Allelic variation in Brassica oleracea CIRCADIAN CLOCK ASSOCIATED 1 (BoCCA1) is associated with freezing tolerance

Abstract

Freezing tolerance is an important horticultural trait in cabbage (Brassica oleracea). Molecular markers for freezing tolerance are needed for marker-assisted breeding of freezing-tolerant cabbage plants. To develop gene-based molecular markers for freezing-tolerance in cabbage, we focused on CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a core circadian clock component that affects metabolic pathways and confers cold tolerance by upregulating C-repeat binding factor (CBF) pathway genes. We cloned and analyzed CCA1 genes (BoCCA1s) from seven inbred lines and one cultivar of B. oleracea ssp. capitata. Two types of BoCCA1 alleles were detected: BN106-type (freezing-tolerant; BoCCA1-1) and BN107-type (freezing-susceptible; BoCCA1-2). Numerous insertions/deletions (InDels), simple sequence repeats, and single nucleotide polymorphisms (SNPs) were found in the exons and introns of BoCCA1s from the ATG start codon at the second exon to the TGA stop codon at the eighth exon. Using InDels and SNPs, we designed PCR primer pairs to distinguish the freezing-tolerant lines, and validated these markers using 102 cabbage lines and cultivars. The inbred lines possessed either the BN106-type or BN107-type allele, but most cultivars had both alleles. Freezing-tolerant cabbage plants had BN106-type InDels and/or BN106-type SNPs regardless of the presence of BN107-type InDels and SNPs, and BN106-type SNPs were more widely detected in the freezing-tolerant cabbage plants than BN106-type InDels. The expression patterns of BoCCA1-1 and BoCCA1-2 were similar under normal versus temperature-stressed conditions (low and high temperatures), suggesting a functional difference at the post-transcriptional level. Cabbage breeders should use several markers derived from different genes and independently established inbred lines from different seed companies.