Natural variation in CIRCADIAN CLOCK ASSOCIATED 1 is associated with flowering time in Brassica rapa

Flowering time is a very important agronomic trait and the development of molecular markers associated with this trait can facilitate crop breeding. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a core oscillator component of circadian rhythms that affect metabolic pathways in plants, has been implicated in flowering time control in species of Brassica. CCA1 gene sequences from three Brassica rapa inbred lines, showing either early flowering or late flowering phenotypes, were analyzed and a high level of sequence variation was identified, especially within the fourth intron. Using this information, three PCR primer sets were designed and tested using various inbred lines of B. rapa. The usage of InDel markers was further validated by evaluation of flowering time and high resolution melting (HRM) analysis. Both methods, PCR and HRM, validated the use of newly developed markers. Additional sequence analyses of Brassica plants with diploid (AA, BB, or CC) and allotetraploid genomes further confirmed a large number of sequence polymorphisms in the CCA1 gene, including insertions/deletions in the fourth intron. Our results demonstrated that sequence variations in CCA1 can be used to develop valuable trait-related molecular markers for Brassica crop breeding.