Fruit Pigment Analysis by HPLC

Three plants were randomly selected from each group of fruit color phenotypes (eight in total) in the 494 F₂ individuals and the parental lines. Five ripe fruit from each selected plant were used for biochemical analysis using high performance liquid chromatography (HPLC). The phytoene, carotenoids, and chlorophylls were separated using reverse-phase columns (Kinetex 2.6 μm, C18 100A, 100  ×  4.60 mm, Phenomenex, Torrance, CA, USA). The whole-fruit extracts were filtered with a 0.2 μm PTFE filter prior to injection. The mobile phase consisted of two solvents, A = 78% methanol and B = 100% ethyl acetate. The carotenoid and chlorophyll analyses were performed using a 1260 Infinity series HPLC instrument (Agilent, Santa Clara, CA, USA) and Chemstation software (Santa Clara, CA, USA). The carotenoids and chlorophylls were identified and quantified based on their retention times and the absorbance between 280 nm and 480nm for carotenoids and 660nm for chlorophylls following the protocol described by Yoo et al. [31 (link)]. The values represented in this study are the mean of three biological replicates.