Expressed sequence markers for genetic analysis of bulb onion (Allium cepaL.)

Sequencing of cDNA clones previously screened for ability to reveal RFLPs i n bulb onion has been completed and a further 128 ESTs from 111 clones have been deposited in public databases. A putative function was assigned to 66 % (84/128) of ESTs by BLASTX searches against public databases and FASTA co mparisons were used to determine similarity among clones, including those w hich detected linked RFLP loci. Cleavage amplified polymorphisms (CAPs) and single-stranded conformation polymorphisms (SSCP) were evaluated as strate gies for converting onion expressed sequence tags (ESTs) into PCR-based ass ays for gene mapping. We screened 14 ESTs with 8 to 12 restriction enzymes and detected two CAPs, which mapped in the 'Brigham Yellow Globe' (BYG15-23 )x'Ailsa Craig' (AC43) mapping population. A wider survey of CAPs for ESTs among eight bulb onion populations with six frequently cutting restriction enzymes detected variation, but too little to be practical for routine gene mapping. By contrast, non-radioactive SSCP of amplicons from 3' UTRs of ES Ts was found to detect useful levels of variation within bulb onion germpla sm. In addition to SSCPs, homo- and hetero-duplex polymorphisms (duplex pol ymorphisms) were also frequently observed on the same gets. Of a total of 3 1 ESTs surveyed, 26 exhibited SSCP/duplex variation among bulb onion popula tions. SSCP/duplex polymorphisms in 11 ESTs were mapped in the 'BYG15-23'x' AC43' family and, of these, ten were linked to an RFLP locus revealed by th e original cDNA. The SSCP/duplex assays of five additional ESTs showed Mend elian segregations in the 'Colossal Grano'x'Pukekohe Longkeeper' (P12) F-2 population. Two of these markers were linked, as predicted from linkage of their corresponding RFLPs in the 'BYG15-23'x'AC43' family. Ninety two perce nt (12/13) of EST PCR products that amplified in Allium roylei exhibited ma rked differences in SSCP patterns from bulb onion. ESTs for invertase and s ucrose-sucrose fructosyltransferase were mapped by SSCP and an ATP sulfuryl ase gene cloned by RT-PCR revealed SSCP/duplex polymorphism within bulb oni on. These results demonstrate that SSCP/duplex is an efficient and economic al technique for exploiting onion EST information for gene mapping in onion .