A genetic linkage map of Physocarpus, a member of the Spiraeoideae (Rosaceae), based on RAPD, AFLP, RGA, SSR and gene specific markers B. G. S UTHERLAND 1 , K. R. T OBUTT 1 , A. M ARCHESE 1 , G. P ATERNOSTER 1 , D. W. S IMPSON 1 and D. J. S ARGENT 1,2 1 East Malling Research (EMR), New Road, East Malling, Kent ME19 6BJ, UK; 2 Corresponding author, E-mail: dan.sargent@emr.ac.uk With 1 figure and 1 table Received October 24, 2007/Accepted January 17, 2008 Communicated by T. Debener Abstract Physocarpus opulifolius is a deciduous shrub native to North America belonging to the Spiraeoideae subfamily of the Rosaceae. The cultivars ÔLuteusÕ and ÔDiaboloÕ are grown in gardens for their ornamental foliage, golden and purple respectively. We developed a linkage map of P. opulifolius with a view to detecting markers for the leaf colour genes, which are under major gene control. A total of 162 molecular markers (128 RAPDs, 27 AFLPs, three RGA, three STS markers and one SSR) and the leaf colour genes Pur and Aur were scored in the Physocarpus progeny and used to create a linkage map covering 586.1 cM over nine linkage groups. There was an average of 18.2 markers per linkage group and a mean linkage group length of 65.1 cM. Both leaf colour genes were mapped. This is the first reported linkage map of a member of the Spireaeoideae and the mapping of a small number of transferable markers has demonstrated its utility to comparative mapping, which will com- plement existing comparative mapping efforts in other rosaceous subfamilies. Key words: comparative mapping — Spireaeoideae — Rosa- ceae — molecular marker Physocarpus opulifolius (L.) Maxim., or ninebark, is a decid- uous shrub native to North America. The common name comes from the appearance of the bark, which peels away in many layers, giving it a flaky appearance. The cultivars ÔLuteusÕ and ÔDiaboloÕ are grown in gardens for their ornamental maple-like foliage, golden and deep purple respectively. Physocarpus belongs to the Spiraeoideae (2n = 2x = 18) subfamily of the Rosaceae, a horticulturally important family. Recently, studies of chloroplast DNA sequences (Morgan et al. 1994, Potter et al. 2002) have indicated that the Spiraeoideae are polyphyletic. The genetics of many of the economically important genera within the three major subfamilies of Rosaceae (Prunoideae, Maloideae and Rosoideae) have been studied in detail at the molecular level and linkage maps based on a wide range of molecular marker systems have been produced with a view to marker-assisted selection. These include maps of soft-fruit genera including Fragaria (Sargent et al. 2006) and Rubus (Graham et al. 2006), tree fruit genera including Malus (Silfverberg-Dilworth et al. 2006), Prunus (Dirlewan- ger et al. 2004) and Pyrus (Yamamoto et al. 2002), and ornamental genera such as Rosa (Yan et al. 2005). The maps of all these major economic genera employ transferable markers such as microsatellites (SSRs), which are transfer- able between species within a genus and often between closely related genera such as Malus and Pyrus (Yamamoto et al. 2004). The Spiraeoideae have been little studied genetically, and no linkage maps or transferable molecular markers are currently available for Physocarpus or closely related genera. Random amplified polymorphic DNA (RAPD) and ampli- fied fragment length polymorphism (AFLP) markers require no prior knowledge about genome sequence of a crop species and produce large numbers of polymorphic markers per primer combination used. As a result, they have frequently been used for generating linkage maps (Pattison et al. 2007), and such markers therefore provide an alternative route to producing saturated maps where no transferable markers exist. As part of a programme to breed woody ornamentals and to clarify the genetics of horticulturally important characters at East Malling, a progeny was raised from the cross P. opulifo- lius ÔDiaboloÕ · ÔLuteusÕ, and two of the seedlings were subsequently intercrossed. The resulting progeny, approximat- ing to an F 2 , has been used to develop a preliminary map for Physocarpus, based primarily on RAPD and AFLP markers, which were supplemented with resistance gene analogues (RGA), known function gene and SSR markers from other genera. Our intention was to construct a linkage map for Physocarpus to allow us to map the genes responsible for golden and purple foliage, and to extend linkage mapping studies using molecular markers in the Rosaceae to a member of the Spiraeoideae. Materials and Methods Mapping population, phenotypic assessment and DNA extraction: An initial progeny (#764) of nine seedlings was raised from the cross ÔDiaboloÕ · ÔLuteusÕ and two of these, 764-3 (purple and golden) and 764-Z (purple) were intercrossed (764-3 · 764-Z) to produce a mapping progeny, Phy-5 of 94 seedlings. Seedlings were scored for leaf colour – green, purple, golden or purple-golden (which appeared as amber or tawny) scored in spring and early summer on older plants grown outdoors. Genomic DNA was extracted from the parents and the 94 seedlings following the CTAB extraction protocol of Doyle and Doyle (1987) with the addition of 1% [v/v] b-mercaptoethanol and 2% [w/v] polyvinyl pyrollidone (PVP 40) to the extraction buffer. RAPD markers: The RAPD primers used in this investigation were selected from the Operon RAPD series A-L (Operon Biotechnologies GmbH, Ko¨ln, Germany). PCR was performed in a final volume of 12.5 ll comprising 1 ng template DNA, 1· PCR buffer, 1.5 mM Mg 2+ , 200 lM dNTPs, 0.4 lM RAPD primer and 0.25 U Taq polymerase (Invitrogen, Paisley, UK). Reactions were then carried out using the following RAPD protocol: an initial denaturation step of 94°C for 3 min, followed by 40 cycles of 94°C for 1 min, 35°C for 2 min 30 s, 44°C for 30 s and 72°C for 2 min 30 s, then a final single extension step of 72°C for 7 min. Plant Breeding doi:10.1111/j.1439-0523.2008.01505.x Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Verlag, Berlin www.blackwell-synergy.com