Blue elderberries were determined to be ripe when the berries in a cyme were deep purple, with or without the white bloom, and had no green berries present. Ripe elderberries were harvested by hand from all four quadrants of the elderberry shrub, totaling approximately 3 kg of elderberries. The berries were placed in clear plastic bags, stored on ice, and transported to the laboratory. A subsample was separated for moisture analysis, while the rest was de-stemmed and stored at -20 °C until analyzed. HPLC grade methanol , acetonitrile , phosphoric acid, ethanol, hydrochloric acid, and sodium hydroxide were purchased from Fisher Scientific . Ascorbic acid was purchased from Acros Organics . Formic acid, gallic acid, sucrose, chlorogenic acid, rutin, and catechin, and Folin-Ciocalteu reagent were purchased from Sigma Aldrich . Cyanidin-3-glucoside was purchased from Extrasynthese . Ultra-pure water was obtained from a Milli-Q water system . From each shrub, about 250 g of frozen berries were thawed in a glass container overnight at 4 °C. The following day, the thawed berries were mashed for 4 min by hand using a plastic pestle, then homogenized , and centrifuged at 3,000 rpm for 7 min. The supernatant was strained, collected, and weighed. A 15 mL aliquot of sample extract was stored in a 15 mL plastic tube at -20 °C for total monomeric anthocyanin analysis. The remaining supernatant was used to determine soluble solids, pH, and titratable acidity . One pooled sample was analyzed from each shrub and analyzed in duplicate analytical repetitions. A refractometer was used to determine soluble solids. It was calibrated with standard solutions of 5°, 10°, vertical gardening in greenhouse and 15° Brix made with sucrose and water. A 150 µL aliquot of elderberry juice was placed on the prism and read using the automatic setting. The pH was determined using a SevenMulti pH meter .

It was calibrated before each use using buffers at pH 4.0, 7.0, and 10.0. To determine TA, 10 mL of elderberry juice was diluted to 100 ml with nanopure water and mixed. This dilute juice was titrated to pH 8.2 using 0.1 N NaOH. The volume of 0.1 N NaOH used to achieve the desired pH was used to calculate the mg citric acid per 100 g fresh weight . For each of these analyses, duplicates were run on each juice. Elderberries were extracted by combining 5 g frozen berries with 25 mL MeOH:formic acid in a conical tube. The contents were homogenized, placed in a shaker without water at speed 7.5 for 20 min, then centrifuged at 3,000 rpm for 7 min. The supernatant was transferred to a 15 mL plastic tube and stored at -80 °C for no more than two weeks prior to analysis. Duplicate extracts were made from each shrub. TPC was determined using the Folin-Ciocalteu method. First, elderberry phenolic extract was diluted 1:4 with water. Each extract was analyzed in duplicate and averaged. In 10 mL glass tubes, 6 mL water was combined with 100 µL sample and 500 µL Folin-Ciocalteu reagent. After mixing and incubating for 8 min at room temperature, 1.5 mL 20% aqueous sodium carbonate was added. The tubes were mixed, covered with foil to avoid light exposure, placed in a water bath at 40 °C for 40 min, then cooled at room temperature for 15 min. The samples were read by a UV visible spectrophotometer at 765 nm and quantified using an external standard curve prepared with gallic acid . TPC is expressed as mg gallic acid equivalents per 100 g FW. Five grams of frozen berries were mixed with 25 mL of in a conical tube, which was then homogenized for 1 min at 7,000 rpm . The mixture was stored at 4 °C overnight, then in the morning, centrifuged at 4,000 rpm for 7 min. The supernatant was used directly for analysis. Three pooled samples were made for each hedgerow, each consisting of even amounts of berries from three distinct shrubs. Eachpooled sample was extracted once to give 3 biological replicates, and each extract was run in duplicate . The concentration of phenolic compounds in blue elderberry followed the method by Giardello et al. with some modifications.

Briefly, samples were analyzed via reversed-phase liquid chromatography on an Agilent 1200 with a diode array detector and fluorescence detector . The column used was a PLRP-S 100A 3 µm 150 x 4.6 mm at 35 °C, and the injection volume was 10.0 µl. Mobile phase A was water with 1.5 % phosphoric acid, while mobile phase B was 80%/20% acetonitrile/ mobile phase A. The gradient used was 0 min 6% B, 73 to 83 min 31% B, 90 to 105 min 6% B. The DAD was used to monitor hydroxybenzoic acids at 280 nm, hydroxycinnamic acids at 320 nm, flavonols at 360 nm, and anthocyanins at 520 nm. The FLD was used to monitor flavan-3-ols, with excitation at 230 nm and emission at 321 nm. External calibration curves were prepared using chlorogenic acid for phenolic acids, rutin for flavonols, and cyanidin-3-glucoside for anthocyanins , at the following concentrations: 200, 150, 100, 75, 50, 25, 10, 5, and 2.5 mg/L. Catechin was used to quantify flavan-3-ols and standards were run at 150, 100, 75, 50, 25, 10, 5, 2.5, and 1 mg/L. Compounds were identified based on retention time and spectral comparisons with standards. Information about the linear equations and lower limits of detection and quantitation can be found in Table S1 in the supplementary material. The LLOD was calculated as 3.3 times the standard deviation of the y-intercept of the curve divided the slope, while the LLOQ was calculated as 10 times those values.Several peaks appeared in the HPLC chromatograms that could not be identified using the above parameters. Chromatographic eluents of these peaks were collected individually and dried under vacuum. These extracts were reconstituted with mobile phase A, and 5 µL were injected into the HPLC- QTOF-MS/MS for accurate mass analysis . A Poroshell 120 EC-C18 column was used at 35 °C. Mobile phase A was 1% formic acid in distilled water, and mobile phase B was 1% formic acid in acetonitrile. The gradient used was 0 min 3% B, 30 min 50% B, 31-32 min 95% B, 33-38 min 3% B. The mass spectrometer was used in negative mode, and the mass range for MS was 100 to 1000 m/z while the range for MS/MS was 20-700 m/z. Collision energies at 10, 20, and 40 V were applied. The drying gas was set to a flow of 12 L/min at 250 °C, while the sheath gas was set to 11 L/min at 350 °C. The nebulizer was set to 40 psig, the capillary voltage was 3500 V, the nozzle was set to 500 V, and the fragmentor was set to 100 V. Data was analyzed using Agilent MassHunter Workstation Qualitative Analysis 10.0 .

Tentative identification was achieved by comparing the mass to charge ratio of the precursor and fragment ions to online libraries of compounds as well as using formula generation for the peaks in the spectra. The composition of blue elderberries is presented for the first time, which is key to understanding how this subspecies of Sambucus nigra compares to commercialized elderberry subspecies, S. nigra ssp. nigra and S. nigra ssp. canadensis. These data help to establish the blue elderberry grown in hedgerows in California as a viable source of berries and bioactive compounds. Data for the compositional assays is presented for the 2018 and 2019 harvest years as the average of all shrubs sampled in Table 2. The average moisture for the blue elderberries was 79.5 ± 1.5% in 2018 and 79.5 ± 1.6% in 2019, which is very similar to the levels found in wild elderberries in Spain 95. The average soluble solids found in blue elderberry ranged from 11.94 ± 2.08 to 14.95 ± 1.02 g per 100 g FW in 2018 and from 12.64 ± 1.86 to 17.09 ± 1.60 g per 100 g FW in 2019. These values are slightly higher than the soluble solids found in S. nigra ssp. cerulea grown in Slovenia29 and American elderberries grown in Ohio52. Compared to European and American elderberries evaluated in other studies, blue elderberries have similar levels of soluble solids 8,18,29,49,50,95. In the present study, the overall average content of soluble solids was significantly different between years, greenhouse vertical farming as blue elderberries harvested in 2019 had significantly higher average soluble solids than the elderberries harvested in 2018 . The pH in the blue elderberry ranged from 3.44 to 3.86 in 2018 and from 3.46 to 3.79 in 2019, with no significant difference found between harvest years. These values are slightly lower than the values found in European elderberry, which ranged from 3.9 ± 0.06 to 4.1 ± 0.04 with an average pH of 3.9 ± 0.2, and American elderberry, which ranged from 3.9 ± 0.04 to 4.5 ± 0.03 with an average pH of 4.2 ± 0.2 49 Another evaluation of pH in American elderberries had a range of 4.5 ± 0.08 to 4.9 ±0.12, higher than those found in the blue elderberry.52 The higher sugar and lower pH levels in blue elderberry could potentially impact taste and performance in food and beverages as compared with the European and American species. The average titratable acidity in blue elderberries ranged from 0.45 ± 0.08 to 0.77 ± 0.03 g citric acid per 100 g FW in 2018 and from 0.54 ± 0.06 to 0.77 ± 0.11 g citric acid per 100 g FW in 2019 with no significant difference found between harvest years. These values are lower than the total acids found by Mikulic-Petkovsek et al. 29 in S. nigra ssp. cerulea , but they are similar to the levels found in European elderberry 8,18,49,50 . Anthocyanins are a class of phenolics that contribute red, purple, and blue hues to fruits and vegetables, act as attractants for pollinators, and are potent antioxidants. European and American elderberries are well-known for containing high levels of anthocyanins 8,18,49. The anthocyanin content of elderberries strongly correlates to the antioxidant potential of the fruit, which may confer health-promoting properties 50,89, which is one reason why elderberries are used in supplements and value-added products. Elderberry is also used as a source of natural food colorants due to the levels of anthocyanins35. Understanding the levels of anthocyanins in the blue elderberry grown in hedgerows is critical towards establishing this native fruit as an additional and more sustainable elderberry. The average TMA measured in blue elderberry ranged from 34.2 ± 9.7 to 113.4 ± 18.2 mg CGE per 100 g FW in 2018 and from 43.1 ± 11.5 to 121.5 ± 11.5 mg CGE per 100 g FW in 2019 . TMA was variable between hedgerows in both years of harvest, with relative standard deviation values between 16% and 30%, yet there was not a significant difference in the overall average TMA between 2018 and 2019 . Furthermore, most hedgerows were not significantly different from the other hedgerows harvested that year despite significant differences in TMA values found between farms in both years . Regarding the age of the elderberry shrub, hedgerows 2 and 14 had two of the three highest concentrations of TMA in 2019 . This suggests that blue elderberries can be harvested from plants as young as two years without a significant loss of TMA concentrations. TMA values for the blue elderberries are lower than those found in other elderberry subspecies. In European elderberries, TMA levels range from 170 ± 12 to 343 ± 11 with an average of 239 ± 94 mg CGE per 100 g FW 49. A study of American elderberry grown in Ohio showed a range from 354 ± 59 to 595 ± 26 mg CGE per 100 g FW.52 In the present study, bare root prerooted cuttings of American elderberries were planted, along with blue elderberries, on Farm 1 in 2018, and three shrubs were harvested in 2019. These American elderberries had an average TMA value of 263 ± 5.4 mg CGE per 100 g FW, which is more similar to what has been observed in other studies on this subspecies. This suggests it is a subspecies difference contributing to the lower anthocyanin concentration in the blue elderberry and not the difference in growing conditions.