Genet Resour Crop Evol (2008) 55:105–114 DOI 10.1007/s10722-007-9218-5 RESEARCH ARTICLE Microsatellite markers and genetic diversity assessment in Lolium temulentum Francis M. Kirigwi Æ John C. Zwonitzer Æ M. A. Rouf Mian Æ Zeng-Yu Wang Æ Malay C. Saha Received: 10 August 2006 / Accepted: 29 January 2007 / Published online: 18 April 2007 Ó Springer Science+Business Media B.V. 2007 Abstract Lolium and Festuca are two important further analysis of genotypes. The TF-EST- and the genera of cool-season forage and turf grasses world- F · L genomic-SSRs identified 10.3 and 9.3 alleles wide. Lolium temulentum L. (darnel ryegrass) has per marker, respectively with an average polymor- been proposed as a model species for genomics phic information content (PIC) value of 0.66. The studies of cool-season forage and turf grasses. A phenogram based on 319 EST-SSR and 296 genomic study with 41 darnel ryegrass, three tall fescue SSR fragments, grouped L. temulentum accessions (Festuca arundinacea Schreb.), two tetraploid fescue into three major clusters except for accession ABY- (F. glaucescens), and two meadow fescue (F. prat- BA 8892.78. Lolium temulentum accession ABY-BA ensis) genotypes was initiated to (i) identify a set of 8892.78 did not cluster with any other accession. The microsatellite (simple sequence repeats) markers Festuca clusters were distantly related with darnel useful for L. temulentum L., and (ii) to utilize such ryegrass clusters with a similarity coefficient of 0.26. markers for assessing the genetic variability of The selected set of tall fescue EST- and F · L L. temulentum accessions collected from different genomic SSRs were useful in assessing L. temulen- geographical regions of the world. A total of 40 tall tum genetic diversity and could benefit the genetic fescue (TF) EST-SSRs and 60 Festuca–Lolium improvement of members of the Festuca–Lolium (F · L) genomic SSRs were screened on a subset of complex. eight genotypes. The selected 30 tall fescue EST- SSRs and 32 F · L genomic SSRs were used for Keywords Festuca spp. Á Genetic diversity Á Lolium temulentum Á Microsatellite markers F. M. Kirigwi Á J. C. Zwonitzer Á M. A. R. Mian Á Z.-Y. Wang Á M. C. Saha (&) Forage Improvement Division, The Samuel Roberts Noble Foundation, Inc., 2510 Sam Noble Parkway, Ardmore, Introduction OK 73401, USA e-mail: [email protected] Members of the Festuca–Lolium complex are widely used as forage and turf, especially in the temperate J. C. Zwonitzer Department of Plant Pathology, North Carolina State regions of the world. They contain highly productive University, Gardner Hall No. 2518, Campus Box 7616, forage grass species that render numerous benefits to Raleigh, NC 27695, USA humans, including providing feed and fodder for millions of dairy and beef cattle, horses, sheep, and M. A. R. Mian Corn and Soybean Research Unit, USDA-ARS, Ohio many wild animals (Wang et al. 2002). These grasses State University, OARDC, Wooster, OH 44691, USA also play a major role as turf in golf courses and 123 106 Genet Resour Crop Evol (2008) 55:105–114 lawns worldwide. These grasses belong to the sub- Moreover, biotic stress genes have been introgressed family Pooideae and tribe Poeae. Among these from Festuca to ryegrass species (Armstead et al. grasses, tall fescue (Festuca arundinacea Schreb.) 2006). Combining the attributes of both genera is is an allohexaploid (2n = 6x = 42) with three feasible through hybridization because chromosomes genomes, PG1G2 (Seal 1983). Other cultivated cool of the two have high homology, hybridize and have a season grasses in the genus Festuca include tetraploid high frequency of recombination resulting in fertile fescue (Festuca glaucescens Hegetschw. & Heer., the offspring (Jauhar 1993; Zwierzykowski et al. 1999; donor of the G1 and G2 genomes) and meadow Terrel 1966). This is possible because hybrids fescue (Festuca pratensis (Hudson) P. Beauv., the between the two undergo promiscuous chromosome donor of the P genome) (Sleper 1985). The genus recombination which enables gene transfer from one Lolium is comprised of the outcrossing Lolium homoeologous chromosomal region to another perenne L., Lolium multiflorum Lam., the self- (Humphreys and Pasˇakinskiene´ 1996; Humphreys pollinated L. temulentum L. subsp. temulentum et al. 2003). Furthermore, introgressed segments of (darnel, darnel ryegrass), and L. persicum Boiss. et chromosomes from one species through interspecific Hohen. ex Boiss. (Persian darnel). It has been hybridization can be distinguished by genomic in situ demonstrated that L. temulentum is closely related hybridization (GISH) (Thomas et al. 2003). Intergen- to other members of the Festuca–Lolium complex as eric hybridization between Lolium · Festuca can be well as a number of other important forage and turf used for the production of androgenic plants that may grass species (Mian et al. 2005). display transgressive resistance for abiotic stresses Darnel ryegrass is widely distributed throughout (Humphreys et al. 2003). the world. It is found growing as mimic weed with Simple sequence repeats (SSRs) (Microsatellite) similar life cycle and morphology in wheat and are among the most variable DNA sequences. They barley fields. It is a long day annual grass that is are highly polymorphic, abundant, easy to use, and classified as a noxious weed in Arkansas and as a have become an important marker system in cultivar plant pest in South Carolina (Kuk et al. 2000). fingerprinting, diversity studies, molecular mapping However, because of the simplicity of the biology of and in marker-assisted selection (Goldstein and darnel ryegrass, it is being used as a model species for Schlo¨terer 1999). The loci of these markers are genetic and genomic studies in forage and turf grasses highly transferable across species (>50%) within a (Wang et al. 2005). As a model species for forage and genus (Ro¨der et al. 1995; Peakall 1998; Thiel et al. turf grasses, darnel ryegrass offers the following 2003; Saha et al. 2004; Eujayl et al. 2004), but that advantages: it is self-fertile, has a short life cycle transferability is low across genera (Peakall 1998; (less than 3 months), is a diploid, is easy to grow, and Roa et al. 2000; Thiel et al. 2003). However, SSR closely related to major grass species in the Festuca– markers derived from expressed sequence tags (EST- Lolium complex and others. In contrast, most impor- SSRs) are likely to be more transferable because they tant forage and turf grasses require vernalization to are a part of the transcribed regions of DNA (Scott flower, many are polyploids, and are obligate out- et al. 2000). Transcribed regions are more conserved breeders with gametophytic self incompatibility sys- across species and genera and EST-SSRs can be used tems largely controlled by the SZ multi- allelic locus for comparative mapping (Saha et al. 2004; Yu et al. (Lundqvist 1962; Fearon et al. 1983). 2004). Interspecific hybridization between species in the Darnel ryegrass has been used as a model in the Festuca–Lolium complex has been exploited in the study of a senescence- induced degradation gene in development of forage germplasm of high-quality F. pratensis through introgression into L. temulentum and winter hardiness, and in the introgression of background (Thomas et al. 1999). In addition to its abiotic stress tolerance traits (Humphreys et al. 2005). demonstrated potential as a model species for a Members of the Lolium genus are high yielding and number of important forage and turf grass species, produce fodder of high quality and digestibility. On darnel ryegrass can also be exploited for introgression the other hand, members of the genus Festuca have of its self-compatibility genes into the out-crossing high resistance to abiotic stresses which include members of the Festuca–Lolium complex (Yamada winter hardiness, drought resistance and persistence. 2001). Understanding the genetic diversity among 123 Genet Resour Crop Evol (2008) 55:105–114 107 accessions and cultivars within a species is important Molecular markers and amplification for their efficient use in genetic and genomic manipulation studies. The Noble Foundation main- A total of 40 tall fescue EST-SSRs (TF EST SSRs tains a large world-wide collection of darnel ryegrass from Saha et al. 2004) and 60 Festuca–Lolium accessions and the genetic relatedness among these (F · L) genomic SSRs (kindly provided by Dr. Marc accessions has not been reported. At present, only Ghesquiere, Lusignan, INRA, France) were screened few SSR markers are available for darnel ryegrass with a subset of 8 genotypes (L6, L7, L18, L25, L30, (Senda et al. 2003). Potential use of tall fescue EST- L39, PI and PI383647) (Table 1). Thirty TF EST SSR markers developed at the Noble Foundation for SSRs and 32 F · L genomic SSRs with clean genetic studies in darnel ryegrass has been demon- amplification products were selected for further strated (Wang et al. 2005;Mianetal.2005). analysis with all genotypes. The PCR reactions were Identification of a large number of cross-species run under standard conditions for all primers using 1 microsatellite markers would be useful for genetic U AmpliTaq Gold with GeneAmp PCR buffer II characterization of darnel ryegrass. The objectives of (Applied Biosystems/Roche, Branchburg, NJ), 3 mM this study were: (a) to identify a set of cross-species MgCl2,200lM dNTPs, 0.2 mM of each primer and SSR markers useful for study of L. temulentum and 20 ng of template DNA in a 10 ll reaction. (b) to assess the genetic diversity among the L. tem- Thermocycler model ABI 9700 was used for PCR ulentum accessions at the Noble Foundation. amplification and the reaction conditions consisted of 10 min at 958C, followed by 40 cycles of 50 s at 958C, 50 s at a temperature between 588C and 648C Materials and methods (optimum annealing temperature for each primer pair), 90 s at 728C, and a final extension step of Plant materials 10 min at 728C.