Initial Fine Mapping of the Spinach Downy Mildew Resistance Locus RPF1

Spinach is an important vegetable crop grown in many parts of the world (Correll et al. 2011). It has become increasingly popular due to its nutritional value as it is very high in antioxidants (Prior, 2003). Spinach production, however, is affected by many biotic stresses. Downy mildew, caused by Peronospora farinosa f. sp. spinaciae (Pfs) , is perhaps the most important biotic constraint for spinach production worldwide (Correll et al, 1994). As of 2012, 14 races of Pfs have been reported (Correll et al., 2011). The rapid increase of new races is likely a direct result of substantial changes in spinach production during the past decade, including a rapid expansion of planted acreage, 12-month per year production cycles, high density plantings, reduced usage of rotation crops, and possibly the global movement of the pathogen either as mycelium in seed or as dormant oospores (Inaba and Morinaka, 1984; Inaba et al., 1983). Very few studies have been published on the genetics of resistance to Pfs . However, several more recent studies on the genetics of resistance have been conducted and it has been hypothesized that there are at least six resistance loci (RPF1-RPF6) which control resistance to Pfs races 1-14 (Correll et al., 2007; 2011; Irish et al., 2008). Irish et al. (2008) also identified a molecular marker, designated Dm-1, which was closely linked to the resistance locus RPF1. The Dm-1 marker was a co-dominant marker and the genetic distance between Dm-1 and RPF1 was estimated to be 1.7 cM. In the current study, efforts were initiated to fine-map the Dm-1 marker relative to the RPF1 locus. To address this objective, a spinach BAC library that was previously developed from line NIL1 containing Dm-1 and RPF1 was evaluated. Southern blot hybridizations identified 19 BAC clones that contained Dm-1. Dm-1 was subsequently confirmed in these 19 clones based on PCR analysis. Out of over 75,000 clones evaluated, BAC-end sequencing was conducted on 14 of the 19 BAC clones and the sequence information was used to develop probes for Southern hybridizations. Hybridizations with the 28 probes developed were conducted to identify the BAC-end furthest from the Dm-1 marker. These activities provided important sequence information about the region surrounding the Dm-1 marker, and refined a protocol to map and ultimately clone the RPF1 locus through BAC-based chromosome walking combined with genetic mapping