|Mccallum, John - CROP&FOOD RES NEW ZEALAND|
|Leite, Daniela - CLIMA TEMP PELOTAS BRAZIL|
|Pither-Joyce, Meeghan - CROP FOOD RES NEW ZEALAND|
Submitted to: Journal of Theoretical and Applied Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 15, 2001
Publication Date: N/A
Interpretive Summary: The genetic analysis of onion is difficult because of a huge amount of DNA in the nucleus and relatively long generation times. Expressed sequence tags (EST) reflect the locations of expressed genes in the nucleus and are useful to assign specific traits to expressed genes. We developed ESTs in onion and assigned putative functions to 35 percent (44/126) using public databases. We identified techniques allowing for the easy and relatively cheap identification of ESTs in onion. These results are useful for identification of specific regions of the onion DNA and assignment of specific traits to variation at known genes. We should be able to use these results to better understand the genetic bases of economically important traits in onion and aid in the development of new value-added onion cultivars.
Technical Abstract: Sequencing of cDNA clones previously screened for ability to reveal RFLP in bulb onion has been completed and a further 126 ESTs from 111 clones has been deposited in public databases. Putative function was assigned to 35 percent (44/126) of ESTs by BLASTX searches against public databases and FASTA comparisons among the sequences was used to clarify similarity of clones that detected linked RFLP loci. Cleavage amplified polymorphisms (CAPS) and single-stranded conformation polymorphisms (SSCP) were evaluated as strategies for converting onion expressed sequence tags (ESTs) into PCR-based assays 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 detected variation, but too little to be practical for routine gene mapping. By contrast, non-radioactive SSCP of amplicons from 3' UTRs of ESTs was found to detect useful levels of variation within bulb onion germplasm. In addition to SSCPs,, homo- and heteroduplex polymorphisms (duplex polymorphisms) were also frequently observed on the same gels. Of a total of 31 ESTs surveyed, 26 exhibited SSCP/duplex variation among bulb onion populations. SSCP/duplex polymorphisms in eleven ESTs were mapped in the 'BYG15-23' x 'AC43' family and of these, ten were closely linked to an RFLP locus revealed by the original cDNA.