A multiplex PCR assay for identification of the ogura male sterile cytoplasm and the Rfo restorer gene among oilseed rape breeding forms
Journal Title: Rośliny Oleiste - Oilseed Crops - Year 2010, Vol 31, Issue 2
Abstract
Due to the fact that cross-pollination controlling male sterility systems have been developed for oilseed rape, breeders’ interest in hybrid varieties of this oil crop permanently increases. In Poland, a total of 111 oilseed rape cultivars are currently registered in the National Register of the Research Centre for Cultivar Testing, including 87 winter varieties – 35 of which are hybrid ones. The F1 restored hybrids and the restorer lines are phenotypically identical with the canola-type (double-low, 00) varieties, hence in order to monitor the presence of the ogura male-sterile cytoplasm (ogura CMS) and the Rfo restorer gene, two genetic markers were applied independently — one of them specific for the ogura CMS (a SCAR marker of about 500 base pairs) and another one — for the Rfo restorer gene (the C02 SCAR of about 1 100 bp). In order to upgrade the effectiveness of the performed analyses and to reduce the costs, the ‘multiplex PCR’ method was adapted based on a simultaneous analysis of both SCAR markers in one PCR amplification. In addition, an internal control comprising PCR amplification of a conservative region of an actin cDNA (a structural protein present in all eukaryotic cells), a 600 bp SCAR marker, was included. As a result, false negatives due to the matrix DNA degradation, bad quality of reagents and solutions or possible manual mistakes were eliminated. Two hundred and eight of DNA samples obtained from oilseed rape F1 hybrids, restorer lines and recombinant breeding forms were genotyped with the use of the multiplex PCR method. Simultaneous analysis by PCR with a single primer set for the two SCAR markers was performed thus confirming the reliability of the multiplex PCR method. It will be further applied for routine analysis in hybrid and recombinant breeding.
Authors and Affiliations
Katarzyna Mikołajczyk, Agnieszka Dobrzycka, Jan Podkowiński, Wiesława Popławska, Stanisław Spasibionek, Iwona Bartkowiak-Broda
Keywords
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