Detecting Genes Expressed at Different Stages of Reproductive Arrest in Brassica oleracea

Abstract

Cauliflower (Brassica oleracea var. italica) and Broccoli (Brassica oleracea var. botrytis) differ mainly in the stage of reproductive arrest. Cauliflower curd is an inflorescence meristem, while broccoli arrests just before anthesis. Arabidopsis studies led to the hypothesis that a mutant BoCAL allele arrested cauliflower earlier. Later, a mutant in BoAP1 was found to have similar effects. These partially redundant genes, and several identified since, are present in multiple copies in B. oleracea. Understanding their role in the arrest requires quantification of transcript abundance analysis by real-time PCR. Designing selective PCR primers is a critical first step in the process. Designs were based on alignment among the genes of interest (MADS-box genes BoCAL, BoAP1, FUL, and the non MADS-box genes LFY and TFL1) and their paralogs. The high sequence similarity (some over 95%) makes the target transcripts difficult to distinguish. Therefore, primers were designed mostly for targets in the 3'UTR region in order to gain specificity. Short amplicons, 68bp to 200bp, were required for the high PCR efficiency required to quantify these low-abundance transcripts. Primers were evaluated by conventional RT-PCR and real-time PCR. By altering temperature, Total RNA was isolated from plants that were arrested at three developmental stages, inflorescence meristem (cauliflower), floral meristem (intermediate), and floral bud (broccoli) by varying temperature. RT-PCR products were single bands of the expected size, despite the high homology between genes under study. Real-time melting curve analysis (fluorescence derivative vs. melting temperature) corroborated the presence of a single amplicon. The identity of products was confirmed by sequencing and restriction enzyme digestion.