Theoretical and Applied Genetics

, Volume 103, Issue 6, pp 979–991

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

  • J. McCallum
  • D. Leite
  • M. Pither-Joyce
  • M. J. Havey

DOI: 10.1007/s001220100630

Cite this article as:
McCallum, J., Leite, D., Pither-Joyce, M. et al. Theor Appl Genet (2001) 103: 979. doi:10.1007/s001220100630

Abstract  

Sequencing of cDNA clones previously screened for ability to reveal RFLPs in 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 comparisons were used to determine similarity among clones, including those which 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)×’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 ESTs was found to detect useful levels of variation within bulb onion germplasm. In addition to SSCPs, homo- and hetero-duplex 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 11 ESTs were mapped in the ’BYG15–23’×’AC43’ family and, of these, ten were linked to an RFLP locus revealed by the original cDNA. The SSCP/duplex assays of five additional ESTs showed Mendelian segregations in the ’Colossal Grano’×’Pukekohe Longkeeper’ (P12) F2 population. Two of these markers were linked, as predicted from linkage of their corresponding RFLPs in the ’BYG15–23’×’AC43’ family. Ninety two percent (12/13) of EST PCR products that amplified in Allium roylei exhibited marked differences in SSCP patterns from bulb onion. ESTs for invertase and sucrose-sucrose fructosyltransferase were mapped by SSCP and an ATP sulfurylase gene cloned by RT-PCR revealed SSCP/ duplex polymorphism within bulb onion. These results demonstrate that SSCP/duplex is an efficient and economical technique for exploiting onion EST information for gene mapping in onion.

Keywords  Expressed sequence tagSingle-stranded conformation polymorphismsHeteroduplex analysisAllium cepa

Copyright information

© Springer-Verlag Berlin Heidelberg 2001

Authors and Affiliations

  • J. McCallum
    • 1
  • D. Leite
    • 2
  • M. Pither-Joyce
    • 1
  • M. J. Havey
    • 3
  1. 1.Crop and Food Research, Private Bag 4704, Christchurch, New Zealand e-mail: mccallumj@crop.cri.nz Tel.: 64-3-325-6400, Fax: 64-3-325-2074NZ
  2. 2.Embrapa Clima Temperado, BR 392 Km 78, 96001-970 Pelotas, RS, BrazilBR
  3. 3.USDA-ARS, Department of Horticulture, 1575 Linden Drive, University of Wisconsin, Madison, WI 53706, USAUS