Isolation and characterization of simple sequence repeat markers for the tetraploid forage grass Dactylis glomerata

Dactylis glomerata (orchardgrass or cocksfoot) is an important forage species in temperate regions and is native to Europe, Asia and North Africa. It has also been introduced into North America. It is used as a hay grass and for pastures because of its high yields, moderate shade tolerance and high sugar content. Among Japan’s cultivated forage species, it is second only to timothy (Phleum pratense) in its cultivated acreage.

Orchardgrass has 28 chromosomes and is a mostly cross-pollinated, auto-tetraploid species with a large genome (4312 Mbp) (Creber et al. 1994, Bennett and Leitch 1997). Because of its auto-tetraploid status and large genome, the genetic markers that were reported for this species are mainly non-species-specific types such as isozymes (Tosun et al. 2002), randomly amplified polymorphic DNA (Kölliker et al. 1999, Tuna et al. 2004), amplified fragment length polymorphisms (AFLP) (Peng et al. 2006, 2008), inter-simple sequence repeats (ISSRs) (Zeng et al. 2006) and sequence-related amplified polymorphisms (Zeng et al. 2008).

The genomes of all eukaryotes contain a class of sequences referred to as simple sequence repeats (SSRs) (Tautz et al. 1986) or microsatellites (Litt and Luty 1989). SSRs with tandem repeats of a basic motif of ≤6 bp have emerged as important sources of ubiquitous genetic markers for many eukaryotic genomes (Wang et al. 1994). SSR markers have the advantages of being polymerase chain reaction (PCR)-based, multi-allelic, co-dominant and highly polymorphic. Especially because of their multi-allelic status, SSR markers will be highly useful in genetic studies of auto-polyploid species. Until the present study, no species-specific orchardgrass SSR markers had been reported, although some cross-species amplification tests using SSR markers from Lolium multiflorum × Festuca glaucescens and tall fescue (Festuca arundinacea) have been conducted (Rouf Mian et al. 2005, Litrico et al. 2009).

Here, we report the isolation and characterization of 969 orchardgrass SSR markers from four SSR-enriched genomic libraries. One screening panel consisting of eight orchardgrass individuals was tested with all 969 SSR markers to determine their amplification ability, detect polymorphism levels and identify a set of loci suitable for framework mapping.

Four SSR-enriched genomic libraries (CA/TG, GA/TC, AAG/TTC and TAGA/TCTA) were constructed by Genetic Identification Services (Chatsworth, CA, USA) from one individual of ‘Akimidori II’, a Japanese orchardgrass variety, and the sequences (about 700 bp long) were obtained at Dragon Genomics (Yokkaichi, Mie, Japan). Detailed methods were described in Cai et al. (2003). After sequencing, the Phred values (Brent et al. 1998) were calculated, and clones with Phred values >15 and longer than 100 bp were used for primer design.

A total of 4000 clones (1000 from each of the four libraries) were sequenced. After excluding a few clones whose sequences flanking the SSR motifs were too short to allow us to design both forward and reverse primers, a total of 969 unique SSR clones (sequences containing at least seven dinucleotide repeats or five trinucleotide repeats or three tetranucleotide repeats or two pentanucleotide repeats) were identified and used for design primers. The efficiency of SSR marker isolation was 24.2% (of the sequenced clones), and it was similar to that in zoysiagrass (19.2%, Cai et al. 2005) but lower than the results obtained in sunflower (49.2%, Tang et al. 2002), Italian ryegrass (25.6%, Hirata et al. 2006) and timothy (33.3%, Cai et al. 2003) through the use of SSR-enriched libraries produced by Genetic Identification Services, perhaps because our libraries have a high level of redundancy. The percentage of unique SSR clones in library B (motif GA/TC, 55.6%) was higher than in the other libraries (10.7–21.2%); this result agreed with that in timothy (Cai et al. 2003) and zoysiagrass (Cai et al. 2005).

The 969 unique SSR sequences were searched against the DDBJ nucleotide databases to identify the relationships between the SSR sequences for known genes. BLASTX searches were run on 22 June 2009, and a bits score >100 and an E-value ≤1 × 10⁻⁵ were considered to be significant. And a total of 63 sequences were found that showed significant homology with the sequences of other plant species. Of these, 34 showed significant homology with genomic or cDNA sequences with unknown functions from rice and other plant species, but 18 showed significant homology with known genes (Table 1), five showed significant homology with transposable elements, five were similar to microsatellite sequences from two other species and one was similar to a chloroplast gene in the Agrostis stolonifera cultivar ‘Penn A-4’ and also had a high and significant homology with rice genomic DNA.

The SSR loci were classified by repeat type and structure into four categories: perfect, imperfect, interrupted or compound repeat defined by Jones et al. (2001). All four libraries contained perfect clones at very high frequencies, with an average of 88.4% (ranging from 83.4% to 93.4%). Compound repeats were contained at higher frequencies in library D (14.4%) than in the others (3.3–8.8%). Imperfect clones were contained at low frequencies (≤1.0%) in each library. In all four libraries, the predominant motif was the expected type (CA/TG for A, GA/TC for B, AAG/TTC for C and TAGA/TCTA for D). In the most frequent perfect clones that contained dinucleotide, trinucleotide and tetranucleotide motifs, the average repeat numbers were 19.1 (CA/TG), 22.3 (GA/TC), 13.7 (AAG/TTC) and 12.0 (TAGA/TCTA).

Although AT/TA is the most common dinucleotide motif in plants (Cardle et al. 2000), this motif is not usually used in SSR-enrichment procedures owing to its self-complementary nature; therefore, we did not use AT/TA in our study. Of 857 perfect SSR clones, the motifs occurring at the highest rates were GA/TC (58.7%), CA/TG (20.6%), AAG/TTC (12.9%) and TAGA/TCTA (7.8%). These results and those in timothy (Cai et al. 2003) and zoysiagrass (Cai et al. 2005) are similar to those reported by Cardle et al. (2000), who reported that the most common dinucleotide motif found in plants is AT/TA, followed by GA/TC and CA/TG, and that the most common trinucleotide motifs were AAT/TAA and ATC/TAG, whereas AAG/TTC was dominant in Arabidopsis thaliana.

A panel consisting of eight orchardgrass individuals was used for testing PCR amplification. The panel included three individuals of ‘Akimidori II’, two individuals of ‘Loke’ (a Swedish variety) and three other orchardgrass individuals collected from natural grassland at Nasushiobara, Tochigi, Japan. The primers design, PCR and fragment analysis were according to Cai et al. (2003). Of the 969 primer pairs tested, 606 (62.5%) amplified polymorphic products in the eight orchardgrass individuals used, 70 (7.2%) amplified monomorphic products, 56 (5.8%) amplified multiple bands, and 237 (24.5%) amplified no bands. The percentage of functional primers (62.5% polymorphic and 7.2% monomorphic) in our study was 69.7%, which is higher than those in wheat (47.1%, Röder et al. 1998), and similar to those in timothy (74%, Cai et al. 2003), but lower than those in barley (83.4%, Ramsay et al. 2000), Italian ryegrass (90.4%, Hirata et al. 2006), perennial ryegrass (81%, Jones et al. 2001), sunflower (87.9%, Tang et al. 2002) and zoysiagrass (89.7%, Cai et al. 2005). The screening panel included three individuals of ‘Akimidori II’, two individuals of ‘Loke’ and three other orchardgrass individuals collected at same region (Nasushiobara, Tochigi, Japan), the close relationship of these individuals may be the reason of low rate of polymorphism.

The percentage of polymorphic primers in library B was 73.8% (398/539)—higher than those observed in the other three libraries (about 50%). Most markers had a large number of alleles, ranging from 1 to 13 (Table S1). The number of primer pairs that produced one allele per individual was 66 (10.9%) vs. 277 (45.7%) for two alleles, 184 (30.4%) for three alleles and 79 (13.0%) for four alleles (Table S1).

A pseudo-testcross F₁ population consisting of 88 individuals derived from a cross between two orchard grass cultivars, ‘Akimidori II’ and ‘Loke’, was used to detect polymorphism of the mapping population. The efficiency of the polymorphic markers in a mapping population was also tested by using the 606 SSR markers in a screening between the two parents of the mapping population and revealed that 53% (321) of the markers were polymorphic.

We isolated and characterized 969 SSR sequences from four SSR-enriched libraries and used them to develop 606 polymorphic SSR markers by screening eight orchardgrass individuals. Orchardgrass is one of the best-documented examples of a natural intraspecific polyploidy complex, but before these SSR markers were developed, most studies were based on the geographical distribution, comparative morphology, cytogenetic structure and interploidal hybridization of the species. When combined with recent isozyme and other non-species-specific molecular markers, the SSR markers developed in this study will be powerful tools for studying old problems such as origin, differentiation, gene flow and gametic non-reduction in Dactylis, the oldest studied grass species. The markers will also provide an ideal marker system for studying new challenges in modern breeding, such as gene targeting, quantitative trait locus mapping, variety or species identification and marker-assisted selection in Dactylis species.