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Kentucky bluegrass (Poa pratensis L.) is an important facultative apomictic temperate perennial grass species used for both forage and cultivated turf. Through apomixis, this species is able to propagate diverse and odd ploidy levels, resulting in many genetically distinct phenotypes. A wide range of diverse cultivars and accessions of kentucky bluegrass have been previously characterized based on pedigree, common turf performance, and morphological characteristics to create a kentucky bluegrass cultivar classification system. The objectives of the current study were to assess the amount of genetic divergence among kentucky bluegrass cultivars, experimental selections, and plant collections and revise/update the original pedigree, turf performance, and morphological characteristics kentucky bluegrass classification system using recently described kentucky bluegrass microsatellite [simple sequence repeat (SSR)] markers. In this study, 247 kentucky bluegrass cultivars, experimental selections, and collections were genotyped using 25 SSR markers. SSR markers showed a strong correlation between genetic relatedness as assessed by molecular markers and the original kentucky bluegrass classification system and also provided justification for a revision/update of the classification system. Traditional classification types that were supported by the current SSR analysis include BVMG, Compact, Compact-America, Julia, Mid-Atlantic, Midnight, and Shamrock types. Newly proposed classification types included Cynthia, Jefferson/Washington, Limousine, P-105, Sydsport, and three Eurasian types. The majority of cultivars, experimental selections, and collections were uniquely identified with the current set of SSR markers. Genetic relationships of individuals as assessed by SSR markers closely matched known pedigrees. The current set of SSR markers can be used to rapidly genotype and assign new cultivars/accessions to kentucky bluegrass classification types and assess genetic relatedness among individuals and should be considered for use in a kentucky bluegrass plant variety protection program. Kentucky bluegrass (Poa pratensis L.) is a facultative apomictic cool-season perennial grass species widely used for forage and turf in the United States and Canada (Huff, 2003, 2010). Although sexuality in kentucky bluegrass can be variable, the species reproduces mainly through apomixis resulting in a high percentage of offspring that are genetically identical to the mother plant (Huff and Bara, 1993; Mazzucato et al., 1996). The chromosome number of kentucky bluegrass is also variable with reports of both polyploidy and aneuploidy ranging from 2n = 28 to 154 (Akerberg, 1939; Grazi et al., 1961; Huff, 2003; Love and Love, 1975; Meyer and Funk, 1989; Muntzing, 1933; Nielson, 1946; Tinney, 1940). This complex polyploidy and aneuploidy can complicate kentucky bluegrass breeding efforts; however, apomixis allows this species to propagate diverse and odd ploidy levels, which results in many genetically distinct individuals (Huff, 2010). This high level of diversity has allowed for the development of numerous unique kentucky bluegrass cultivars directly selected from promising apomictic plants from natural populations (Bashaw and Funk, 1987; Bonos et al., 2000; Pepin and Funk, 1971). Additional cultivars have been developed from the improvement of intraspecific hybridization techniques (Pepin and Funk, 1971). A classification system, based on varying combinations of pedigree information, common turf performance characteristics, and morphological traits, was developed to characterize the large number of diverse kentucky bluegrass cultivars and accessions (Bara et al., 1993; Bonos et al., 2000; Murphy et al., 1997; Shortell et al., 2009). This classification system was developed to group cultivars and accessions into various classification types to provide an overview of the similarities and differences between cultivars and cultivar types and as a guide to help turf managers develop blends of cultivars that are genetically diverse yet still uniform in morphological and performance characteristics (Shortell et al., 2009). A detailed discussion of the original classification scheme, the description of the classification types as well as subsequent updates are available in previous publications (Bara et al., 1993; Bonos et al., 2000; Murphy et al., 1995, 1997; Shortell et al., 2004, 2009). A brief summary and description of the most recent version of the Pedigree, Turf performance, and Morphological (PTM) kentucky bluegrass classification system (adapted from Shortell et al., 2009) is shown in Supplementary Table 1. The PTM kentucky bluegrass classification system has been useful to turf researchers and turf managers; however, the use of morphological traits and cultivar performance characteristics to distinguish varieties has several limitations. Morphological characterization requires large reference collections for comparative analyses, oftentimes with a limited number of morphological descriptors available to distinguish cultivars. Additionally, assessment of morphological and cultivar performance characteristics requires field-based measurements of large numbers of samples and replicates with the potential for the expression of these traits to be influenced by environmental conditions (Giancola et al., 2002; Ibanez et al., 2009; Kwon et al., 2005; Lombard et al., 2000; Roldan-Ruiz et al., 2001). Finally, common morphological or performance characteristics may not necessarily equate to genetic relatedness. As a result of these drawbacks, numerous researchers have proposed using molecular markers for variety discrimination, genetic diversity studies, and Plant Variety Protection (Cooke and Reeves, 2003; Gunjaca et al., 2008; Ibanez et al., 2009; Tommasini et al., 2003). Molecular markers offer a number of advantages over morphological characters and the assessment of common performance characteristics including a high degree of polymorphism, ease of scoring, a large potential number of characters for discrimination, and the fact that molecular markers are unaffected by environmental conditions (Lombard et al., 2001; Smykal et al., 2008; Tommasini et al., 2003). Kentucky bluegrass cultivars and accessions have previously been characterized using random amplified polymorphic DNA (RAPD) markers (Curley and Jung, 2004; Huff, 2001; Johnson et al., 2002); however, these studies either did not attempt to correlate (Huff, 2001; Johnson et al., 2002) or failed to find a strong correlation (Curley and Jung, 2004) between genetic diversity assessed by RAPD markers and the PTM kentucky bluegrass classification system. In the current study we used microsatellite, or SSR, markers to study the genetic relationships of 247 kentucky Received for publication 20 Mar. 2012. Accepted for publication 21 June 2012. This work was supported by the Rutgers Center for Turfgrass Science and the New Jersey Agricultural Experiment Station. To whom reprint requests should be addressed; e-mail honig@aesop.rutgers.edu. 1356 HORTSCIENCE VOL. 47(9) SEPTEMBER 2012 bluegrass cultivars and accessions. The specific objectives of this study were to assess the amount of genetic divergence between kentucky bluegrass cultivars, experimental selections, and plant collections, and revise/update the original PTM kentucky bluegrass classification system using recently described kentucky bluegrass SSR markers (Honig et al., 2010). Materials and Methods Simple sequence repeat markers and genotyping The development of 88 kentucky bluegrass SSR markers was previously described by Honig et al. (2010). Primer sequences, characteristics of the SSR repeat motifs, number of alleles per SSR marker, and polymorphism information content (PIC) of the SSR marker alleles for all 88 SSR markers are available in Honig et al. (2010). In the current study, 25 SSR markers from Honig et al. (2010) (Supplementary Table 2), with the highest average PIC values across all alleles in a given SSR marker, were used to genotype a new set of 247 kentucky bluegrass cultivars, accessions, and collections and one wild ecotype of Poa annua L. Although the calculation of PIC is not strictly accurate in the case of polyploidy, it is still a useful method for discriminating appropriate primers. To minimize polymerase chain reaction (PCR) artifacts, only alleles with a PIC value greater than 0.15, for any SSR marker, were used in the current analysis. Plant genomic DNA was isolated from the 247 entries of the current study using Sigma GenElute Plant Genomic DNA Miniprep Kit (St. Louis, MO) according to the manufacturer’s instructions. PCR genotyping reactions followed the protocol outlined in Honig et al. (2010). PCR products were run on an ABI 3130xl capillary electrophoresis genetic analyzer (Applied Biosystems, Foster City, CA), sized using the LIZ 500(-250) size standard, and analyzed using Genemapper 3.7. SSR markers can generate codominant data; however, problems may arise during allele scoring of polyploid individuals because of difficulties in assigning alleles and inferring allelic dosage over two or more homoeoloci (George et al., 2006; Liao et al., 2008; Markwith et al., 2006; Saltonstall, 2003); thus, DNA banding patterns at any given locus are more accurately scored as ‘‘allele phenotypes’’ (Becher et al., 2000). Individual alleles of the 25 SSR markers used in the current study of polyploid kentucky bluegrass were treated as ‘‘allele phenotypes’’ and scored as dominant markers to create a binary data matrix (band absence = 0; band presence = 1). Plant material Two hundred forty-seven kentucky bluegrass cultivars, experimental selections, and collections were evaluated in this study and are listed in Table 1 according to the newly revised/updated version of the kentucky bluegrass classification system based on SSR markers. A duplicate list is provided in Supplementary Table 3, organized according to previous ver

Classification of Kentucky Bluegrass (Poa pratensis L.) Cultivars and Accessions Based on Microsatellite (Simple Sequence Repeat) Markers