Crop Science 41:1184-1189 (2001)
© 2001 Crop Science Society of America
TURFGRASS SCIENCE
Comparative DNA Profiling of U-3 Turf Bermudagrass Strains
M. P. Anderson*,a,
C. M. Taliaferroa,
D. L. Martinb and
C. S. Andersona
a Dep. Plant and Soil Sciences, Oklahoma State Univ., Stillwater, OK 74078
b Dep. Horticulture and Landscape Architecture, Oklahoma State Univ., Stillwater, OK 74078
* Corresponding author (mpa{at}mail.pss.okstate.edu)
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ABSTRACT
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Long-term maintenance of genetic fidelity of clonally propagated bermudagrass (Cynodon spp.) cultivars is difficult. Contamination may arise through mechanical mixtures, the presence of viable seed, and somatic mutations. DNA amplification fingerprinting (DAF) was used to compare genetically putative ‘U-3’ bermudagrass [Cynodon dactylon (L). Pers.] selected in the early 1930s with bermudagrass currently produced in Oklahoma and sold as U-3. Four samples of putative U-3 and seven samples of Oklahoma U-3 were tested. Foundation class ‘Tifway’ (C. dactylon x C. transvaalensis Burtt-Davy), along with two other commercially labeled Tifway strains, were included as reference standards to gauge genetic diversity. Six DAF primers were used to differentiate the 14 bermudagrass strains used in this study. The DAF analysis of all bermudagrass strains produced an average of 56.0 ± 5.9 SE polymorphic bands/primer. Comparisons among the putative and Oklahoma grown U-3 strains resulted in 37.1 ± 5.1 SE polymorphic bands/primer. Phenetic analyses using both the UPGMA algorithm and principal coordinate analysis revealed a wide separation between the putative U-3 and the Oklahoma strains identified as U-3. Putative U-3 samples collected from three mid-Atlantic golf courses clustered tightly as did all U-3 strains from Oklahoma. A putative U-3 strain from Illinois clustered with putative U-3 from the mid-Atlantic golf courses, but was distinct. The Oklahoma U-3 strains were equally distant from Foundation class Tifway as they were from putative U-3. The Oklahoma U-3 collections appear genetically identical and likely resulted from mechanical contamination of a true U-3 nursery plot that served as a source of planting stock for sod growers.
Abbreviations: bp, base pairs • DAF, DNA amplification fingerprinting • NC, Northcutt Farms • RFLP, restriction fragment length polymorphisms • RAPD, random amplified polymorphic DNA • SIU, Southern Illinois University • TGS, Tulsa Grass and Sod Co. • USGA, United States Golf Association
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INTRODUCTION
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U-3 BERMUDAGRASS was derived from a collection of plants selected from golf course putting greens in the early 1930s (Hanson, 1972). D. Lester Hall, a greenskeeper at the Savannah Golf Club near Savannah, GA, identified U-3 in 1936 as a superior plant in his collection (Juska and Hanson, 1964). It was subsequently evaluated in United States Golf Association (USGA) tests at Arlington, VA (Burton, 1977), and propagating stock was increased at the USDA Plant Industry Station, Beltsville, MD (Juska and Hanson, 1964). U-3 was widely distributed by USGA and USDA-ARS turfgrass agronomists in 1946–1947 to turfgrass scientists at public research institutions and to commercial users. Hanson (1972) described U-3 as moderately fine-leaved, cold hardy, and rapidly spreading cultivar that was adapted to a wide range of soil and climatic conditions. Suggested uses of U-3 included lawns, playing fields, park areas, and golf course tees and fairways.
U-3 was largely replaced as the variety of choice in the southern USA by ‘Tifgreen’ and ‘Tifway’ (both C. dactylon x C. transvaalensis Burtt-Davy), released in 1956 and 1960, respectively (Burton, 1977). The relatively greater cold hardiness of U-3 resulted in its continued use in the northern half of the transition zone between the subtropical and cool temperate climatic zones. In Oklahoma, bermudagrass labeled as U-3 presently continues as a popular standard of the turfgrass sod production industry. However, the identity of the grass presently produced by several Oklahoma sod growers and marketed directly or through distributors as U-3 has been questioned. Oklahoma sod growers requested research to resolve this concern.
DNA profiling techniques provide a reliable means of measuring the genetic relatedness of plant genotypes. The RFLP and RAPD procedures are used extensively for assessing intraspecific comparisons in turfgrasses (Huff, 1997) and tomato, Lycopersicon spp., (Villand et al., 1998). A powerful technique termed DNA amplification fingerprinting (DAF) has been used successfully to distinguish variations within species of centipedegrass [Eremochloa ophiuroides (Munro) Hack] (Weaver et al., 1995), sweet potato (Ipomoea batatas L. Lam) (Trigiano et al., 1995), and petunia (Petunia spp.) (Cernay et al., 1996). The DAF procedure uses shorter oligonucleotide primers and higher primer concentration to produce a more robust fingerprint pattern than other conventional PCR-based techniques like RAPD. The DAF procedure has also been used to distinguish between interspecific bermudagrass hybrids and between original and mutant genotypes such as Tifway and its irradiated mutant ‘Tifway II’ (C. dactylon x C. transvaalensis) (Elliott, 1995).
The objective of this research was to assess the verity of U-3 bermudagrass produced in Oklahoma by DAF profiling.
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MATERIALS AND METHODS
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Plant Material
U-3 labeled bermudagrass strains from Oklahoma were obtained from Tulsa Grass and Sod (TGS) and the Northcutt Farms (NC) (Table 1). One representative sample was taken from a 9-m2 TGS demonstration plot cloned from a 49-ha production field. The TGS production field was uniform in appearance with no evidence of contamination. Two NC U-3 fields were sampled. One was the company's original field (7.7 ha) that had been maintained under certification until 1987. The second field (8.1 ha) was established later in time than the first and is currently used to supply the company's demand for U-3 planting stock. Uniform stands existed in both fields with no visual or morphological evidence of contamination by other bermudagrasses. However, because of the age differences in the fields and because the older one was the last U-3 field in Oklahoma that had been certified we took three random samples from each field. Samples from the original certified fields are labeled NC #1 through NC #3 and those from the production field are labeled NC #4 through NC #6 (Table 1). Both NC and TGS represent a majority source of U-3 sprigs grown in Oklahoma.
Breeder, Foundation, Registered, or Certified class propagating stock of U-3 no longer exist. Our putative U-3 (NY #1, NY #2, and PA) comes from remnant plantings on three mid-Atlantic golf courses (Table 1). Mr. James Snow, USGA Greens Section Director, identified these sites where U-3 was planted in the late 1950s by Fred Grau, deceased USGA Agronomist. USGA records and testimony by long-term employees of the three respective golf courses indicate that U-3 was the only bermudagrass ever planted on these courses. The U-3 strain from Southern Illinois University (SIU) was from an original plot planted and maintained by Herb Portz, deceased long-time turfgrass scientist at SIU. The U-3 stocks from the New York and Pennsylvania golf courses have presumably been genetically isolated from all other bermudagrass since the original plantings in the 1950s. Barring somatic mutation or contamination emanating from viable seed, this plant material should represent the original U-3 developed and distributed by Fred Grau. U-3 is a tetraploid (2n = 4x = 36) clonally propagated plant that produces few viable seed. We included in this study three Tifway labeled strains as outgroups. Tifway is a triploid (2n = 3x - 27) interspecific hybrid and should show major differences when compared with U-3. Personnel from Tulsa Grass and Sod (TGS) indicated that TGS Tifway and TGS Astro were not likely to be true Tifway. All plants were grown in a greenhouse at the Oklahoma State University Agronomy Research Station in 15-cm pots containing a standard soil mix. Plants were regularly supplied a balanced fertilizer sufficient to maintain healthy growth (Peter's No-Stain 20:20:20 Formula; Grace-Sierra, Milpitas, CA).
DNA Isolation
Genomic DNA from leaf tissue was isolated according to the hexadecyltrimethyl ammonium bromide (CTAB) procedure. Leaves were cut from the plants, placed on ice, ground in liquid nitrogen, and extracted in CTAB buffer consisting of: 2% (w/v) CTAB, 100 mM TRIS at pH 8.0, 20 mM EDTA, and 1.4 M NaCl. The homogenate was incubated at 65°C for 30 min, shaken for 20 min, emulsified with an equal volume of chloroform: isoamyl alcohol (24:1 v/v) and centrifuged for 30 min at 1000 g and 4°C. To the supernatant was added 2 mL of 10% (w/v) CTAB and 0.7 M NaCl with an equal volume of chloroform: isoamyl alcohol. The solution was gently mixed and centrifuged as before. Two milliliters of CTAB precipitation buffer, consisting of 1% (w/v) CTAB, 50 mM TRIS-HCl at pH 8.0 and 10 mM EDTA, was added to the supernatant, gently mixed and centrifuged as before. The pellet was dissolved in 5 mL of 1 M NaCl, treated with ribonuclease A at 37°C for 30 min and incubated for 2h at 56°C to dissolve the DNA. The DNA was precipitated with two volumes of ethanol, hooked with a glass rod, and rinsed several times with 70% (v/v) ethanol. The DNA concentration was measured by an ethidium bromide spot assay against DNA standards (Sambrook et al., 1989). Prior to PCR amplification, genomic DNA was diluted to a final concentration of 100 ng/µL and 3 µg of DNA was digested with two units of TaqI restriction endonuclease for 3 h at 65°C (Life Technologies, Rockville, MD). Digested DNA was quantitated by the ethidium bromide spot assay as before.
PCR Amplification
Six primers (Table 2) were used to fingerprint the14 bermudagrass strains used in this study (Table 1). The PCR amplification mixture consisted of: 2.5 units of Qiagen Taq polymerase (Qiagen Inc., Valencia, CA), 6 mM MgCl2, 250 µM dNTP, 3 µM primer, and 12.5 ng of digested genomic DNA in a total volume of 25 µL. Amplification of DNA proceeded by means of a "touchdown" PCR program to increase specificity during the first six cycles. The PCR mixtures were first denatured at 94°C for 2 min, denatured at 94°C for 15 s, annealed at 30°C for 20 s, and extended at 72°C for 1 min. The program recycled six times, decreasing the annealing temperature by 0.5°C each cycle. After touchdown PCR the program proceeded with a denaturation step at 94°C for 20 s, annealing step at 24°C for 20 s, and an extension step at 72°C for 1 min, followed by a repetition of the last three cycles 32 times. After PCR, the DNA was quantitated with a silver stained spot assay prior to electrophoresis to normalize the DNA load between lanes. Two microliters of each PCR product was spotted in triplicate onto a polyacrylamide denaturing gel, incubated under vacuum overnight, and stained in silver (Promega, Madison, WI) following the procedure of Bassam and Caetano Anolles (1993). The spots were quantitated with a Bio-Rad GS-700 densitometer (Bio-Rad, Richmond, CA) and the samples normalized by dilution to obtain equal silver staining potential prior to electrophoresis.
Denaturing Electrophoresis
Equal volumes of sample and loading buffer, consisting of 4 M urea with bromphenol blue, were mixed, and 10 µL was loaded on the gel. The PCR fragments were separated on a 4% polyacrylamide gel containing 1x TBE and 7 M urea. Long Ranger acrylamide (FMC Corp., Rockland, ME) was used to separate large DNA fragments up to 2 kilobases. One side of the glass plate was treated with Bind Silane (Promega) and the other side Sigmacote (Sigma Chemical Co., St. Louis, MO) to make sure the gel would stick to one glass plate upon disassembly. The 20-cm gels were loaded and run on a Bio Rad Protean II apparatus at 100 V until the bromphenol blue strain reached three quarters of the length of the gel. The gel was removed and stained by means of a Promega silver staining kit as described above. After staining, the gel was equilibrated in 10% (v/v) glycerol and 20% (v/v) ethanol, covered with cellophane, and air-dried overnight at room temperature.
Data Profiling and Analysis
A total of six PCR primers were used in this study (Table 2) to profile all 14 bermudagrass strains. All 14 PCR products were run on the same gel to facilitate accurate comparisons between lanes. Electrophoretic bands were scored for presence (1), absence (0) or ambiguous (9) in each bermudagrass strain throughout the gel profile. Data were compiled for each replicate experiment and analyzed phenetically by the NTSYS version 2.0 program (Exeter Software, New York, NY). Similarity coefficients were computed by the SIMQUAL module. Cluster analysis was performed according to the unweighted pair-group mean algorithm (UPGMA) within the SAHN module of the NTSYS program. A principal component analysis to construct a three-dimensional array of eigenvectors was performed using the DCENTER module of the NTSYS program. The PCR reaction, electrophoresis separation, staining of gel, data profiling, and analysis was replicated for each sample two to four times with near identical results.
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RESULTS AND DISCUSSION
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The six DAF primers produced 336 bands, averaging 56 ± 5.9 SE bands/primer (Table 2). A DAF gel with PCR products resulting from primer 4267 is shown in Fig. 1. Overall, of the 336 bands scored, 331 (99%) were polymorphic, meaning that they were absent in at least one of the 14 bermudagrass strains tested. For the nine U-3 strains, a total of 223 bands were detected, averaging 37.1 ± 5.1 SE bands/primer. Of the 138 total bands scored within the Oklahoma U-3 strains, 44 (32%) were polymorphic. Of the 44 polymorphic bands, two were unique to ‘TGS U-3’, two to NC certified #1, one to NC certified #3 and two to NC from the sprig-digging field. One particular 163-bp band was present in all Oklahoma strains including the TGS U-3 (Fig. 1).
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Fig. 1. DAF profile on denaturing polyacrylamide gel stained with silver. PCR products were generated from the 4267 primer (GAAACGCC). Molecular marker lane references the nucleic acid fragment size in number of base pairs. The 163-bp fragment was shown to be unique to the Oklahoma U-3.
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Cluster analysis separated the 14 bermudagrasses into four distinct groups comprising: (i) Oklahoma U-3 strains, (ii) putative U-3 strains from New York, Pennsylvania, and Illinois, (iii) Foundation class Tifway, and (iv) TGS Astro and TGS Tifway (Fig. 2). Similarity and matching coefficients (Table 3) averaged 0.664 for all U-3 strains tested. Similarity and matching coefficients for the Oklahoma U-3 and putative original U-3 strains averaged 0.942 and 0.925, respectively. The U-3 strain from SIU clustered with the three putative U-3 strains from New York and Pennsylvania, but was clearly distinguishable from them. Within the putative U-3 and SIU U-3 group, 65 (56%) of 147 bands scored were polymorphic. Of the 65 polymorphic bands, 36 (24%) were unique to SIU U-3. The large number of unique bands from SIU U-3 is indicative of its genetic separation from the Northeastern U-3. Only two bands were unique to the Port Washington, NY, and Norristown, PA, bermudagrasses. Several of these polymorphic bands could be useful as DNA signatures for variety identification. Similarity coefficients averaged 0.662 for all Tifway strains indicating substantial dissimilarity within that group (Table 3).
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Fig. 2. Similarity coefficients derived from phenetic analysis of DAF gels using the UPGMA algorithm. Note the clustering into four distinct groups comprising the putative U-3 strains from New York and Pennsylvania and the Illinois as one group, the Oklahoma Tifway strains, the Oklahoma U-3 strains, and Foundation class Tifway.
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Principal coordinate analysis clearly indicated that the Oklahoma U-3 strains were very closely related and as a group widely separated from the putative U-3 strains and from the Tifway strains (Fig. 3). In fact, the Oklahoma U-3 strains were as different from the putative U-3 strains as they were from Foundation class Tifway. Collectively, the DAF profile analyses definitively point to wide genetic difference between the U-3 strains from Oklahoma and the putative U-3 strains, and to strong genetic similarity among strains within these groups. Similarly, the results point to clear genetic difference between Foundation Class Tifway and the two TGS Tifway strains, the latter being genetically similar. These results confirm the field observations or TGS personnel that the TGS Tifway and TGS Astro are not true Tifways lines. Observations of plants in the greenhouse and field plots indicated that the Oklahoma U-3 strains were more aggressive, formed quicker groundcover, had coarser textured leaves, and had lighter green color than the putative U-3 strain bermudagrasses.
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Fig. 3. Bermudagrass strains separated by principal coordinate analysis from data generated from DAF gels. As in Fig. 2 note the distinct clustering into four groups.
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While these results clearly demonstrate the wide genetic difference between Oklahoma U-3 strains and putative U-3, they do not provide information on the origin of the former. Probable major sources of these variabilities may be attributed to mechanical contamination with vegetative propagules, contamination from viable U-3 seed and possibly genetic mutations. The DAF analyses of the sterile, triploid (2n = 3x = 27) interspecific hybrids Tifway, Tifgreen, and ‘Tifdwarf’(C. dactylon x C. transvaalensis) and off-type plants derived from these may provide insight into the origins of off-type plants. Analysis of Tifway pointed to its genetic fidelity and the probable source of off-types as mechanical contamination in source planting stock fields (Caetano-Anolles et al., 1997). Conversely, the detection of subtle genetic differences between Tifdwarf and Tifgreen, and off-type accessions from the two cultivars provided strong evidence of genetic mutation as a probable source of many of the off-type plants (Caetano-Anolles, 1998a, b). Indeed, Tifdwarf has long been considered a probable somatic mutant of Tifgreen (Hanson, 1972). The wide divergence of genetic relationship between putative U-3 and the Oklahoma bermudagrass strains labeled as U-3 suggests the latter to be due to mechanical contaminants or viable seeds. The very close genetic relationship of the Oklahoma strains suggests that they are clonal propagules of a single plant. We hypothesize that the Oklahoma U-3 strain originated as a mechanical contaminant or viable seed in a U-3 nursery plot that served as a source of planting stock for sod growers. The more aggressive contaminant plant could easily have become dominant in the source nursery and derivative plantings from this nursery.
It is often very difficult to prevent cross-contamination of bermudagrasses in the field. The transfer of vegetative propagules and viable seed from one site to another by natural forces, mowers and other mechanical maintenance practices, constitutes a major challenge in maintaining the purity of plantings. Additionally, clonally propagated bermudagrasses like U-3 are capable of producing some seed that fall to the soil surface and under favorable conditions produce contaminant plants. Stringent standards are needed to prevent and/or control contamination from these sources. The practice of such stringent standards is especially important in Breeder and Foundation and Registered class plantings that serve as source nurseries for propagating Certified stock. Regardless of the origin of the Oklahoma U-3 bermudagrass strain, it clearly is genetically and morphologically different from putative U-3 bermudagrass.
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ACKNOWLEDGMENTS
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We appreciate the financial support provided by the Oklahoma State Agricultural Experiment Station. In addition, we are appreciative of the cooperation of personnel and the facilities of the National Science Foundation EPSCORE funded Recombinant DNA/Protein Facility in the Department of Biochemistry and Molecular Biology on the OSU campus. We appreciate the assistance of Mr. James Snow, Director of the USGA Green Section, and the cooperating Golf Course Superintendents in New York and Pennsylvania for providing the putative U-3 material. Finally, we would like to acknowledge the cooperation of Ken Diesberg for providing the U-3 strain from Southern Illinois University. We also appreciate the financial contribution of the Oklahoma Sod Producers Association and the efforts of Dave and Doug Northcutt of Northcutt Sod Farm and Ray Volentine, Sr., of Tulsa Grass and Sod, for permitting the collection of representative Oklahoma varieties on their commercial farms. The experiments described in this paper are in accordance with current laws of the United States of America.
Received for publication June 21, 2000.
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REFERENCES
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- Bassam B.J., and G. Caetano-Anolles. 1993. Silver staining of DNA in polyacrylamide gels. Appl. Biochem. and Biotech. 42:181–187.
- Burton, G.W. 1977. Better turf means better golf: The bermudagrasses-past, present, and future. U.S. Golf Assoc. Green Section Record, March 1977, p. 5–7.
- Caetano-Anolles, G. 1998a. DNA analysis of turfgrass genetic diversity. Crop Sci. 38:1415–1424.[Abstract/Free Full Text]
- Caetano-Anolles, G. 1998b. Genetic instability of bermudagrass (Cynodon) cultivars ‘Tifgreen’ and ‘Tifdwarf’ detected by DAF and ASAP analysis of accessions and off-types. Euphytica 101:165–173.
- Caetano-Anolles, G., L.M. Callahan, and P.M. Gresshoff. 1997. The origin of bermudagrass (Cynodon) off-types inferred by DNA amplification fingerprinting. Crop Sci. 37:81–87.
- Cernay, T.A., G. Caetano-Anolles, R.N. Trigiano, and T.W. Starman. 1996. Molecular phylogeny and DNA amplification fingerprinting of petunia taxa. Theor. Appl. Genet. 92:1009–1016.
- Elliott, M.L. 1995. DNA amplification fingerprinting analysis of bermudagrass (Cynodon) genetic relationships between species and interspecific crosses. Theor. Appl. Genet. 91:228–235.
- Hanson, A.A. 1972. Grass varieties in the United States. USDA-ARS, Agric. Handbook No. 170. U.S. Gov. Print. Office, Washington, DC.
- Huff, D.R. 1997. RAPD characterization of heterogeneous perennial ryegrass cultivars. Crop Sci. 37:557–564.[Abstract/Free Full Text]
- Juska, F.V., and A.A. Hanson. 1964. Evaluation of bermudagrass varieties for general purpose turf. USDA-ARS, Agric. Handbook No. 270, U.S. Gov. Print. Office, Washington, DC.
- Miller, G.L., and R. Dickens. 1995. DNA amplification fingerprinting and hybridization analysis of centipedegrass. Crop Sci. 35:881–885.[Abstract/Free Full Text]
- Sambrook, J., E.F. Fritsch, and T. Maniatis.1989. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
- Trigiano, R.N., G. Caetano Anolles, B.J. Bassam, and M.T. Windham. 1995. Analysis of genetic diversity in a sweetpotato (Ipomoea batatas) germplasm collection using DNA amplification fingerprinting. Genome 38:938–945.[Medline]
- Villand, J., P.W. Skroch, T. Lai, P. Hanson, C.G. Kou, and J. Nienhuis. 1998. Genetic variation among tomato accessions from primary and secondary centers of diversity. Crop Sci. 38:1339–1347.[Abstract/Free Full Text]
- Weaver K.R., L.R. Callahan, G. Caetano-Anolles, P.M. Gresshoff. 1995. DNA amplification fingerprinting and hybridization analysis of centipedegrass. Crop Sci. 35:881–885.
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ABSTRACT
INTRODUCTION
