucky bluegrass and other plant species is flow cytometry. Flow cytometry is an analytical technique that can be used to study many different aspects of cytology and genetics. For example, it can be used to study the cell cycle by determining the proportions of cells in G₁, S, and G₂/M stages of the cell cycle, thus allowing the calculation of cell cycle times (Marie and Brown, 1993). It is also useful in performing tasks such as chromosome sorting and quantification of DNA content. Thus, flow cytometry enables a quick estimation of DNA amount by quantifying nuclear DNA content. Flow cytometry has the advantage of being faster and less labor intensive than other methods of DNA content estimation, such as the microdensitometer technique and cytological chromosome counts.

Flow cytometry has been used to quantify the DNA content in many different plant species (Arumuganathan and Earle, 1991a), including a variety of turfgrass species. This technique determines DNA content in terms of the C value, which refers to the DNA amount of the haploid genome of an individual (Bennett and Leitch, 1995). Thus, DNA/2C refers to the DNA in the nucleus of a diploid individual. Arumuganathan et al. (1999) used flow cytometry to quantify the DNA content in 13 species of turfgrass. They reported that the genomes of warm-season turfgrasses were smaller than the genomes of cool-season turfgrasses regardless of the ploidy level. Flow cytometry was also used to quantify DNA content in buffalograss [Buchloe dactyloides (Nutt.) Engelm. (=Bouteloua dactyloides (Nutt.) Columbus)] (Johnson et al., 1998) and 10 species of fine fescue (Festuca spp.) (Huff and Palazzo, 1998) as a means of distinguishing between ploidy levels and species, respectively.

For Kentucky bluegrass, Huff and Bara (1993) compared flow cytometry with silver-stained RAPD (random amplified polymorphic DNA) markers for the ability to determine the origins of aberrant progeny from a facultative apomictic reproduction system ranging from nearly obligate apomixis to complete sexuality. Huff and Bara (1993) suggested a strong correlation between DNA content and chromosome number in Kentucky bluegrass. This correlation was expressed in a linear regression of three data points in which 11 pg of DNA/2C corresponded with 82 to 90 chromosomes, 5 pg corresponded with 38 to 44 chromosomes, and 16 pg corresponded with at least 125 chromosomes. Recently, Barcaccia et al. (1997) also used flow cytometry to estimate the DNA content of Kentucky bluegrass. They correlated DNA content with chromosome number and found that 4.76 pg DNA/2C corresponded to 40 chromosomes, 5.04 pg corresponded to 44 chromosomes, 6.54 pg corresponded to 56 chromosomes, and 6.69 pg corresponded to 58 chromosomes.

Although valuable, the studies performed by Huff and Bara (1993) and Barcaccia et al. (1997) focused primarily on the comparison of flow cytometry with DNA marker based techniques. Furthermore, they only considered a limited number of Kentucky bluegrass cultivars and genotypes, and only accounted for a few of the numerous possible ploidy levels in Kentucky bluegrass. The establishment of an accurate correlation between DNA content and ploidy levels would be of great value in estimating the ploidy level of unknown samples using flow cytometry. Therefore, our research objectives were focused on estimating the variation of ploidy level in a larger, genetically diverse group of Kentucky bluegrass cultivars by virtue of DNA content using flow cytometry, and subsequently establishing a precise regression equation between the DNA content and the ploidy levels.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Plant Materials
Twenty-two genotypes representing each of 22 Kentucky bluegrass cultivars were selected for analysis in our study (Table 1). Genotypes were selected based on two factors. First, we selected cultivars to represent each of 12 morphologically classified types of cultivars based on a system of classification that was developed by Murphy et al. (1997) and updated by Bonos et al. (2000). This system of classification groups cultivars together based on morphological characteristics as well as resistance to certain diseases. We selected two cultivars as representatives of each type, with the exceptions of the Other and Common types, for which we only had one cultivar available from each (Table 1). The second factor that we used to select cultivars was genetic relatedness estimates, which facilitated choosing two cultivars with the most genetic diversity within each type. The consideration of both morphology and genetic relatedness was necessary because Johnson et al. (2002) demonstrated that molecular and agronomic characterization was needed for a comprehensive assessment of Kentucky bluegrass diversity.

The seedlings of each genotype were allowed to reproduce vegetatively through tillers and rhizomes to provide enough tissue for the flow cytometry analysis. Consequently, the genotypes used to represent a cultivar had originated from a single seed of that cultivar. However, because of DNA content variation within each cultivar, the DNA amounts obtained for these genotypes are not necessarily the same for all genotypes within the respective cultivar. This variation can result from mitotic and meiotic chromosome number aberrations, as well as production of nonapomictic off-type seedlings. Most of the genotypes used in the flow cytometry analysis were included in the genetic relationship study with the exceptions of the genotypes representing the Caliber, Julia, and Shamrock cultivars. These three cultivars were included so their respective morphological types were represented with two cultivars.

Flow Cytometry
All plants used were cultured in a greenhouse where they were watered twice weekly and fertilized at a rate of 0.03358 kg N/m²/yr. Young leaf tissue was excised from each Kentucky bluegrass genotype. Leaf tissue was also excised from ‘Belle’ soybean and used as the standard. A standard has a known DNA content, and the flow cytometer uses the standard as a reference for determining the DNA content of an unknown sample. Soybean was selected because it had been previously analyzed and determined to have a DNA content of 2.31 pg DNA/2C (Arumuganathan and Earle, 1991a), which falls below the range of DNA content for Kentucky bluegrass. Should the standard have a DNA content similar to that of any of the Kentucky bluegrass genotypes, the standard will interfere with the generation of data for that particular genotype.

The nuclear DNA content of the genotypes was determined using the flow cytometry protocol described by Arumuganathan and Earle (1991b). To reduce the amount of debris generated from the soybean tissue, we made a slight modification to the protocol by preparing each sample with 5 mg of soybean leaf tissue and 55 mg of leaf tissue from the respective Kentucky bluegrass genotype. This modification uses less standard tissue and more Kentucky bluegrass tissue than the original protocol, yet maintains the same amount of total tissue (60 mg). The generation of data by the flow cytometer was not impacted by this change in protocol.

Respective tissue was placed in a plastic Petri dish with 1 mL of propidium iodide-MgSO₄ chopping buffer [10 mM MgSO₄, 15 mM KCl, 5 mM HEPES (N-2-hydroethylpiperazine-N'-2-ethansulfonic acid)], 1.0 mg/mL dithiothreitol, 20 µL propidium iodide stock (5 mg/mL), and 25 µL Triton X-100 stock (10% w/v). The leaf tissue was sliced into small pieces with a scalpel. The solution containing the pieces of tissue was next filtered through a 30-µm nylon mesh and centrifuged at 14000 rpm for 20 s. The pellet was resuspended in 400 µL of the chopping buffer and 0.93 µL of DNase-free RNase. Each sample consisted of a solution containing intact nuclei from soybean and Kentucky bluegrass. The samples were incubated in a 37°C water bath for 15 min and then analyzed at the Flow Cytometry Facility of the University of Wisconsin Comprehensive Cancer Center using a FACSScan flow cytometer (Becton Dickinson Immunocytometry System, San Jose, CA) with a 15-mW, 488-nm laser. For each sample, a total of 1000 nuclei were collected and analyzed. The data were represented as a histogram consisting of two peaks which were referred to as G₀/G₁ peaks. The nuclear DNA content of the Kentucky bluegrass genotypes was determined by taking the ratio of the positions of the G₀/G₁ peaks for the soybean standard and the Kentucky bluegrass genotype and then multiplying the subsequent ratio by the DNA content of the standard. For each genotype, three samples were prepared independently and analyzed on the flow cytometer. The mean DNA content and standard deviation was calculated for each genotype and regressed against chromosome number.

Cytology
Chromosome counting was conducted on six genotypes that cover a wide range of DNA content values and represent the Brilliant, Caliber, Eclipse, Kenblue, Northstar, and Rugby II cultivars. Chromosome numbers were visually determined using the protocol described by Cheng et al. (2001). In brief, cells were isolated by first excising root tips from the six genotypes. Root tips were placed in a 0.002-M hydroxyquinoline solution for 2 h and were then transferred into Carnoy solution (3 parts methanol to 1 part glacial acetic acid). Next, cells were lysed in a solution of 2% cellulase and 1% pectolyase and kept at 37°C for 40 min. Cells were then fixed on a precooled microscope slide and chromosomes were viewed using phase contrast microscopy. For each genotype, three cells showing distinct chromosomes were selected for visual chromosome counts (Fig. 1) . The mean chromosome number of the three cells from each genotype was used for subsequent analyses.

View larger version (102K): [in this window] [in a new window]   Fig. 1. Photographs showing mitotic metaphase chromosomes prepared from the meristematic area of root tips of each genotype representing the following Kentucky bluegrass cultivars: (A) ‘Eclipse’, (B) ‘Rugby II’, and (C) ‘Caliber’, with 39, 65, and 101 chromosomes, respectively.

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Our first objective was to evaluate the range of DNA content (pg/2C) in Kentucky bluegrass using flow cytometry (Table 1). The values for DNA content range from 5.39 ± 0.03 pg DNA/2C in the genotype representing the Eclipse cultivar to 17.69 ± 0.65 pg DNA/2C in the genotype representing the Shamrock cultivar. However, it is likely that these values vary between individuals with each cultivar. These results support previous observations that Kentucky bluegrass has large variations in DNA content (Huff and Bara, 1993; Barcaccia et al., 1997) and in chromosome number (Speckmann and Vandijk, 1972). Interestingly, 91% of the genotypes tested fall in the range of 7 to 13 pg DNA/2C. One possible explanation is that seed-producing cultivars that are highly apomictic and have the optimal morphological traits might also have DNA contents that fall within this range. As mentioned previously, DNA content has been demonstrated to affect numerous plant traits. Therefore, plant breeders may have indirectly selected cultivars within this range of DNA content while searching for cultivars with desired agronomical traits. A statistically significant difference in the 2C DNA content was observed among the genotypes within each morphologically based type with the exceptions of the Compact, Compact-Midnight, and Compact-America types (Table 1). More cultivars under those types need to be evaluated to validate these findings.

Our second objective was to establish a reliable regression line between DNA content and chromosome number. If a correlation were found, then chromosome numbers of unknown germplasm from either plant introductions or naturalized populations could be estimated using flow cytometry. Chromosome counts demonstrated that the mean chromosome numbers for six genotypes representing the six cultivars of Eclipse, Kenblue, Brilliant, Rugby II, Northstar, and Caliber were 37.3 ± 1.5, 53.3 ± 3.1, 53.3 ± 3.2, 63.7 ± 4.2, 91.0 ± 4.6, and 108.0 ± 6.2, respectively (Fig. 1). These values probably vary within each cultivar. A simple linear regression of these data against the respective DNA contents for these genotypes suggests a strong linear correlation (R² = 0.973, P < 0.001) between DNA content and chromosome number, as expected (Fig. 2) . The linear regression equation estimated is Y (nuclear DNA content, pg/2C) = 0.102 x (number of chromosomes) + 1.898. This correlation supports previous findings that DNA content as determined by flow cytometry can be used as a means for determining ploidy level in Kentucky bluegrass.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Regression analysis of nuclear DNA content (pg/2C) estimated using flow cytometry and mean chromosome number of each genotype representing the following six Kentucky bluegrass cultivars: Eclipse, Kenblue, Brilliant, Rugby II, Northstar, and Caliber.

Since Löve and Löve (1975) reported euploid chromosome numbers of 28 to 140 for Kentucky bluegrass, we attempted to perform a linear regression of the estimated number of chromosomes based on DNA content against the expected chromosome number in a polyploidy series with x = 7 (Fig. 3) . The estimated number of chromosomes was determined by using the regression equation from Fig. 2 to estimate the chromosome number of each Kentucky bluegrass genotype based on DNA content. The expected chromosome numbers in a polyploidy series with x = 7 would simply be multiples of seven. We found that complete euploid chromosome numbers within a range of 35 to 105, except for 42, are predicted by our results (R² = 0.988, P < 0.001). However, some level of variation in chromosome number exists within each ploidy level, suggesting that some genotypes in our study may be aneuploids. The observed variation has been reported previously (Speckmann and Vandijk, 1972) and may be attributable to mitotic and meiotic aberrations (Porceddu et al., 2002).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Regression line between estimated number of chromosomes based on DNA content (pg/2C) and expected number of chromosomes in a series of euploids with x = 7 in Kentucky bluegrass.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Comparison of regression lines of nuclear DNA content (pg/2C) and chromosome number as reported by Huff and Bara (1993), Barcaccia et al. (1997), and the current study.

We tested two independent regression lines (the current study and Barcaccia et al., 1997) with one combined line by forcing both intercepts to be the same but with different slopes, as well as by forcing both slopes to be the same but with different intercepts. The results indicated that an extra slope estimated from the combined data did not improve the regression when two intercepts were forced to be the same. In other words, the slopes in both lines were not significantly different from each other. However, if both lines were forced to have the same slope, the fit of the regression was improved by having the different intercepts. We can therefore conclude that if we force both lines to have the same slope, then allowing them to have different intercepts indeed improves the fit. On the other hand, if we force both lines to have the same intercept, then allowing them to have different slopes really does not improve the fit. Using the combined data from both studies, another regression equation (Y, DNA content = 0.122x + 0.226) was estimated. This equation was used to estimate the known chromosome number (x) of the Midnight cultivar of Kentucky bluegrass. When the combined regression line is used to estimate the chromosome number for the DNA content value (8.48 pg), the estimated chromosome number is 67.7. This suggests that our original estimated regression equation predicts a chromosome number of the cultivar closer to the actual number of chromosomes than the combined one.

In our current study, morphologically and genetically similar genotypes grouped into the Compact-Midnight type did not demonstrate a significant difference in DNA content. This indicates that genotypes within this type are likely to be similar in terms of genetics and ploidy level. Since the Compact-Midnight type was only represented by the genotypes for the Award and Rugby II cultivars in our study, we supplemented our results by using flow cytometry data for cultivar Midnight from Arumuganathan et al. (1999). As mentioned previously, the Midnight cultivar along with Award and Rugby II were genetically and morphologically grouped in the Compact-Midnight type (Bonos et al., 2000; Curley and Jung, 2004), and additionally the Midnight cultivar had a DNA content of 8.48 pg DNA/2C. This DNA content value is similar to the DNA contents (8.60 and 8.77 pg/2C) of the Award and Rugby II genotypes, respectively, calculated in our study. These results suggest a significant similarity between the DNA contents of the individual cultivars representing the Compact-Midnight type. Interestingly, in our study the chromosome number for Award was estimated to be 66 and that for Rugby II was visually estimated to be 63.7 ± 4.2, based on chromosome counts. All of these results are significant to Kentucky bluegrass research because genetic distance derived from RAPD markers suggests that the cultivars within the Compact-Midnight type are genetically similar (Curley and Jung, 2004). Thus, these techniques could be used to determine if all of the cultivars in the Compact-Midnight type are similar in terms of DNA content and molecular marker-derived genetic distance.

In conclusion, flow cytometry provides a powerful and rapid tool for Kentucky bluegrass breeders to make early selection of true hybrids derived from intraspecific hybridization in Kentucky bluegrass and interspecific hybridization between Kentucky bluegrass x Texas bluegrass. In addition, initial germplasm from either plant introductions or naturalized populations can be efficiently screened and selected for use as breeding materials. The reliable regression equation estimated here will help researchers estimate ploidy level of unknown samples via flow cytometry. This study provides valuable information that may be used by researchers investigating the genetics and cytogenetics of Kentucky bluegrass.

	ACKNOWLEDGMENTS

 
The authors of this paper thank the members of the Jung and Williamson labs who contributed their time and effort in processing all of the flow cytometer samples. The authors are also grateful to Dr. Kathiravetpilla Arumuganathan at the Benaroya Research Institute in Seattle, WA, for instruction and advice concerning flow cytometry, and Karen Bresee in the Department of Horticulture at the University of Wisconsin-Madison for providing aid with the chromosome counts. The authors also express their appreciation to Jacklin Seed; Lesco, Inc.; Olds Seed Solutions; Pickseed West, Inc.; The Scotts Company; and Turf-Seed, Inc. for providing us with Kentucky bluegrass seed. Finally, the authors thank the technicians at the Flow Cytometry Facility of the University of Wisconsin Comprehensive Cancer Center for the use of their flow cytometer. This research was fully funded by the United States Golf Association.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Research funded by a grant from the United States Golf Association.

Received for publication December 30, 2003.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Related articles in Crop Science Similar articles in this journal Similar articles in Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Eaton, T. D. Articles by Jung, G. Search for Related Content PubMed Articles by Eaton, T. D. Articles by Jung, G. Agricola Articles by Eaton, T. D. Articles by Jung, G. Related Collections Turfgrass Crop Cytology