CgABR1-CgFAD negatively regulates the fruit neck length in citrus.

Pummelo (Citrus maxima) is a fundamental species of Citrus which contributes to most of the cultivated citrus, including sweet orange, lemon and etc. The fruit neck is a structural feature of pummelo, and a long fruit neck reduces the edible rate of the fruit. In this study, we assembled a telomere-to-telomere (T2T) gap-free reference genome for the typical short fruit neck cultivar, 'Pingshan' pummelo, and a chromosome-level genome for the typical long fruit neck cultivar, 'Shatian' pummelo. Here, we used a segment population derived from a cross between a long fruit neck cultivar ('Guanxi' pummelo) and a short fruit neck ('Pingshan' pummelo) cultivar to map the determinant controlling the fruit neck length. We identified a strong peak on chromosome 1 within the 27.5-30.5 Mb physical region and found a 52 bp deletion linked with the fruit neck length. Moreover, by combining RNA sequencing data of the fruit neck development and variation analysis, we identified two genes, one encodes ethylene-responsive transcription factor (CgABR1) and the other encodes FAD-dependent urate hydroxylase (CgFAD). Genetic transformation confirmed that overexpression of CgABR1 and CgFAD can inhibit fruit neck length. DNA affinity purification sequencing, electrophoretic mobility shift assays and dual-LUC reporter assays demonstrated that CgABR1 can activate the expression of CgFAD by directly binding to its promoter. In summary, we assembled a T2T gap-free genome for pummelo and identified the key genes for fruit neck length in citrus, offering an important resource and new genes for citrus genetic improvement and breeding programs.