Changes in Anthocyanin Production during Domestication of Citrus

Abstract

Mandarin (Citrus reticulata), citron (Citrus medica), and pummelo (Citrus maxima) are important species of the genus Citrus and parents of the interspecific hybrids that constitute the most familiar commercial varieties of Citrus: sweet orange, sour orange, clementine, lemon, lime, and grapefruit. Citron produces anthocyanins in its young leaves and flowers, as do species in genera closely related to Citrus, but mandarins do not, and pummelo varieties that produce anthocyanins have not been reported. We investigated the activity of the Ruby gene, which encodes a MYB transcription factor controlling anthocyanin biosynthesis, in different accessions of a range of Citrus species and in domesticated cultivars. A white mutant of lemon lacks functional alleles of Ruby, demonstrating that Ruby plays an essential role in anthocyanin production in Citrus Almost all the natural variation in pigmentation by anthocyanins in Citrus species can be explained by differences in activity of the Ruby gene, caused by point mutations and deletions and insertions of transposable elements. Comparison of the allelic constitution of Ruby in different species and cultivars also helps to clarify many of the taxonomic relationships in different species of Citrus, confirms the derivation of commercial varieties during domestication, elucidates the relationships within the subgenus Papeda, and allows a new genetic classification of mandarins.

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Figure 2.

Functionality of different Ruby alleles. N. benthamiana leaves were infiltrated with cDNA constructs corresponding to three different Ruby alleles. The phenotypes of leaves, the color of the leaf extracts, and the HPLC scans at 525 nm 7 d after infiltration are shown. A, Transient overexpression of Ruby cDNA encoding a full-length protein isolated from the flesh of Moro blood orange. B, Transient overexpression of a cDNA construct encoding a protein with a terminal deletion of 116 amino acids corresponding to the alleles found in some pummelo and mandarin accessions and hybrids derived from them. C, Transient overexpression of Ruby cDNA isolated from the fruit peel of M. australasica var Sanguinea encoding a protein with a 27-amino acid (aa) subterminal deletion. D, Evidence of the identity of delphinidin-3-rutinoside in sample C by positive mode electrospray MS1 and MS2. For this compound, the expected mass is 611.1607 (found mass, 611.1633; error, 4.25 ppm). Note the fragment representing delphinidin (expected mass, 303.0499) and the losses of a hexose moiety (expected loss, 162.0528) and a rhamnose moiety (expected loss, 146.0579).

Figure 3.

Schematic representation of different Ruby alleles identified in primary species of Citrus and related genera. Functional alleles (R) associated with anthocyanin pigmentation are highlighted in purple.

Figure 4.

Classification of mandarins based on the analysis of the Ruby gene. Mandarins can be classified into three groups (A, B, and C) on the basis of two deletions and an introgression from pummelo. Many mandarin varieties are hybrids between members of these three groups or interspecific hybrids involving other Citrus species. C. indica, recognized by both Tanaka and Swingle as a mandarin species, is not related to mandarins. Roman numbers indicate the five taxonomic groups recognized by Tanaka (Hodgson, 1967). The gradation in color shading indicates the contribution from genomes of other species. Common names for the mandarin accessions are provided in Table I.

Figure 5.

Segregation analysis of Ruby in some of the commercially important Citrus species and hybrids considered in this study. This simplified diagram shows a single existing variety of pummelo and mandarin as potential parents of both sweet and sour orange, which is highly unlikely. Light purple boxes indicate species and hybrids that are able to accumulate anthocyanins and that are always associated with active Ruby alleles from citron (R^C) or papeda (R^Pap2). Pink and blue arrows indicate the maternal and paternal contributions to the generation of a hybrid based on specific mitotypes and chlorotypes as determined in previous studies. The gradation in color shading indicates the contribution from different species to the derivation of the hybrids.

Figure 6.

Analysis of the wild type (WT) and the white mutant of lemon. A, HPLC scans recorded at 525 nm of methanol extracts from young leaves of Femminello wild-type (purple line) and Zagara Bianca (green line) lemons. Peaks marked with roman numbers indicate the main compounds identified in Femminello and absent in Zagara Bianca: Ia, cyanidin 3-glucoside; Ib, cyanidin 3-rutinoside; II, cyanidin 3-(6″-malonyl)-β-glucoside; and III, peonidin 3-(6′′-malonylglucoside). The identification of peaks is based on ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis (Supplemental Figs. S8–S10) and comparison with literature data. B, Relative quantitative reverse transcription-PCR quantification of Ruby expression in flower buds and young leaves of Femminello and Zagara Bianca lemons. Error bars represent se. C, Schematic diagram of the two alleles of Ruby identified in lemon, R^C and r^STOPp. The different symbols are explained in Figure 3 and Supplemental Figure S2A. The HindIII restriction sites and the genomic region used as a probe are shown. D, Southern-blot analysis of genomic DNA from different lemon varieties digested with HindIII and probed with a ³²P-labeled probe of the Ruby gene. Zagara Bianca, the only variety completely unable to produce anthocyanins, does not display the hybridization band corresponding to the active allele R^C.

Figure 7.

Phylogenetic relationship between primary species of Citrus and related genera based on the sequence of Ruby. The phylogenetic trees were generated using the Phylemon software program. Branch support is given at all branches. The different Ruby alleles are color coded as in Table I. The sequences of the Ruby gene and its promoter used for phylogenetic analysis have been deposited in GenBank under accession numbers KT591672 to KT591689.