A knockout mutation in the lignin biosynthesis gene CCR1 explains a major QTL for acid detergent lignin content in Brassica napus seeds

Seed coat phenolic compounds represent important antinutritive fibre components that cause a considerable reduction in value of seed meals from oilseed rape (Brassica napus). The nutritionally most important fibre compound is acid detergent lignin (ADL), to which a significant contribution is made by phenylpropanoid-derived lignin precursors. In this study, we used bulked-segregant analysis in a population of recombinant inbred lines (RILs) from a cross of the Chinese oilseed rape lines GH06 (yellow seed, low ADL) and P174 (black seed, high ADL) to identify markers with tight linkage to a major quantitative trait locus (QTL) for seed ADL content. Fine mapping of the QTL was performed in a backcross population comprising 872 BC₁F₂ plants from a cross of an F₇ RIL from the above-mentioned population, which was heterozygous for this major QTL and P174. A 3:1 phenotypic segregation for seed ADL content indicated that a single, dominant, major locus causes a substantial reduction in ADL. This locus was successively narrowed to 0.75 cM using in silico markers derived from a homologous Brassica rapa sequence contig spanning the QTL. Subsequently, we located a B. rapa orthologue of the key lignin biosynthesis gene CINNAMOYL CO-A REDUCTASE 1 (CCR1) only 600 kbp (0.75 cM) upstream of the nearest linked marker. Sequencing of PCR amplicons, covering the full-length coding sequences of Bna.CCR1 homologues, revealed a locus in P174 whose sequence corresponds to the Brassica oleracea wild-type allele from chromosome C8. In GH06, however, this allele is replaced by a homologue derived from chromosome A9 that contains a loss-of-function frameshift mutation in exon 1. Genetic and physical map data infer that this loss-of-function allele has replaced a functional Bna.CCR1 locus on chromosome C8 in GH06 by homoeologous non-reciprocal translocation.