In the present study, the fruit physiology and sugar-acid ratio of 24 pomelo cultivars grown in ten different locations of the subtropical region of China were measured. The contents of soluble sugars and organic acids were quantified using high-performance (HPLC-MS) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), respectively. The results revealed that the physiological and basic quality attributes of 24 pomelo cultivars, including fruit weight, fruit width, fruit length, peel thickness, number of segments, pulp weight, pulp color, soluble solids, and Vitamin C, ranged between 264.63–1945.85 g, 8.60–19.56 g, 7.40–20.70 g, 0.46–3.33 mm, 11–18.66, 210.25–1351.66 g, 8.59–15.14 Brix°, and 34.79–84.58 mg/100 g, respectively. Soluble sugars, i.e., fructose, glucose, and sucrose, ranged between 16.25–24.25, 16.17–24.22, and 19.90–55.28 mg/g, respectively. Similarly, Organic acids, i.e., pyruvate, fumaric acids, succinic acid, tartaric acid, quinic acid, citric acid, malic acid, and cis-aconitic acid, in 24 pomelo cultivars ranged between 0.48–1.84, 0.02–0.45, 0–0.05, 0.01–0.1, 0–0.14, 3.01–11.85, 0.18–1.42, and 0.01–0.16 mg/g, respectively. The pomelo cultivars ‘Hongzuanmi’, ‘Minihong’, and ‘Hangwanmi’ exhibited maximum contents of citric acid and pyruvate and showed ultimately excessive organic acids. Overall, the ‘Guanximi’ and its budding cultivars, i.e., ‘Hongroumi’, ‘Huangjinmi’, and ‘Sanhongmi’, had the best quality fruits having maximum sugar-acid ratio. Correlation analysis showed that total soluble sugars had a significantly positive correlation with sucrose contents, while citric acids, malic acid, and pyruvate were positively correlated with total organic acids. The determined sugar-acid profile of pomelo cultivars provides the basis for future elucidation of key mechanisms regulating sugars and acids biosynthesis in pomelo.
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