Molecular tools for understanding and predicting flowering in cauliflower

Synchronous flowering between parent lines is essential for pollination and seed set in commercial hybrid cauliflower seed production. However, weather-related variation in reproductive development and attempts to produce novel hybrids by crossing parents with markedly different flowering phenology make synchronous flowering difficult to achieve. Molecular technology presents an opportunity to understand and manage this variability. Prospects for understanding the molecular control of flowering time in cauliflower have benefited greatly from recent work with the closely-related model species Arabidopsis thaliana. Over the last decade, genetic analysis in Arabidopsis has identified a large number of flowering genes, enabling the development of a model describing the molecular pathways that regulate floral induction through environmental cues such as photoperiod and vernalisation. Although elements of this model are conserved in B. oleracea, the scenario has been complicated by ancient genome triplication and rearrangements within the genome. The degree of genetic diversity generated by such events has given rise to a wide range of flowering habits within the species. In this study, we investigated the contribution of the BoFLC gene family to flowering time variation and examined the vernalisation-regulated expression of key flowering genes. The flowering behaviour of 54 homozygous cauliflower lines ranging from tropical to winter varieties was characterised and plants were screened for polymorphisms in flowering time candidate genes including members of the vernalisation-mediated BoFLC family. A functional allele of the BoFLC2 gene was identified for the first time in an annual brassica variety, along with an allele disrupted by a frameshift mutation in exon 4. A strong correlation between flowering time and BoFLC2 genotype was observed in field and glasshouse trials. In a segregating F\(_2\) population derived from a cross between late-flowering (functional BoFLC2) and early-flowering (mutated BoFLC2) cauliflower lines, the BoFLC2 gene behaved in a dosage-dependent manner and BoFLC2 genotype was found to account for up to 65% of flowering time variation. The identification of this gene as an important determinant of flowering time within brassicas grown as annual crops is potentially valuable to plant breeders, with possible applications as a genetic input for QTL-based models of flowering time, as a marker for screening and classifying flowering time in cauliflower germplasm, or as a target for genetic modification. RT-qPCR was used to study the effects of vernalisation on the expression of several key flowering time candidate genes in a wide range of flowering types. For the first time in cauliflower, it was shown that vernalisation reduced levels of BoFLC2 and BoFLC3 transcript and upregulated expression of the flowering signal integrator BoFT, with colder temperatures and increased vernalisation duration amplifying these effects and hastening curd initiation. BoFT was not significantly upregulated in vernalised plants less than four weeks old, whilst the final level of BoFLC2 and BoFLC3 downregulation was independent of plant age. BoFLC2 gradually increased with plant age in unvernalised plants, possibly reflecting the transition from reproductive juvenility to maturity. In early-flowering lines with mutated BoFLC2, overall expression of BoFLC2 was lower and BoFT expression significantly higher supporting the idea that BoFLC2 plays a key role in maintaining the vegetative state. A homologue of Arabidopsis VIN3 was also isolated for the first time in a brassica crop species, and was found to be upregulated in ten-week-old plants by as little as two days of vernalisation at 5°C, in contrast to findings in Arabidopsis, where prolonged exposure to cold was required to elicit upregulation. Minor differences in the transcriptional dynamics of these genes were observed between apex and leaf tissue. Devernalisation and floral reversion have been reported to contribute to asynchronous or uneven flowering in cauliflower seed production, forcing growers to avoid warm and dry areas that would otherwise be conducive to high-yielding crops. The effects of devernalising conditions on curd development and gene expression were investigated. In contrast to findings in Arabidopsis, the vernalisation-induced downregulation of BoFLC2 was unstable, with expression returning to pre-vernalisation levels after return to warm conditions. By contrast, BoFT was stably upregulated, possibly suggesting the involvement of a BoFLC-independent process. The strong correlations observed between gene expression and flowering time in controlled-environment experiments were validated with gene expression analysis of field-grown cauliflower parent lines and cauliflowers grown outdoors in pots. "Natural" outdoor vernalising temperatures were capable of downregulating BoFLC2 and upregulating BoFT, and a strong correlation was found between both BoFLC2 and BoFT expression and curding and flowering time. These findings indicate a need for further investigation of the potential for this molecular tool to be used as a predictive assay for curd initiation and flowering time, or as a management tool for assessing the effect of crop treatments such as growth regulator applications.