Towards a Framework PCR-Based Map of Onion (Allium cepa L.)

Genetic analysis of onion has been hampered by a lack of portable codominant markers based on the polymerase chain reaction (PCR). The public release of a relatively large set of non-redundant onion expressed sequence tags (ESTs) in 2003 has provided the opportunity to develop such markers for use in Allium research and industry. We have mined this collection for simple sequence repeats (SSRs) and screened over 200 primer sets designed to flank SSRs for their ability to amplify low-copy polymorphic products in Allium cepa and A. roylei. Screening to date confirms that these are a rich source of polymorphic markers in Allium. Half of the sets reveal polymorphism between onion and A. roylei, ~40% reveal putative allelic variation in a small set of onion germplasm and 20% reveal polymorphism in a single F2 family in the cross ‘W202A’ x ‘Texas Grano 438’. Mapping common markers has permitted alignment with the RFLP map developed in the ‘Brigham Yellow Globe 15-23’ x ‘Alisa Craig 43’ population. Preliminary studies show these markers are effective in distinguishing variation in allelic diversity among a set of inbred and open-pollinated onion varieties. Our results suggest that these markers will enable effective genome scanning and analysis of genetic diversity and identity in cultivated onion. INTRODUCTION Genetic understanding of onion (Allium cepa L.) is significantly less developed than other crops due to the practical difficulties of developing and maintaining genetic stocks and a lack of DNA sequence and marker resources. Existing Allium genetic maps based on RFLP (King et al., 1998) and AFLP markers (van Heusden et al., 2000b) have permitted the tagging of genome regions associated with economic traits but have not provided portable genetic markers based on the polymerase chain reaction (PCR) that can be widely applied to exploit or extend these findings. Until 2003, sequence resources in Allium were limited to small sets of onion expressed sequence tags (ESTs) generated by projects at USDA-ARS (King et al., 1998), Plant Research International (Unpublished) and Crop & Food Research (McCallum et al., 2002). We previously showed that standard mutation-scanning methods such as single-stranded conformation polymorphism (SSCP; McCallum et al., 2001) or cleaved amplified polymorphic sequence (CAPS; van Heusden et al., 2000a) analyses could be used to convert some EST sequences into useful PCRbased genetic markers. The USDA Asparagales genome project has recently released a set of over 18,000 onion ESTs (Kuhl et al., 2004). We describe recent progress in exploiting this resource for simple sequence repeat (SSR) marker development and preliminary results in using such markers in genetic map development and diversity studies. RESULTS AND DISCUSSION Genetic Resources The key genetic resource to date has been the set of 58 F3 families from the cross Proc. IV IS on Edible Alliaceae