Carotenoids and apocarotenoids act as phytohormones and volatile precursors that influence plant development and confer aesthetic and nutritional value critical to consumer preference. Citrus fruits display considerable natural variation in carotenoid and apocarotenoid pigments. In this study, using an integrated genetic approach we revealed that a 5 0 cis-regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C 30 apocarotenoids responsible for red coloration of citrus peel. Functional analyses demonstrated that in addition the known role in synthesizing b-citraurin, CCD4b is also responsible for the production of another important C 30 apocarotenoid pigment, b-citraurinene. Furthermore, analyses of the CCD4b promoter and transcripts from various citrus germplasm accessions established a tight correlation between the presence of a putative 5 0 cis-regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C 30 apocarotenoid-rich red-peeled accessions. Phylogenetic analysis provided further evidence that functional diversification of CCD4b and naturally occurring variation of the CCD4b promoter resulted in the stepwise evolution of red peels in mandarins and their hybrids. Taken together, our findings provide new insights into the genetic and evolutionary basis of apocarotenoid diversity in plants, and would facilitate breeding efforts that aim to improve the nutritional and aesthetic value of citrus and perhaps other fruit crops.

Summary Hongkong kumquat (Fortunella hindsii) is a wild citrus species characterized by dwarf plant height and early flowering. Here, we identified the monoembryonic F. hindsii (designated as ‘Mini‐Citrus’) for the first time and constructed its selfing lines. This germplasm constitutes an ideal model for the genetic and functional genomics studies of citrus, which have been severely hindered by the long juvenility and inherent apomixes of citrus. F. hindsii showed a very short juvenile period (~8 months) and stable monoembryonic phenotype under cultivation. We report the first de novo assembled 373.6 Mb genome sequences (Contig‐N50 2.2 Mb and Scaffold‐N50 5.2 Mb) for F. hindsii. In total, 32 257 protein‐coding genes were annotated, 96.9% of which had homologues in other eight Citrinae species. The phylogenomic analysis revealed a close relationship of F. hindsii with cultivated citrus varieties, especially with mandarin. Furthermore, the CRISPR/Cas9 system was demonstrated to be an efficient strategy to generate target mutagenesis on F. hindsii. The modifications of target genes in the CRISPR‐modified F. hindsii were predominantly 1‐bp insertions or small deletions. This genetic transformation system based on F. hindsii could shorten the whole process from explant to T1 mutant to about 15 months. Overall, due to its short juvenility, monoembryony, close genetic background to cultivated citrus and applicability of CRISPR, F. hindsii shows unprecedented potentials to be used as a model species for citrus research.

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In plants, the carotenoid cleavage dioxygenase 4 (CCD4) could target on plastoglobules and cleave specific carotenoids, producing apocarotenoids and volatile compounds. These compounds are important for color and aroma formation in fruits and flowers. In this study, five CCD4 gene members (CCD4a, b, c, d, and e) were investigated in different citrus species including mandarin, pummelo, and sweet orange. Sequence analysis showed that the CCD4 genes from all the species examined exhibited extensive allelic variability (including SNPs and frame-shift mutations). Furthermore, the distribution of the CCD4 allelic mutation sites supported our previous hypothesis that the sweet orange originated from the hybridization of mandarin and pummelo. A derived cleaved amplified polymorphic sequence (dCAPs) marker was then successfully developed based on the allelic polymorphism of CCD4c, providing an ideal molecular marker for studying the genetic relationship between citrus species. Quantitative RT-PCR analysis identified differential expression patterns for the CCD4 genes in tissues/organs, and CCD4b was shown to have a high-level expression in citrus fruit flavedos (especially those with a deep orange-reddish color). HPLC-based detection of a key component (i.e., β-citraurin) for orange-reddish flavedo formation in different citrus revealed a positive correlation between CCD4b expression levels and the presence of β-citraurin, suggesting that CCD4b may be responsible for β-citraurin biosynthesis in flavedo. In summary, this study not only reinforced the anticipated roles of CCD4 genes in flavedo color formation in citrus, but also provided new information about gene expression patterns, allelic polymorphism characteristics, and sequence variability for this gene subfamily.