Plant anthocyanin content detection kit

Total anthocyanin content was measured via the micromethod using the Plant Anthocyanin Content Detection Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The absorbance of the samples were determined at 500 nm. A standard curve was obtained according to the kit instructions, with the equation y = 0.1108x + 0.0171 (R² = 0.9935). A sample mass of 0.1 g was used to calculate ∆A = A measuring tube—A blank tube. ∆A was used as y in the standard curve to obtain x. The total anthocyanin content was calculated as content (mg/g) = x/0.1. All operations were conducted with three repeats.

In 2015 and 2016, the parental lines, the F₁ (E3316) and F₂ populations were sown in a greenhouse, sequentially. The seedlings were planted in the field at the four-true leaf stage in the Tianhe District (113.35^°N, 23.12°E), Guangdong, China. Although the degree of anthocyanin pigmentation can be affected by light, temperature, and other environmental factors, the presence or absence of anthocyanins was not affected by these environmental factors, and this phenotype was stable and consistent in different planting seasons. Thus, the fruit color phenotype of all the F₂ progeny was recorded as anthocyanin pigmentation (scored as 1) or non-anthocyanin pigmentation (scored as 0) at the marketable fruit stage.
nthocyanins were extracted from peels of fruits of the parents and F₁ (E3316) and then quantified using the Plant Anthocyanin Content Detection Kit (Solarbio, Beijing, China). The anthocyanin contents were calculated using the formula [45 ]: TA = [(A530nm-A620nm)-0.1(A650nm-A620nm)]/  × (V/m) × M × 100, where TA is the total anthocyanin content (mg/100 g FW), is the molar absorptivity, V is the total volume (ml), m is the sample weight (g), M is the molecular weight. Three biological replicates were used for each sample.

For ‘HB’, ‘RB-4’and ‘HY-1’, 10 g of flesh tissue per sample was ground and then extracted in a solution consisting of anhydrous ethanol, hydrochloric acid and water (volume ratio-2:1:1) twice by ultrasonic techniques. The anthocyanin components were qualitatively and quantitatively analyzed using UPLC-MS/MS (ultra-performance liquid chromatography coupled with tandem mass spectrometry) (Agilent, Palo Alto, CA, USA). Cyanidin (CAS: 528-58-5; chromatographic purity >98%; Chromadex), delphinidin (CAS: 528-53-0; chromatographic purity >96%), cyanidin-3-O-galactoside (CAS: 27661-36-5; chromatographic purity >98%; Chromadex) and delphinidin-3-O-galactoside (CAS: 28500-00-7; chromatographic purity >95%; Chromadex) were used as authentic standard samples for constructing a standard curve and for single point quantitation. Total anthocyanin content was measured using Plant Anthocyanin Content Detection Kit (Solarbio, Beijing, China) following manufacture’s recommendations. Each sample was analyzed in triplicate.