Theor Appl Genet (2005) 111: 795–803 DOI 10.1007/s00122-005-2065-x ORIGINAL PAPER N. Chakraborty Æ J. Bae Æ S. Warnke T. Chang Æ G. Jung Linkage map construction in allotetraploid creeping bentgrass (Agrostis stolonifera L.) Received: 3 December 2004 / Accepted: 2 May 2005 / Published online: 25 June 2005 Ó Springer-Verlag 2005 Abstract Creeping bentgrass (Agrostis stolonifera L.) is Introduction one of the most adapted bentgrass species for use on golf course fairways and putting greens because of its high Agrostis, or bentgrass, is a large genus of over 200 species tolerance to low mowing height. It is a highly out- in the Poaceae family (Hitchcock 1951). Only five species crossing allotetraploid species (2n=4x=28, A and A 2 3 are used as turfgrass in the United States: colonial subgenomes). The first linkage map in this species is (Agrostis capillaris L.), velvet (Agrostis canina L.), dry- reported herein, and it was constructed based on a land (Agrostis castellana Boiss. and Reut.), redtop (Ag- population derived from a cross between two heterozy- rostis gigantea Roth) and creeping (Agrostis stolonifera gous clones using 169 RAPD, 180 AFLP, and 39 het- L.). These species are perennial, outcrossing cool-season erologous cereal and 36 homologous bentgrass cDNA grasses used for lawns, athletic fields, and golf courses. RFLP markers. The linkage map consists of 424 map- Currently, the stoloniferous, allotetraploid creeping ped loci covering 1,110 cM in 14 linkage groups, of bentgrass (2n=4x=28, A and A subgenomes) is the which seven pairs of homoeologous chromosomes were 2 3 most adapted species for use on golf course fairways and identified based on duplicated loci. The numbering of all greens because of its fine texture and high tolerance to seven linkage groups in the bentgrass map was assigned low mowing height (Warnke 2003). This demand has according to common markers mapped on syntenous created $50 million in sales in the turf seed industry, chromosomes of ryegrass and wheat. The number of making creeping bentgrass an economically important markers linked in coupling and repulsion phase was in a turfgrass species in the United States (Cook 1996). 1:1 ratio, indicating disomic inheritance. This supports a Molecular marker-based genetic linkage maps have strict allotetraploid inheritance in creeping bentgrass, as been developed in a number of economically important suggested by previous work based on chromosomal cereal crops such as wheat (Triticum aestivum L.), rice pairing and isozymes. This linkage map will assist in the (Oryza sativa L.), oat (Avena spp.), barley (Hordeum tagging and eventually in marker-assisted breeding of spp.), and maize (Zea mays L.) (Rayapati et al. 1995), economically important quantitative traits like disease and also in pasture and turfgrass species like perennial resistance to dollar spot (Sclerotinia homoeocarpa F.T. ryegrass [(Lolium perenne L.) Hayward et al. 1994; Jones Bennett) and brown patch (Rhizoctonia solani Kuhn). et al. 2002; Warnke et al. 2004; Sim et al. 2005], Italian ryegrass [(Lolium multiflorum Lam.) Inoue et al. 2004], Keywords Agrostis stolonifera L. Æ Creeping bentgrass Æ and meadow fescue [(Festuca pratensis Huds.) Alm et al. Allotetraploid Æ Linkage map Æ Homoeologous 2003]. Plant breeders and geneticists are increasingly chromosomes Æ Molecular markers using molecular markers to study genome structure and to manipulate genes. Furthermore, many Poaceae spe- cies possess significant chromosomal regions of con- Communicated by P. Langridge served synteny between their genetic maps constructed using a common set of heterologous RFLP probes (Gale & N. Chakraborty Æ J. Bae Æ T. Chang Æ G. Jung ( ) and Devos 1998). Since gene order of perennial ryegrass Department of Plant Pathology, University of Wisconsin, Madison, WI 53706, USA and Triticeae cereals are highly conserved, their indi- E-mail: [email protected] vidual maps have been merged (Jones et al. 2002; Sim S. Warnke et al. 2005). Such map integration and comparative map USDA–ARS US National Arboretum, analysis can be used for the improvement of less well- Washington, DC 20002, USA studied species like bentgrass. 796 Linkage maps have been constructed in allotetraploid available, 94 progenies along with the parental clones species such as cotton (Gossypium sp.), tef [Eragrostis tef were randomly selected and used in mapping in an initial (Zucc) Trotter], white clover (Trifolium repens), and RAPD analysis, and subsequently, 90 progeny of the Brassica napus L. (Osborne et al. 1997; Brubaker et al. original 94 were used in AFLP and RFLP analysis for 1999; Zhang et al. 2001; Barrett et al. 2004). However, map construction. these species are self-compatible, and therefore, mapping populations have a maximum of two alleles per locus, whereas creeping bentgrass is a self-incompatible allo- RAPD analysis tetraploid consisting of two diploid genomes with a maximum of four alleles per locus (Warnke et al. 1998). DNA extraction for RAPD analysis was performed Moreover, in some species like Brassica sp., double according to Scheef et al. (2003). Initially, 523 primers haploid lines have been developed that reduce the (Operon Technologies, Alameda, Calif. USA) were number of alleles to two per locus. Maps have also been screened against the two parents and 65 primers with the constructed from known diploid progenitors, which highest number of polymorphic bands were chosen to be reduce the complexity of interpreting codominant tested on the 94 progenies. RAPD reactions were per- banding patterns in segregating progeny. This linkage formed in 96-well plates with a total volume of 10 llin map in creeping bentgrass will be the first one con- an MJ PTC-100 thermal cycler (MJ Research, Water- structed for an outcrossing, allotetraploid monocot town, Mass., USA) with the same thermal cycling con- species with no available diploid progenitors. ditions as described by Scheef et al. (2003)of91°C for In this paper, our objectives were to construct a denaturation, 42°C for annealing, and 72°C for elon- genetic linkage map from a cross between two highly gation. PCR products were run in a 1.5% (w/v) agarose heterozygous creeping bentgrass clones using RAPD, gel and stained in ethidium bromide (1.5 lg/ml) for AFLP, and heterologous cereal and homologous bent- 30 min and subsequently destained in distilled water for grass cDNA RFLP markers, and to designate each of 20–30 min. the seven pairs of homoeologous chromosomes in Each segregating RAPD marker was named using bentgrass respective to syntenous chromosomes of its approximate size in base pairs, combined with the wheat and ryegrass. We also discuss whether creeping Operon primer name. Three banding pattern classes bentgrass follows a disomic segregation pattern. were identified: (1) bands present in both parents and segregating 3:1 in progeny (indicated by marker names beginning with D); (2) band present only in the female Materials and methods parent (549) and segregating as 1:1 in the progeny (name of the marker beginning with 5); and (3) band Plant material and full-sib mapping population present only in the male parent (372) and segregating as 1:1 in the progeny (name of the marker beginning Two highly heterozygous parental creeping bentgrass with 3). clones, 372 and 549, were selected from 700 clones col- lected from golf course fairways and putting greens throughout Wisconsin that are at least 75 years old AFLP analysis (Casler et al. 2003). Both clones are from fairways, and clone 372 was collected from Lake Forest Golf Course, DNA digestions, adaptor ligations, and pre-selective Eagle River, Wis., and clone 549 was obtained from and selective amplifications were performed according to Ojibwa Golf Course, Chippewa Falls, Wis. (Wang et al. instructions provided with an AFLP analysis kit pur- 2005). These two creeping bentgrass clones exhibit dra- chased from Invitrogen (Gathersburg, Md., USA), and matic differences in leaf color, shoot density, root depth, standard AFLP procedures (Vos et al. 1995). The and disease response to snow mold (Typhula spp.) selective amplifications with three selective bases per (Wang et al. 2005) and dollar spot (Sclerotinia homo- primer were carried out using 6-carboxy fluorescein eocarpa F.T. Bennett) (Chakraborty et al. 2003). Clone fluorescent primers labeled on the 5¢ nucleotide. The 372 is lighter green in leaf color with higher shoot den- amplified fragments were detected with an ABI3730xl sity, shorter internodes, and narrower leaves than the instrument (PE Applied Biosystems, Foster City, Calif., dark-green clone 549. USA). Each sample lane included the GeneScan 500- During the spring of 2002, a full-sib mapping popu- LIZ internal lane standard (Larson et al. 2001). The lation named 549 · 372 consisting of 697 progeny was capillary electrophoresis procedures were performed by developed in a greenhouse, Madison, Wis., from a single the Bovine Functional Genomics Lab (Beltsville, Md., controlled cross between the creeping bentgrass clones USA). Fluorescent fragments between 50 nucleotides 372 and 549. Seeds were collected in late June from the (nt) and 500 nt were identified by GeneScan 3.1 software maternal parental clone 549. Seeds were sown in a 4-in. (PE Applied Biosystems). GeneScan trace files were then round pot, and after germination and growth in the analyzed for the presence or absence of AFLP products, greenhouse, individual seedlings were transferred to 3-in. in 1-nt intervals using the computer program Genogra- square pots and assigned a number. Of 697 progeny pher (Benham et al. 1999). 797 RFLP analysis Parental clones were screened with 140 bentgrass cDNA probes (termed ‘‘Ast’’ markers for Agrostis spp.), Young leaf tissue from greenhouse-grown plants was and a subset of 20 polymorphic probes were chosen for harvested, lyophilized, and genomic DNA was extracted progeny evaluation based on the criteria detailed above from 500 mg of ground tissue using a modified cetylt- for heterologous probes.