**Background:** Blueberry production has increased by over 600% in the last thirty years, driven in part by studies on the health benefits of antioxidant compounds such as phenolic acids. Chlorogenic acid (CGA, 5-O-caffeoylquinic acid) is the major hydroxycinnamic acid in blueberries, constituting 10–16% of total acids, with concentrations of 98–208 mg/100g FW in V. corymbosum cultivars. Other phenolic acids include acetyl-caffeoylquinic acid isomers (ACQA1, ACQA2) and caffeoylarbutin (CA). Despite the importance of these compounds, only one prior study (Mengist et al.) had mapped phenolic acid content in blueberry, identifying a QTL on Vc02 in a tetraploid population. The present study aimed to map the genetic control of phenolic acid content using a newly developed interspecific diploid mapping population, which offers advantages over tetraploid populations for genetic analysis.
**Methods:** An F1 population (BNJ16-4) of 1025 full-sib individuals was developed from crosses between two wild V. corymbosum var. caesariense plants (OPB-8, OPB-15) and two wild V. darrowii plants (NJ88-12-41, NJ88-14-03), with two resulting F1 hybrids (BNJ05-218-9, BNJ05-237-8) crossed reciprocally. Plants were maintained in a greenhouse in Chatsworth, NJ. A subset of genotypes was phenotyped: 185 genotypes in both 2019 and 2020, 45 only in 2019, and 48 only in 2020. Fully ripe fruit was harvested at 7–14-day intervals. Phenolic compounds were extracted using 80% aqueous acetone with 0.1% acetic acid and analyzed by HPLC-PDA at 366 nm and LC-MS-MS for identification. Compounds were quantified as chlorogenic acid equivalents. Genotyping-by-sequencing (GBS) libraries were prepared using MspI and PstI-HF double digestion, sequenced on an Illumina HiSeq (2×150 bp), and processed with STACKS v2. Reads were aligned to the W85 V. corymbosum var. caesariense reference genome. Map construction was performed in JoinMap v5 using regression mapping with the Kosambi mapping function. QTL mapping was conducted in R using the package qtl with Haley-Knott regression and 1000 permutations for significance thresholds. Best linear unbiased predictions (BLUPs) were calculated using lme4.
**Key Results:** The final genetic map contained 2090 markers across 12 chromosomes, with a total length of 1591.6 cM and an average marker spacing of 0.8 cM. Collinearity between physical and genetic maps was very strong (Spearman's rho > 0.999). Mean CGA concentration in the BNJ16-4 population was 0.25 ± 0.15 mg/g FW in 2019 and 0.23 ± 0.17 mg/g FW in 2020. ACQA1 means were 0.14 ± 0.07 mg/g FW (2019) and 0.13 ± 0.07 mg/g FW (2020). ACQA2 means were 0.11 ± 0.04 mg/g FW (2019) and 0.09 ± 0.04 mg/g FW (2020). CA means were 0.05 ± 0.02 mg/g FW in both years. Parental species showed distinct profiles: V. corymbosum var. caesariense had higher ACQA1/ACQA2, while V. darrowii had higher CGA and CA. ACQA1 and ACQA2 showed strong correlation (0.89–0.98, p < 0.001). CGA and CA showed moderate correlation (0.26–0.37, p < 0.001). QTL mapping identified overlapping peaks on Vc02 for CGA, ACQA1, and ACQA2 BLUPs. The peak for ACQA1 and ACQA2 BLUPs was at 8.8 Mb with LOD scores of 26.8 and 23.6, explaining 48.7% and 44.4% of variation, respectively. The CGA BLUP peak was at 7.8 Mb with LOD 17.65, explaining 35.6% of variation. CA BLUPs mapped to Vc07 (peak LOD 5.55, 12.9% variation) and Vc12 (peak LOD 7.02, 16.0% variation). Broad-sense heritability was high: 0.894 (CGA), 0.945 (ACQA1), 0.948 (ACQA2), 0.891 (CA). Within the significant region on Vc02 (252,772–24,241,116 bp), nine candidate genes were identified: seven with homology to hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) from tea and coffee, and two with homology to UDP-glucose:cinnamate glucosyl transferase (UGCT) from poplar. Additionally, 15 MYB transcription factor gene models were located in this region.
**Clinical Implications:** This study provides the first genetic map of phenolic acid content in a diploid interspecific blueberry population, identifying strong QTL that explain up to 48.7% of phenotypic variation. The identified markers can be used in marker-assisted breeding to develop blueberry cultivars with enhanced antioxidant potential. CGA is known to be bioavailable in humans and associated with reduced risk of oxidative stress, type 2 diabetes, cardiovascular disease, neurodegenerative disease, and cancer. CA, also identified in this study, has shown tyrosinase-inhibitory (skin-whitening) and anticancer properties. The overlapping QTL on Vc02 suggest a common genetic basis for biosynthesis of these phenolic acids, with HCT and UGCT as strong candidate genes. The diploid nature of this population allows for direct application to tetraploid breeding programs through unreduced gametes or polyploidization, potentially accelerating the development of nutritionally improved blueberry varieties.