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Chunxian Chen and William R. Okie

Several available Prunus chloroplast genomes have not been exploited to develop polymorphic chloroplast microsatellites that could be useful in Prunus phylogenetic analysis and maternal lineage group (MLG) categorization. In this study, using available bioinformatics tools, 80, 75, and 78 microsatellites were identified from the chloroplast genome of P. persica (CPpe), P. kansuensis (CPka), and P. mume (CPmu), respectively. The genome features and polymorphism status of these microsatellites were characterized. The genomic locations and motif types of most chloroplast microsatellites were conserved in CPpe, CPka, and CPmu. Of the 67 microsatellites with primer sequences and names, 57 were polymorphic for their in silico motif, amplicon lengths, or both among the three genomes. Based on the genotyping data of eight most polymorphic microsatellites, eight unique MLGs were found among the 736 peach materials in a breeding program. Most peach cultivars (111 of 161 genotyped) belong to MLG-1, the Chinese Cling-derived group reflecting the heavy use of this germplasm in early peach development. Forty-one cultivars belong to MLG-2, the European-derived group of peaches. MLG-3 consists of ornamental accessions. MLG-4 and MLG-5 contain only ‘Flordaking’ and ‘Reliance’, respectively. MLG-6 to MLG-8 consists of selections derived from P. tangutica, P. davidiana, and P. mira, respectively. These amplicons from the representative material for each MLG were sequenced, revealing additional single nucleotide polymorphisms (SNPs) within the amplicons. With the polymorphism status and amplification reliability validated, these new polymorphic chloroplast microsatellite markers may be useful in Prunus phylogenetic analysis.

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Chunxian Chen and William R. Okie

Several new peach (Prunus persica) flower types were discovered in an F2 segregating population from an open-pollinated, non-showy-flowered F1 seedling of ‘Helen Borchers’, a double-flowered ornamental cultivar. The novel flower types were white and red single-flowered, non-showy blooms, as well as double-flowered, non-showy red, pink, white, and yellow phenotypes. The double, non-showy flowers were very attractive, and resembled pom-pom chrysanthemums. Yellow flower color is unknown in peach. Flower type in the F2 family segregated ≈3:1 for non-showy (Sh_) vs. showy (shsh), for anthocyanin-present vs. anthocyanin-absent, and for pink (R_) vs. red (rr), independently. Flower petal number segregated at about 9:3:4 for classes single:semi-double:double. Although both parents were late flowering, the F1 was not. The F2 seedlings showed a wide range in time of flowering. Higher petal number was correlated with later bloom, although it is unclear whether this is due to linkage or developmental differences in the flowers with extra petals. These novel flower types might be useful as ornamentals, and for use in genetics and breeding studies. Microsatellite analysis of possible pollen donors revealed that ‘Oldmixon Free’, a non-showy-flowered peach cultivar, was likely the pollen parent of the F1.

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Chunxian Chen, Paul Cancalon, Carl Haun and Fred Gmitter Jr.

Furanocoumarins are organic chemical components in grapefruit (Citrus paradisi) juice that have been shown to induce potentially deleterious drug interactions. In this study we measured seven furanocoumarins (FCs) [bergamottin, 6′,7′-dihydroxybergamottin (6,7-DHB), paradisin C, bergaptol, isoimperatorin, 5′,8′-dimethylallyloxypsoralen (5,8-DMP), and epoxybergamottin (EBM)] in fruit of three grapefruit cultivars [Foster (Fos), Low Acid Foster (LAF), and Hudson (Hud)], one pummelo (C. maxima) cultivar [Hirado Buntan (HBP)], 17 randomly selected hybrids from HBP× Hud, and 31 other triploid hybrids. Bergamotton, 6,7-DHB, and paradisin C were not detected or extremely low in HBP (0.00, 0.11, and 0.00 mg·L−1) and LAF (0.40, 3.83, and 0.00 mg·L−1) compared with Hud (13.03. 9.58, and 6.11 mg·L−1) and Fos (6.48, 14.38, and 6.11 mg·L−1). In these hybrids, 6,7-DHB, bergamottin, and paradisin C obviously cosegregated in an approximate rate of 1:1. The three FCs in eight hybrids were not detected or extremely low, like HBP, the maternal parent; those in the other nine were as high as or higher than Hud, the paternal parent. The same segregation tendency was also observed in these triploid hybrids. Based on all the cultivars and hybrids, strong correlations existed among 6,7-DHB, bergamottin, and paradisin C (coefficient up to 0.909). Such strong correlations may reflect their metabolic links in the bergamottin pathway. The 1:1 cosegregation and strong correlation among the three FCs suggested that the trait of FCs is likely controlled by one single enzymatic or regulatory gene in the pathway. The FC profiles and inheritance may lead to a genomic and breeding solution to the grapefruit FC–drug interaction issue. Selection of FC-low or FC-free seedless grapefruit cultivars is underway.

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Chunxian Chen, William R. Okie and Thomas G. Beckman

Peach fruit set is affected by cumulative chill and spring frost. A spring frost occurred on 29 Mar. 2015 at the U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS) Byron station after 3 weeks of bloom, reducing fruit set and resulting in many buttons (abnormally small fruit with dead embryos). Fruit set was rated in 2014, 2015, and 2016 and button set rated in 2015 using the same scale (0 = no fruit/button to 9 = 1–2 fruit/button at every node). The overall fruit set rating was substantially different in the 3 years, averaging 5.61 in 2014, 2.61 in 2015, and 6.04 in 2016. Buttons and skin-damaged fruit in 2015 varied among peach genotypes. Comparison of fruit and button set ratings showed that there was no difference between cultivars and selections, but some significant differences in fruit set for four ripening months, among the 3 years, and among the nine chilling classes, respectively. Among the cultivars, the most common button set rating was 0–3. For example, ‘Sunprince’, ‘Loring’, and ‘Carored’ trees had a high button set rating, whereas ‘Flameprince’, ‘Julyprince’, and ‘Contender’ trees were low. As for peach selections, BY04P1690n was among those with the highest button set rating. In the population derived from a cross of button-prone BY04P1690n and button-free BY99P3866w, fruit and button counts from 10 long fruiting shoots ranged from 4 to 53 fruit (21.63 on average) and 2 to 27 buttons (10.39 on average). The peach button rate ranged from 5.36% to 87.10% (30.70% on average). The range, distribution, and percentage of the button counts suggested that, if buttoning was genetically controlled, it appeared quantitative. Further assessment is needed.

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Milica Ćalović, Qibin Yu, Vladimir Orbović, Frederick G. Gmitter Jr, Jude W. Grosser and Chunxian Chen

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.

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Jude W. Grosser, Hyun Joo An, Milica Calovic, Dong H. Lee, Chunxian Chen, Monica Vasconcellos and Frederick G. Gmitter Jr

Somatic hybridization through protoplast fusion has proven to be a valuable technique in citrus for producing unique allotetraploid breeding parents that combine elite diploid selections. Many citrus somatic hybrids are now flowering and being used in interploid crosses to generate triploid hybrids that produce seedless fruit, a primary objective of citrus breeding programs. Most of the early somatic hybrids produced for mandarin improvement combined sweet oranges with mandarins, because the performance of sweet oranges in tissue/protoplast culture generally exceeds that of most mandarin selections. However, a high percentage of triploid progeny from interploid crosses using sweet orange + mandarin somatic hybrids as the tetraploid parent produce fruit that are difficult to peel. We report nine new allotetraploid somatic hybrids and five new autotetraploids from somatic fusion experiments involving easy-peel mandarin parents. These tetraploids can be used in interploid crosses to increase the percentage of seedless triploid progeny producing easy-to-peel fruit. Ploidy level of the new tetraploids was determined by flow cytometry and their genetic origin by expressed sequence tag–simple sequence repeat marker analysis.

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Chunxian Chen, Jude W. Grosser, Milica Ćalović, Patricia Serrano, Gemma Pasquali, Julie Gmitter and Fred G. Gmitter Jr

Somatic hybridization is a powerful tool for the genetic improvement of citrus rootstocks, and it is part of an efficient in vitro-based breeding system described here. An essential component of the system is the requirement of confirming tetraploidy and the combination of the two donor genomes. Expressed sequence tag–simple sequence repeat (EST-SSR) markers provide a means to accomplish both of these objectives, and their application to a population of pummelo [Citrus grandis (L.) Osbeck] + mandarin (C. reticulata Blanco) somatic hybrids developed for the specific purpose of providing alternative rootstocks for sour orange (Citrus aurantium L.) is detailed. Nineteen new somatic hybrids were produced from various mandarin and pummelo parents, and their ploidy level and the complementation of their nuclear genomes were confirmed using four EST-SSR markers. These markers were selected from markers previously mapped in sweet orange [C. sinensis (L.) Osbeck] and trifoliate orange [Poncirus trifoliata (L.) Raf.] and prescreened for suitable allelic polymorphism within the mandarin and pummelo lines used. After polymerase chain reaction amplification of sequences from the parents and putative hybrids, the products were separated on a genetic sequencer and visualized electronically. Additionally, EST-SSR markers identified the unexpected zygotic origin of a presumed nucellar embryogenic callus line. Integration of EST-SSR techniques for high-throughput genotyping with previously developed approaches to somatic hybrid creation increases substantially the effectiveness and efficiency of this in vitro-based breeding system for citrus rootstock improvement.

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Yuan Yu, Chunxian Chen, Ming Huang, Qibin Yu, Dongliang Du, Matthew R. Mattia and Frederick G. Gmitter Jr.

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.

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Chunxian Chen, Qifa Zheng, Xu Xiang, Jaya R. Soneji, Shu Huang, Young A Choi, Madhugiri Nageswara Rao and Fred G. Gmitter Jr.

Eight new green fluorescent protein (GFP) binary vectors were developed by inserting gfp reporter gene cassettes into pGreen vectors. We chose one of them, pG52KF, with the nptII selection and gfp reporter gene and one recombinant construct, pG52KFp, for a preliminary evaluation in citrus using Agrobacterium-mediated transformation. High-transformation efficiency was observed, whereas green fluorescence greatly facilitated the early in vivo screening and categorizing of the transformants. These pGreen-derived GFP binary vectors, freely available on request, provide more and flexible options for genetic transformation in citrus and other woody plants.

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Aditi D. Satpute, Chunxian Chen, Fredrick G. Gmitter Jr., Peng Ling, Qibin Yu, Melinda R. Grosser, Jude W. Grosser and Christine D. Chase

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.