St. augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is a coarse-textured, warm-season, perennial turfgrass species well adapted for home lawns and commercial landscapes across the southern United States and upward into the southern regions of the transition zone. The species is commonly used in the sod industry for its superior shade tolerance and stoloniferous growth habit (Busey, 2003). A lack of cold tolerance, however, restricts the widespread use of st. augustinegrass in the northern range of its adaptation. In the early 1980s, the cultivar Raleigh was released for its superior cold tolerance (Busey et al., 1982) and has since become the industry’s standard for this trait. ‘Raleigh’ was collected from a home lawn in Raleigh, NC, in 1964, and released (NC Foundation Seed Producers certified number 101649) by Dr. W.B. Gilbert at North Carolina State University in 1980 (Bateman, 1980). During that year, Dr. Gilbert planted foundation fields of Raleigh at the North Carolina State University (NCSU) Faculty Club Research Laboratory (Raleigh, NC) and four North Carolina sod farms. Severe winterkill during an unusually cold winter in 1981 destroyed all of the foundation material (including NCSU’s breeder stocks) except from one location, Oakland Plantation Sod Farm (Council, NC). Harvest of foundation stock of ‘Raleigh’ began in July 1982 and continued until 1999. In 2000, this last foundation field was terminated as a result of poor appearance. Currently, only registered and certified fields of ‘Raleigh’ remain in production.
Vegetative propagation of turfgrass cultivars ensures uniformity and predictable performance. The quality and integrity of commercial cultivars can be compromised by the presence of off-types, which often arise in clonally propagated cultivars through random somatic mutation, contamination by unintended cultivars/genotypes, and seedling infestation (Anderson et al., 2001; Caetano-Anolles, 1998a). Elevated mutation rates and contamination issues have been reported in several clonally propagated crops including agaves (Agavaceae sp.) (Infante et al., 2006), bermudagrass (Cynodon sp.) (Anderson et al., 2001; Caetano-Anolles, 1998b), grapevines (Vitis vinifera L.) (Dangl et al., 2001), and tea plants [Camellia sinesis (L.)] (Singh et al., 2004). Minor mutations that occur in the process of asexual reproduction often produce morphologically indistinguishable changes. Although some changes may be phenotypically unidentifiable, they could still affect turfgrass performance and reduce the acceptance of the cultivar. If off-types go undetected, the vegetative nature of production can perpetuate the genetic variants and lead to very large contamination problems (Busey, 2009).
Many approaches to assess variability in clonal plants have been reported, but indistinguishable morphology has been difficult to characterize. Molecular markers have the ability to detect genetic variation and provide a reliable method to ensure the integrity of a cultivar’s genetic constitution. Anderson et al. (2001) found that different sources of common ‘U-3’ bermudagrass separated into unique unweighted pair group method with arithmetic averaging (UPGMA) clusters according to DNA amplification fingerprinting primers. Molecular markers can provide an empirical estimate of allelic diversity to determine how similar varieties are to one another, which not only aids in maintaining the integrity of the genotype, but also in pursuing varietal protection in the market. Simple sequence repeats (SSRs) are highly informative markers that can provide excellent resolution of genotypic variation within and between species (Powell et al., 1996). In st. augustinegrass, however, SSR marker development has been limited to only a few studies transferring a limited number of SSRs from other crops such as buffelgrass [Pennisetum ciliare (L.) Link] (Genovesi et al., 2009), maize (Zea mays L.), and rice (Oryza sativa) (Kimball et al., 2010). AFLP (Vos et al., 1995) is a multilocus molecular marker technique that is capable of amplifying numerous loci and has high levels of polymorphism. AFLPs are commonly used for assessing genetic diversity and population structure in species with little to no sequence information (i.e., st. augustinegrass) because they do not require prior sequence information to adequately evaluate the genetic diversity of a species or group of species (Meudt and Clarke, 2007). Other benefits of AFLPs include their high resolution and reproducibility.
More than 25 years after its release, ‘Raleigh’ is still widely used and considered the industry’s standard for cold tolerance in st. augustinegrass. Therefore, it is important to investigate whether st. augustinegrass currently produced across the southern United States and sold as ‘Raleigh’ is genetically true to the original stock released in the early 1980s. Molecular markers can be a valuable tool for plant identification and preserving this highly valued cultivar. The objective of this study was to assess the genetic variability of ‘Raleigh’ st. augustinegrass produced across the southern United States using AFLP markers.
Anderson, M.P., Taliaferro, C.M., Martin, D.L. & Anderson, C.S. 2001 Comparative DNA profiling of U-3 turf bermudagrass strains Crop Sci. 41 1184 1189
Bateman, D.R. 1980 Notice to sod producers and growers relative to the naming and release of the new st. augustine cultivar ‘Raleigh’. NC Ag. Res. Serv., Raleigh, NC
Busey, P. 2003 St. augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze, p. 309–330. In: Casler, M.D. and R.R. Duncan (eds.). Biology, breeding, and genetics of turfgrasses. John Wiley & Sons, Inc., Hoboken, NJ
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
Dangl, G., Mendum, M., Prins, B., Walker, M., Meredith, C. & Simon, C. 2001 Simple sequence repeat analysis of a clonally propagated species: A tool for managing a grape germplasm collection Genome 44 432 438
Genovesi, A., Jessup, R., Engelke, M.C. & Burson, B. 2009 Interploid st. augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] hybrids recovered by embryo rescue In Vitro Cell. Dev. Biol. 45 659 666
Infante, D., Molina, S., Demey, R. & Gamez, E. 2006 Asexual genetic variability in Agavaceae determined with inverse sequence-tagged repeats and amplification fragment length polymorphism analysis Plant Mol. Biol. Rpt. 24 205 217
Kimball, J., Zuleta, M. & Milla-Lewis, S.R. 2010 Transferring informative cereal SSRs to warm-season turfgrasses for germplasm characterization and evaluation. Proc. Amer. Soc. Agron. Intl. Ann. Mtg., Long Beach, CA. 31 Oct. to 4 Nov
Kirkland, E. & Wagner, T. 1995 St. augustinegrass ‘SS-100’. U.S. Patent 9,395, filed 6 Apr. 1994 and issued 5 Dec. 1995
Milla, S.R., Isleib, T.G. & Stalker, H.T. 2005 Taxonomic relationships among Arachis sect. Arachis species as revealed by AFLP markers Genome 48 1 11
Milla-Lewis, S.R., Zuleta, M.C., Van Esbroeck, G.A., Quesenberry, K.H. & Kenworthy, K.E. 2010 Molecular and cytological assessment of genetic diversity in Stenotaphrum germplasm. Proc. Amer. Soc. Agron. Intl. Ann. Mtg., Long Beach, CA. 3 Oct. to 4 Nov
Rohlf, F.J. 2000 NTSYS-PC: Numerical taxonomy and multivariate analysis system, Version 2.2. Exeter Software, Setauket, NY
Schneider, S., Roessli, D. & Excoffier, L. 2002 ARLEQUIN Version 2.001: A software for population genetics data analysis. Genetics and Biometry Laboratory, University of Geneva, Switzerland. 19 May 2004. <http://lgb.unige.ch/arlequin/software>
Singh, M., Saroop, J. & Dhiman, B. 2004 Detection of intra-clonal genetic variability in vegetatively propagated tea using RAPD markers Biol. Plant. 48 113 115
Stein, N., Herren, G. & Keller, B. 2001 A new DNA extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum Plant Breed. 120 354 356
Swofford, D.L. 1998 PAUP* Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, MA
Varshney, R.K., Chabane, K., Hendre, P.S., Aggarwal, R.K. & Graner, A. 2007 Comparative assessment of EST-SSR, EST-SNP and AFLP markers for evaluation of genetic diversity and conservation of genetic resources using wild, cultivated, and elite barleys Plant Sci. 173 638 649
Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Friters, A., Pot, J., Paleman, J., Kuiper, M. & Zabeau, M. 1995 AFLP: A new technique for DNA fingerprinting Nucleic Acids Res. 23 4407 4414
Wu, Y., Taliaferro, C., Bai, G. & Anderson, M. 2004 AFLP analysis of Cynodon dactylon (L.) Pers. var. dactylon genetic variation Genome 47 689 696
Zhang, L.H., Ozias-Akins, P., Kochert, G., Kresovich, S., Dean, R. & Hanna, W.W. 1999 Differentiation of bermudagrass (Cynodon spp.) genotypes by AFLP analyses Theor. Appl. Genet. 98 895 902