*2.7. Candidate Gene Analysis*

The annotations of the candidate genes underlying or flanking (±5 kb) the SNPs associated with a trait were obtained from the International Wheat Genome Sequencing Consortium (IWGSC) [62]. If annotations for underlying genes were not available, then a default BLASTN search was performed using the wheat gene coding sequence (CDS) against the Ensembl Plants database of monocot and dicot sequences [63]. The BLAST search was performed to determine the likely annotation of the candidate genes. The BLAST search was limited to the top hit with an E-value cut o ff of less than 1 × 10−<sup>40</sup> and sequence identity greater than 50%. The annotations for the underlying or flanking genes were compared against the trait of interest to determine the candidate genes that were likely important for bran functionality traits.

### *2.8. Linkage Disequilibrium between Trait-Associated SNP Markers on Chromosome 5D and Pin Locus*

The Pina-D1, Pinb-D1b, and Pinb-Wild molecular marker information on 44 lines in the HWWAMP was obtained from the regional breeding programs, Northern Regional Performance Nursery (NRPN) and Southern Regional Performance Nursery (SRPN) from 2005–2009 dataset [64]. The "cor.test" procedure was conducted in R using "ggpubr" package to calculate correlation coefficients (as a measure of linkage disequilibrium (LD)) between trait associated SNP markers on chromosome 5D and Pin markers.
