QTL Analysis for the Identification of Loci Controlling Antioxidant Capacity in Brassica oleracea L. Var. Italica

This research has identified regions of genetic control over the antioxidant capacity of broccoli (Brassica oleracea L. var. italica). 92 F_2:3 families from the mapping population BNC x VI158 (Brown et al., 2007), grown in 2009 and 2010 in Salisbury, NC, were assayed for 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging capacity and 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity according the protocol described by Ku et al. (2010). Briefly, 75 mg of freeze-dried tissue was extracted with 1.5 mL of ddH₂O for 24 hrs at room temperature. 10 µL of extract was added to 190 µL of ABTS or DPPH solution and incubated at room temperature for six minutes or thirty minutes, respectively.  The radical-species extinction was measured spectophotometrically against a Trolox standard curve. All tests were performed in triplicate.  Multiple-QTL-mapping (MQM) analysis of the data for each year individually and the average of the two years was conducted in the software package MapQTL 5 using a step size of 0.5cM.  The LOD-score significance threshold was set at the 95% genome-wide confidence level, determined by permutation testing (1000 iterations), and a 1-LOD drop-off interval was used to determine QTL position.  The linkage map used in this analysis was developed by Brown et al. (2014) using a 60K Illumina (single-nucleotide polymorphism) SNP array of Brassica napus, and was anchored to the genomic sequence of the rapid cycling Brassica oleracea T01000.  It is a dense linkage map with 547 non-redundant SNP marker loci, spanning 948.1 cM over nine chromosomes, with an average interval of 1.7 cM and an estimated 96% coverage of the B. oleracea genome. 11 QTL were identified that were significant in at least one year by radical scavenging assay combination. The three most commonly identified QTL were located on chromosome three at 28.4 - 30.5 cM, chromosome six at 69.5 - 71.5 cM, and chromosome eight at 35.4 - 40.2 cM.  These loci explained 7.6 – 10.5%, 12.4 – 24.9%, and 12.6 – 15.9% of the phenotypic variation, respectively.  The results of this study will provide useful information for the identification of genes involved in the antioxidant activity of broccoli and support the breeding of Brassica cultivars that can serve as functional foods.