Poa annua L. is a ubiquitous cool-season turfgrass that is reported to have originated on the European continent (Tutin, 1957). It is an allotetraploid (2n = 4x = 28) thought to be derived from a chance cross between Poa infirma Kunth and Poa supina Schrader (Darmency and Gasquez, 1997; Nannfeldt, 1937; Tutin, 1952, 1957). P. infirma is an erect, bunch-type, diploid (2n = 2x = 14) annual, whereas P. supina is a prostrate, stoloniferous, diploid (2n = 2x = 14) perennial (Tutin, 1957). P. annua has a continuum of life-cycle types that include true annuals to long-lived perennials as well as numerous variants between the two (Gibeault, 1971; Heide, 2001; Johnson et al., 1993; Timm, 1965). The life cycle forms of P. annua at its extremes have been divided into two types: the annual, designated P. annua var. annua (L.) Timm, and the perennial, P. annua var. reptans (Hauskn.) (Timm, 1965). The common name for P. annua, annual bluegrass or annual meadowgrass, is a misnomer for the perennial type considering it is not an annual. To alleviate the confusion, the perennial type, P. annua var. reptans, was assigned the common name creeping bluegrass (Beard, 1999), as reptans means “creeping” (Bailey, 1948), while annual bluegrass remains the common name for P. annua var. annua. In addition to the tetraploid form, P. annua also exists as a sterile diploid but generally only on putting greens. Velguth and White (1993), in Minnesota, discovered that 24% of randomly collected P. annua plants from selected putting greens were diploid.
P. annua is an economically important turfgrass that has often been ignored even though it is the predominate turfgrass on many of the most prestigious golf courses around the world, including many of the courses that have hosted U.S. Open golf tournaments (Baker et al., 1995; Huff, 1999; Wu et al., 1987). It is particularly well adapted to extremely low heights of cut (3–4 mm), compacted soil, and shade; conditions common to golf course greens and tees. P. annua has long been considered an unwanted weed in highly maintained turfgrass surfaces (Beard, 1970; Kamp, 1981; Sprague and Burton, 1937); however, others view P. annua as a cultivated invader that has performed as well as, if not better than, the planted species. While the former have long attempted to rid the turf of this so-called weed, the latter have believed it best to manage the species most favored in a particular ecosystem (Beard et al., 1978).
Researchers have recognized the attributes of creeping bluegrass that make it a desirable turfgrass surface: perennial, stoloniferous, high tiller density, shade-tolerant, rapid germination, dark color, and limited flowering period (Adams and Bryan, 1980; Johnson et al., 1993; Sprague and Burton, 1937; Timm, 1965). Plant breeders are now accumulating breeding populations, making crosses, and developing cultivars for commercial release (Kind, 1997). To improve breeding and selection for desirable traits, a technique that would accurately differentiate and identify closely related genotypes would be useful. Such a technique could prove useful for legal protection of cultivars in addition to phenotypic data, which can be affected by environmental factors, and could also find utility for marker-assisted selection.
The biological, morphological, and ecological characteristics that influence the distribution of different P. annua types have been investigated (Law et al., 1977; Warwick and Briggs, 1978a, 1978b). Others have examined the genetic relatedness of various populations in differing environments through the use of molecular markers. Darmency et al. (1992) and Darmency and Gasquez (1997) investigated the inheritance of isozyme patterns, and Cline (2001), Mengistu et al. (2000), and Sweeney and Danneberger (1995) used random amplified polymorphic DNA (RAPD) markers to study the relationship of wild P. annua collections. These studies looked at diversity or population relationships of wild P. annua collections within a given environment and different environments on a single golf course; however, no one has studied the genetics of closely related genotypes within a breeding program.
PCR analysis using anchored SSR or ISSR has been reported to be a useful and reliable method of detecting genetic polymorphisms between accessions (Zietkiewicz et al., 1994) and has been used in cultivar identification in many crops, including wheat (Nagaoka and Ogihara, 1997), maize (Kantety et al., 1995), potatoes (Prevost and Wilkinson, 1999), Douglas-fir (Tsumura et al., 1996), and barley (Fernandez et al., 2002). The ISSR PCR method uses di-, tri, tetra-, or pentanucleotide repeats with a one-, two-, or three-base tag attached to the 3′ or 5′ end of the repeat. The tag assures that the primer anneals to the end of the simple sequence repeat and thus provides greater specificity.
The benefit of using ISSR PCR over other methods include greater specificity and repeatability than RAPD, reduction in preparation time over AFLP because there is no need to use restriction enzymes, no requirement for prior knowledge of the target sequence as with SSRs, visualization of PCR products on an agarose gel, and relatively low cost.
This study examined the utility of ISSR PCR for genotyping creeping bluegrass accessions within the University of Minnesota breeding program. Previous molecular marker work with Poa annua has only been done with wild plant collections, while no one has looked at closely related genotypes. The objectives of this study were to test ISSR primers for production of polymorphic fragments and to ascertain the applicability of ISSR PCR to distinguish closely related genotypes.
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