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Published online 23 February 2005
Published in Crop Sci 45:772-777 (2005)
© 2005 Crop Science Society of America
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DNA Fingerprinting of Seeded Bermudagrass Cultivars

Praveen Nagh Yerramsettya, Michael P. Andersona,*, Charles M. Taliaferroa and Dennis L. Martinb

a Dep. of Plant and Soil Sci., Oklahoma State Univ., Stillwater, OK 74078
b Dep. of Hortic. and Landscape Architecture, Oklahoma State Univ., Stillwater, OK 74078

* Corresponding author (mpa{at}mail.pss.okstate.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Bermudagrasses (Cynodon spp.) are important for turf and forage in temperate and tropical climates, with cultivars historically propagated clonally. Over the past two decades the number of seed-propagated commercial cultivars has dramatically increased, but information is lacking on the extent of the genetic diversity among these new cultivars. Accordingly, this research was undertaken to assess the genetic relatedness of 17 seed-propagated turf-bermudagrass cultivars using DNA amplification fingerprinting (DAF). Four DAF and four Minihairpin-DAF (MHP-DAF) primers were used in this study. The DAF and MHP-DAF primers amplified 90 and 131 amplicons, respectively. A total of 13 out of the 17 cultivars were practically indistinguishable using the DAF primers with an average similarity [similarity coefficient (SC)] of 0.982 while the MHP-DAF primers distinguished all cultivars readily. Results from the DAF and MHP-DAF analysis indicated that 14 out of the 17 cultivars were related to Arizona common germplasm with average SC of 0.833 in the MHP-DAF analysis. Arizona common germplasm is naturalized to the Colorado River Valley production areas of Arizona and California. The three most distinct cultivars—‘Princess 77’, ‘Yukon’, and ‘SWI-11’—had an average SC of 0.668. The most distinct cultivar was Yukon with an average SC of 0.604. Yukon showed 59 DNA signatures not observed in the other cultivars studied with DAF and MHP-DAF. These results indicated that a majority of seeded-type bermudagrasses developed over the past two decades depend on a narrow genetic base and that several recent cultivars are markedly genetically distinct, indicating a recent and significant broadening of the germplasm.

Abbreviations: DAF, DNA amplification fingerprinting • MHP-DAF, Minihairpin-DNA amplification fingerprinting • NTEP, National Turf Evaluation Program • PCR, polymerase chain reaction • SC, similarity coefficient


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
BERMUDAGRASS (Cynodon dactylon L. Pers) is a perennial sod-forming turf and forage grass, native to India and eastern Africa (Beard, 1973, p. 132–593; Correll and Johnson, 1970). This grass is extensively used in temperate and subtropical regions of the world for agricultural, recreational, and residential use. Historically, the highest quality turf bermudagrass cultivars have been sterile F1 hybrid plants from crosses between plants of tetraploid (2n = 4x = 36) C. dactylon and diploid (2n = 2x = 18) C. transvalensis Burtt-Davy. These cultivars are commercially propagated by planting either sprigs or sod. Over the past two decades, there has been a dramatic increase in the number of seed-propagated cultivars. National Turf Evaluation Program (NTEP) data (NTEP, 2002) indicate some of the recently developed seeded-type bermudagrasses rival the clonal-standard bermudagrass cultivars in turfgrass quality and other performance characteristics.

Several studies have been conducted to examine the genetic relatedness among vegetative propagated bermudagrass cultivars (Caetano-Anolles et al., 1995; Caetano-Anolles, 1998a; Zhang et al., 1999), but no information has been published concerning diversity among seeded-type bermudagrasses. Several seeded-type bermudagrass cultivars appear to have originated from the naturalized common form of bermudagrass grown in Yuma County, AZ, and the California Imperial Valley and are generally referred to as "Arizona Common." This bermudagrass is thought to have been introduced to the U.S. Southwest desert region at least by the middle of the 19th century (Kneebone, 1966). Baltensperger et al. (1993) indicated that a bermudagrass seed industry started soon after 1900 from bermudagrass naturalized to a region along the Colorado river in Arizona and California. The degree to which current commercial seeded-type bermudagrass cultivars are genetically interrelated is unknown. Accordingly, an estimation of genetic diversity of the seeded-type bermudagrass cultivars would provide important information relative to the need for genetic diversification in breeding programs.

Many techniques have been used to determine genetic relationships, including DAF (Caetano-Anolles et al., 1997), amplified fragment length polymorphism (AFLP) (Zhang et al., 1999), and randomly amplified polymorphic DNA (RAPD) (Huff, 1997). All these take advantage of the natural variations inherent in plant DNA. While all are capable, there are some advantages to each. Amplified fragment length polymorphism is a very powerful and reproducible technique and is readily adaptable to automation. However, the technique is fairly expensive in terms of reagent cost and equipment and requires additional steps to perform when compared with DAF. The DAF technique is a reliable, low-cost, high-resolution method that is capable of revealing many DNA polymorphisms. The DAF method when compared with the similar technique known as RAPD produces a many-fold increase in polymorphism per primer (de Vienne et al., 2003).

A variant of DAF that utilizes short minihairpin primers further increases the resolving power of the DAF technique. In one study, the MHP primers detected five times as many bermudagrass polymorphisms as conventional DAF primers (Caetano-Anolles et al., 1995). The MHP-DAF primers contain palindromic sequences, which hybridize through intraprimer interactions, creating a hairpin and a small looped priming structure (Caetano-Anolles and Gresshoff, 1994). The MHP-DAF technique uses previously amplified DAF amplicons as template to generate further banding pattern diversity.

The DAF technique has been used successfully to determine the phylogenetic relationships among bermudagrass species (Assefa et al., 1999), provide information on the origin of off-type bermudagrass cultivars (Caetano-Anolles, 1998b), and determine the fidelity of bermudagrass commercially sold as ‘U-3’ (Anderson et al., 2001), a cultivar originally developed in the early 1930s. Accordingly, this project was undertaken with the objective of determining the genetic relatedness of selected seeded-type bermudagrass cultivars. In this study, we analyzed 17 seeded cultivars from different backgrounds using DAF.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Plant Materials
The seeds of bermudagrass cultivars were obtained from the suppliers listed in Table 1. Approximately 4500 seeds of each cultivar were planted in a 15-cm-diam. pot containing Metro Mix 250 (Scotts-Sierra, Marysville, OH). The high seeding rate was used to ensure that the resulting plant populations would be representative of the cultivars. Plants were fertilized with Peters Professional Peat-Lite (Scotts-Sierra, Marysville, OH) and Iron Chelate (Miller Chemical and Fertilizer Corp., Hanover, PA). The plants were fungicide-treated with chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) (trade name: Daconil, Ortho Group, Columbus, OH) at a rate of 4.2 mL/L and with aldecarb [2-methyl-2-(methylthio)propionaldehyde O-(methylcarbamoyl oxime] (trade name: Temik, Rhone-Poulenc Ag Company, Research Triangle Park, NC).


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  		Table 1. Seeded-type bermudagrass cultivars used in this study and their source.

 
DNA Isolation
A total of 2 g of leaf tissue was harvested from a single pot containing each cultivar. The leaf tissue was frozen in liquid N and ground in a mortar and pestle to a fine powder. Genomic DNA was isolated from 100 mg of powdered leaf tissue using the DNeasy plant mini-extraction kit (Qiagen Inc., Valencia, CA) according to directions provided by the supplier. The DNA concentration was assessed spectrophotometrically at 260 nm, and quality was assessed by the 260/280 ratio (Sambrook et al., 1989). If one or more DNA extracts of the batch of 17 cultivars showed a 260/280 ratio less than 1.8, the entire batch was extracted again. The DNA was suspended to a final concentration of 5 ng/L in 0.5x TE and stored at 4°C. The DNA quality was further assessed by TBE agarose gel electrophoresis. All samples showed no sign of DNA degradation.

PCR Amplification
Four DAF and four MHP-DAF primers (Table 2) were used to fingerprint the 17 bermudagrass cultivars used in this study. The polymerase chain reaction (PCR) amplification mixture consisted of 2.5 U of Qiagen Taq polymerase (Qiagen Inc., Valencia, CA) 10x PCR buffer, which included MgCl2 for a final concentration of 1.5 mM, 250 µM dNTP, 1.5 µM DAF primers (Integrated DNA Technologies Inc, Corelville, IA), and 0.5 ng of template DNA, with the final volume made to 20 µL with sterile distilled water. The DNA template was initially denatured at 94°C for 60 s. Following denaturation, PCR proceeded at 94°C for 30 s and then 30°C for 30 s and 72°C for 30 s, cycling back 39 times. A final extension at 72°C for 60 s at the end of the 39 cycles was performed. The PCR products were visualized on a 1% TBE agarose gel impregnated with ethidium bromide at a final concentration of 0.5 µg/mL.


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  		Table 2. Sequence of the DNA amplification fingerprinting (DAF) and Minihairpin-DAF (MHP-DAF) primers used in this study.

 
The gel was examined to assure that the overall fingerprint intensity was nearly equal among all lanes. If PCR failed to amplify a fingerprint in any one of the 17 reactions, then the entire set was re-run until the fingerprints were near equally amplified. Conditions for MHP-DAF were the same as for DAF except that 1 µL of DAF PCR product was used instead of the genomic DNA template. We also found that adding 6 mM MgCl2 improved performance of the MHP-DAF.

Denaturing Polyacrylamide Electrophoresis
Polymerase chain reaction products were separated on a 20-cm-long 6% acrylamide denaturing PAGE gel using a Bio Rad Protean II apparatus (Bio Rad, Richmond, CA). The gel was made with Long Ranger Acrylamide (Cambrex Bio Science Inc., Rockland, ME) 1x TBE and 7.1 M urea. A total of 7 µL of PCR products with 3 µL of loading buffer containing the tracking dye bromophenol blue were mixed and loaded onto the gel. Molecular markers were loaded on either side of the lanes containing the PCR amplicons. Electrophoresis continued at 80 V until the bromophenol blue strain reached three-quarters of the length of the gel. The gel was removed and stained with silver using a Bioneer silver-staining kit (BioNexus, Oakland, CA) according to manufacturer directions. After staining, the gel was equilibrated in 10% (v/v) glycerol and 20% (v/v) ethanol, covered with cellophane, and air-dried at room temperature for a week before analysis. All 17 PCR products were run on the same gel to facilitate accurate band-to-band comparisons.

Data Profiling and Analysis
After silver staining, electrophorectic bands of less than 1.5 kDa were scored for their presence (1) or absence (0) for each cultivar. The data were compiled in an Excel spreadsheet and imported into the NTSYS software version 2.0 (Exeter Software, New York) for cluster analysis. Similarity coefficients (Table 3) 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. The PCR, electrophoresis separation, staining of gels, data profiling, and analysis was replicated two to three times. Comparisons showed that there were either no differences, or only very minor differences, between replicate experiments.


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  		Table 3. Similarity coefficient table using Minihairpin DNA amplification fingerprinting (MHP-DAF) analysis.

 

   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
A total of 90 and 131 bands were scored for DAF and MHP-DAF, respectively (Fig. 1) . Over 87% (78 bands) and 79% (103 bands) were found to be polymorphic in the bulked samples using DAF and MHP-DAF, respectively, meaning that the bands were present in at least one cultivar but were not observed in others.


Figure 1
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  		Fig. 1. Minihairpin-DNA amplification fingerprinting (MHP-DAF) electrophoresis gel stained with silver containing polymerase chain reaction (PCR) amplicons from 17 cultivars of bermudagrass.

 
The DAF results indicated that 13 out of the 17 bermudagrass cultivars were very closely related to each other (Fig. 2a) with an average SC of 0.982 (data not shown). The other four cultivars—Riveria, Princess, SWI-11, and Yukon—were easily distinguishable using DAF. The technique of DAF alone could not resolve differences between Arizona Common and ‘CD90160’ or differences among ‘Mohawk’, ‘Savannah’, ‘Southern Star’, ‘Sundevil’, and ‘Numex Sahara’ (Fig. 2a, SC = 1.000). In contrast, the MHP-DAF analysis clearly differentiated among all 17 cultivars (Fig. 2b). The differences between DAF and MHP-DAF were even more dramatic with 14 of the most closely related cultivars in the MHP-DAF analysis showing an average SC of 0.833 while in the DAF analysis, these same cultivars showed an average SC of 0.975 (data not shown). The results from the MHP-DAF and DAF analysis indicated that 14 of the 17 cultivars in this study were closely related to Arizona Common. This group included Arizona Common, CD90160, ‘Jackpot’, ‘Majestic’, Savannah, Southern Star, Sundevil, Mohawk, ‘Riviera’, ‘Mirage’, ‘Sydney’, ‘Pyramid’, Numex Sahara, and ‘Transcontinental’.


Figure 2
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  		Fig. 2. Dendrograms from (a) DNA amplification fingerprinting (DAF) and (b) Minihairpin-DNA amplification fingerprinting (MHP-DAF) analysis of 17 seeded-type bermudagrass cultivars.

 
According to MHP-DAF analysis, the most closely related cultivars grouped into three clusters, including: Arizona Common and CD90160 (Group 1, SC 0.901); Savannah, Southern Star, and Sundevil (Group 2, average SC 0.913); and Numex Sahara and Transcontinental (Group 3, SC 0.901). The two most similar cultivars were Savannah and Southern Star with a SC of 0.924. The pedigree information available for Savannah (Fraser and Rose-Fricker, 1998) and Southern Star (Samudio and Brede, 2002) indicates that bermudagrass germplasm from Walla Walla, WA, collected by the respective developers, contributed to the parentage of both cultivars. The use of additional markers may even better differentiate the closely related Arizona Common-type bermudagrasses.

Yukon, Princess 77, and SWI-11 were least genetically related to Arizona Common of all the cultivars studied. Furthermore, all three cultivars showed little relationship to each other. Yukon was the most distinct cultivar in this study with an average SC of 0.604 across all cultivars. The least similar cultivar to Yukon was SWI-11, and the most similar was Transcontinental, with SCs of 0.511 and 0.649, respectively. These low SCs indicate that Yukon was the most divergent seeded-type bermudagrass cultivars of those studied. Furthermore, 36 bands from Yukon were not observed in other cultivars tested, and 23 bands were found in all other bermudagrasses studied except Yukon. Combining those bands not observed with those uniquely observed in Yukon totaled 59 potential DNA signatures representing over 27% of the bands scored. Yukon is a new cultivar recently released by Oklahoma State University. Two other distinct cultivars, Princess 77 and SWI-11, had average SCs of 0.689 and 0.712, respectively. Both Princess 77 and SWI-11 showed seven signatures not observed in other cultivars in the combined DAF and MHP-DAF studies, or 3% of all bands scored. These DNA signatures may be useful for cultivar maintenance and identification purposes.

The close clustering of the 14 out of 17 cultivars with DAF indicated that most seeded-type bermudagrass cultivars are very closely related. Included in this group is Arizona Common, indicating that many of the cultivars likely originated from breeding populations originally constituted solely, or substantially, from Arizona Common. A second potential reason for some cultivars showing close similarity to Arizona Common relates to mechanical contamination of seed production fields leading to genetic contamination. Seed of many of the cultivars in the study were produced in Yuma County, AZ, or the Imperial Valley, CA, where bermudagrass seed production has been concentrated for nearly a century. Preventing the Arizona Common bermudagrass ubiquitous to this region from mechanically contaminating unique cultivar seed production fields and hybridizing with plants of the unique cultivars is difficult. Seed production fields of cultivars that are less well adapted to the region than Arizona Common can quickly become dominated by the latter. Arizona Common growing as an impurity in seed production fields, or growing in adjacent areas, may hybridize with the cultivars, resulting in genetic contamination of the desired cultivar. One of the authors (C.M. Taliaferro) has observed seed production fields of cultivars that were less well adapted to the region than Arizona Common become dominated by the latter within 1 to 3 yr contingent on the amount of initial contaminant Arizona Common in the stand. Arizona Common growing as contaminant in cultivar seed production fields, or growing in adjacent areas, has the potential of hybridizing with the cultivars. Hoff (1967) demonstrated natural crossing between Arizona Common and giant bermudagrass (C. dactylon var. aridus), the two major forms of bermudagrass traditionally grown in the region. However, the progeny resulting from the hybridization of tetraploid Arizona Common and diploid giant bermudagrass plants were sterile triploids. Such hybridization between tetraploid cultivars could produce fertile progeny leading to genetic contamination. Relative to the usually sterile vegetatively propagated bermudagrass cultivars, the potential for genetic changes in seeded-type bermudagrass cultivars is greater and warrants additional actions to maintain their genetic fidelity.

It should be noted that significant differences exist among the cultivars grouped with Arizona Common for turf quality, cold tolerance, and other performance traits (NTEP, 1997, 2002). Notably, Riviera, though loosely grouped with Arizona Common on the basis of SC values, has much higher turf quality and broader adaptation due to greater cold tolerance. None of the seed-propagated cultivars in the 1992 NTEP trial had turfgrass quality ratings as high as the vegetatively propagated standard cultivars in the test. Results from the 1997 NTEP bermudagrass test indicated that the development of Princess and Riviera represented a major gain in turfgrass quality for seeded-type bermudagrasses relative to industry-standard clonal cultivars. The development of these two cultivars suggests that major gains in performance can be achieved by breeding in relatively diverse germplasm pools with the desired result of maintenance of genetic diversity among cultivars.


   ACKNOWLEDGMENTS
 
We appreciate the financial support provided by the United States Golf Association (USGA). We appreciate the technical support of Carole Anderson. Special thanks to Gary Williams, Sharon Williams, and Rose Edwards for maintenance of the bermudagrass cultivars at the Oklahoma State University greenhouse. We gratefully acknowledge the support of colleagues Janice Hironaka, James Enis, and Madhavi Dhulipala.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Approved for publication by the Director, Oklahoma Agric. Exp. Stn. Research supported by the Oklahoma Agric. Exp. Stn. and the United States Golf Association.

Received for publication February 24, 2004.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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