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Six mandarin cultivars, Ponkan (Citrus reticulata), Willowleaf (Citrus deliciosa), Kinnow (Citrus nobilis × C. deliciosa), Murcott (purported C. reticulata × Citrus sinensis), W. Murcott [purported (C. reticulata × C. sinensis) × C. reticulata)], and Snack (purported C. reticulata hybrid), were used in protoplast fusion with different parental combinations to generate somatic hybrids. Sixty-five somatic regenerants were obtained using optimized formulation of enzymes and molecular weight of polyethylene glycol for improved protoplast yield and heterokaryon fusion rate, respectively. Flow cytometry was used to determine the ploidy level of somatic regenerants, and nuclear expressed sequence tag–simple sequence repeat (EST-SSR) markers to determine their parental source. Of the 65 somatic regenerants, 46 were identified as autotetraploids, 18 allotetraploids, and one undefined. The EST-SSR markers also revealed that some ‘W. Murcott’ embryogenic callus lines that were presumed to be of nucellar origin were actually derived unexpectedly from individual ovules of zygotic origin. These mandarin-derived tetraploids are valuable as potential breeding parents for interploid crosses with an aim at seedlessness and easy-peeling traits.

Citrus (Citrus sp.) germplasm collections are a valuable resource for citrus genetic breeding studies, and further utilization of the resource requires knowledge of their genotypic and phylogenetic relationships. Diverse citrus accessions, including citron (Citrus medica), mandarin (Citrus reticulata), pummelo (Citrus maxima), papeda (Papeda sp.), trifoliate orange (Poncirus trifoliata), kumquat (Fortunella sp.), and related species, have been housed at the Florida Citrus Arboretum, Winter Haven, FL, but the accessions in the collection have not been genotyped. In this study, a collection of 80 citrus accessions were genotyped using 1536 sweet orange–derived single nucleotide polymorphism (SNP) markers, to determine their SNP fingerprints and to assess genetic diversity, population structure, and phylogenetic relationships, and thereby to test the efficiency of using the single genotype-derived SNP chip with relatively low cost for these analyses. Phylogenetic relationships among the 80 accessions were determined by multivariate analysis. A model-based clustering program detected five basic groups and revealed that C. maxima introgressions varied among mandarin cultivars and segregated in mandarin F₁ progeny. In addition, reciprocal differences in C. maxima contributions were observed among citranges (Citrus sinensis × P. trifoliata vs. P. trifoliata × C. sinensis) and may be caused by the influence of cytoplasmic DNA and its effect on selection of cultivars. Inferred admixture structures of many secondary citrus species and important cultivars were confirmed or revealed, including ‘Bergamot’ sour orange (Citrus aurantium), ‘Kinkoji’ (C. reticulata × Citrus paradisi), ‘Hyuganatsu’ orange (Citrus tamurana), and palestine sweet lime (Citrus aurantifolia). The relatively inexpensive SNP array used in this study generated informative genotyping data and led to good consensus and correlations with previously published observations based on whole genome sequencing (WGS) data. The genotyping data and the phylogenetic results may facilitate further exploitation of interesting genotypes in the collection and additional understanding of phylogenetic relationships in citrus.

In cybridization, new combinations of nuclear and cytoplasmic genes result in a unique genotype that may bring cellular, physical, physiological, and biochemical changes to the plant. This has been demonstrated in the unexpected cybrids generated from the fusion of citrus (Citrus sp.) protoplasts in two independent experiments. The first experiment was conducted to generate potentially seedless triploids by fusing diploid protoplasts of embryogenic ‘Dancy’ mandarin (Citrus reticulata) suspension culture cells with haploid ‘Ruby Red’ grapefruit (C. paradisi) protoplasts derived from tetrad-stage microspores. After multiple attempts, only one triploid was recovered, but several diploid plants with typical grapefruit morphology were also regenerated. In the second experiment, protoplasts derived from embryogenic ‘Dancy’ mandarin suspension culture were fused with nonembryogenic protoplasts from ‘Duncan’ grapefruit leaves in an effort to produce an allotetraploid somatic hybrid. The fruit from the resulting trees resembled grapefruit in morphology and type, and maintained excellent quality throughout the summer, when commercial grapefruit rapidly loses quality. Fruit on these trees remained firm with exceptional sweetness and good flavor into August, and without seed germination. The regenerants obtained in the protoplast fusion experiments were confirmed as cybrids by genetic marker analyses. The test grapefruit were identical to commercial ‘Ruby Red’ grapefruit at six nuclear simple sequence repeat (SSR) marker loci, but identical to ‘Dancy’ with respect to a mitochondrial intron marker. The plastid genomes of individual trees originated from either fusion partner. In the first experiment, haploid ‘Ruby Red’ protoplast preparations must have also contained contaminant diploid protoplasts. Apart from the value of altered fruit quality attributes in the marketplace, these plants provide an opportunity to understand the contributions of cytoplasmic organelle genetics to important citrus fruit-breeding objectives.