`Flying Dragon' Poncirus trifoliata L. Raf. is a dwarfing rootstock for citrus. Inheritance of dwarfing ability was studied in a population of open-pollinated seedlings of `Flying Dragon'. Molecular marker genotypes suggest that all seedlings originated from selfing. Progeny seedlings were budded with `Cutter Valencia' orange and planted in the field to evaluate the dwarfing effect of the seedling rootstock. At 5 years after planting, rankit analysis of the frequency distributions of trunk cross-sectional area and canopy volume suggested the presence of two overlapping distributions of 34 dwarf trees and 7 nondwarf. This ratio is consistent with inheritance of rootstock dwarfing as a single dominant gene for which `Flying Dragon' is heterozygous. Two morphological characteristics of `Flying Dragon', curved thorns and twisted trunk growth, were closely linked to, or pleiotropic effects of, the dwarfing gene. Bulked segregant analysis was used to identify three RAPD markers linked to the dwarfing gene. `Flying Dragon' was identical to nondwarfing cultivars of trifoliate orange at 40 homozygous and heterozygous isozyme and RFLP markers; therefore, it is likely that `Flying Dragon' originated as a mutant of a nondwarfing genotype and has not undergone sexual recombination since this event.
Frank Suozhan Cheng and Mikeal L. Roose
Robert R. Krueger and Mikeal L. Roose
New potential citrus germplasm accessions may be received as seed rather than budwood, thereby reducing phytosanitary risks. However, trueness-to-type may be an issue with seed materials because many varieties produce both apomictic (nucellar) and sexual (zygotic) embryos and most citrus is fairly heterozygous. To identify nucellar seedlings of polyembryonic types and to retain these as representing the type, we screened 1340 seedlings from 88 seed sources for markers amplified with two inter-simple sequence repeat (ISSR) primers. Sixteen seed sources produced no seedlings classified as being of nucellar origin. Among the remaining seed sources, seedlings classed as nucellar were identified for potential addition to the collection. In 37 accessions, both nucellar and zygotic seedlings were detected, and in some cases both types were retained. Inclusion of established accessions of the same cultivar group in the analysis allowed an initial assessment of similarity to existing accessions. This technique improved the efficiency of acquiring new germplasm of polyembryonic types by seed. The method identifies those seed sources that produce few or no nucellar seedlings, but it is not useful for determining which seedlings of monoembryonic types should be retained in collections.
David C. Jarrell and Mikeal L. Roose
We report a preliminary genetic map of citrus based on segregation of 8 isozyme and at least 33 RFLP loci. The segregating population consisted of 60 plants from a cross of two citrus rootstock, `Sacaton' citrumelo × `Troyer' citrange. This cross represents an intergeneric F2 since `Sacaton' is Citrus paradisi (grapefruit) × Poncirus trifoliata (trifoliate orange) and `Troyer' is C. sinensis (sweet orange) × P. trifoliata. RFLPs were identified using anonymous probes from both cDNA and genomic DNA libraries of citrus. About 20% of the loci deviated significantly from Mendelian segregation. Two-point linkage analysis identified 8 linkage groups in which pairs of loci were within 30 centimorgans. This suggests that we have markers on most of the 9 chromosomes of Citrus. A map based on multipoint linkage estimates will be reported. Evidence for structural rearrangements between Citrus and Poncirus and extension of the map to additional marker and disease resistance loci will be discussed.
Camilo Canel, Julia N. Bailey-Serres, and Mikeal L. Roose
The acidless phenotype of the pummelo 2240 [Citrus maxima [Burro.] Merrill] is caused by a mutation affecting a key element of the citric acid accumulation pathway. To test the functionality of the tonoplast citrate transport mechanism, we obtained a tonoplast-enriched membrane fraction from juice tissues of acidless fruit by centrifugation through a discontinuous Ficoll gradient. The isolated tonoplast vesicles incorporated radioactively labeled citrate at a higher rate than vesicles from similarly fractionated high-acid fruit juice. Uptake of [14C]citrate occurred against a concentration gradient was stimulated by nitrate-sensitive ATP hydrolysis, but not by hydrolysis of PPi, and was not affected by the ionophore nigericin. Uptake was not inhibited by malate and only slightly by isocitrate. We did not find evidence of a defective citrate transport mechanism at the tonoplast of juice cells of acidless fruit. We propose that citric acid accumulation in the fruit of citrus is mediated by a carrier that uses energy produced during hydrolysis of ATP to transport citrate into the vacuole actively and specifically.
Mikeal L. Roose, Frank Suozhan Cheng, and Claire T. Federici
The `Flying Dragon' cultivar of Poncirus trifoliata L. Raf. is a strongly dwarfing rootstock for Citrus cultivars, reducing canopy volume of 9 year-old `Valencia' orange trees to 1/3 that of trees on standard rootstocks Open-pollinated seed of `Flying Dragon' was screened with isozyme markers to distinguish zygotic from nucellar (apomictic) seedlings. All zygotics had genotypes consistent with an origin by self-pollination. Zygotic seedlings were budded with `Valencia' orange scion and planted in the field. Of 46 progeny evaluated as rootstocks, 35 produced small trees similar to those on nucellar `Flying Dragon' and 11 produced large trees. This ratio is consistent with the 3:1 segregation expected for a single dominant gene. The dwarfing gene was closely linked, or pleiotropic with a gene causing curved thorns and stems. Several RAPD markers close to the dwarfing gene were identified with bulked segregant analysis. `Flying Dragon' apparently originated as a mutation because it had au identical genotype to non-dwarfing strains of trifoliate orange at all 38 isozyme and RFLP markers tested
Mikeal L. Roose, Claire T. Federici, and Gregory P. Copenhaver
To assess genetic diversity in the UC Riverside Citrus Variety Collection, all accessions of pummelo (59), citron (24), and trifoliate orange (48) were studied for RFLP variation using 11-18 cDNA probes that had previously been shown to reveal polymorphism in a broad range of citrus germplasm. Inheritance studies have shown that these probes hybridize to at least 20 loci. The taxa studied are believed to represent biological species rather than hybrids. Citrons were nearly monomorphic and most appeared homozygous at all of the loci studied. Pummelos were very polymorphic and highly heterozygous. Trifoliate orange, an important source of disease resistance in rootstock breeding, was nearly monomorphic but moderately heterozygous (17% of loci). Most accessions of trifoliate orange have evidently differentiated only by mutation. One multilocus probe separated trifoliate orange accessions into 3 groups. Two new trifoliate orange accessions had novel alleles at some loci. The use of genetic markers to recognize hybrid accessions classified as members of species will be discussed.
Deqiu Fang, Robert R. Krueger, and Mikeal L. Roose
ISSR markers were analyzed to study phylogenetic relationships among 46 Citrus L. accessions representing 35 species. A dendrogram based on the unweighted pair-group method, arithmetic average cluster analysis was constructed using a similarity matrix derived from 642 polymorphic ISSR fragments generated by 10 primers. These 46 accessions could be classified into five major groups: 1) C. indica Tan.; 2) C. maxima (Burm.) Merrill; 3) lemon [C. limon (L.) Burm.] or lime [C. aurantifolia (Christm.) Swingle] type accessions; 4) C. halimii B. C. Stone; and 5) sour orange (C. aurantium L.), mandarins and their hybrids. Group 5 was further divided into three subgroups. Although some previous work had grouped it with mandarins, C. indica appeared to be a distinct genotype or species that was not close to mandarins. C. tachibana Tan. grouped closely to mandarins. C. vulgaris Risso was not related to sour orange but was similar to accessions usually classified in the lime or lemon group. Sour orange and its hybrids, C. nippokoreana Tan., C. hanayu Hort. ex Shirai, C. sudachi Hort. ex Shirai, and C. yuko Hort. ex Tan. had close phylogenetic relationships with mandarins. Although the mandarin accessions studied were divergent in morphology, the genetic distances among them were relatively small. Relationships among these Citrus accessions revealed by ISSR markers were generally in agreement with previous taxonomic classifications.
Ed Stover, Chandrika Ramadugu, Mikeal Roose, Joseph Krystel, Richard F. Lee, and Manjunath Keremane
Asiatic citrus canker (ACC) foliar lesions were evaluated on progenies of 84 seed-source genotypes (“parent genotypes”) from the Citrus Variety Collection (CVC) of the University of California at Riverside (UCR) of Citrus trifoliata and hybrids between C. trifoliata and other Citrus species and hybrids. Trees were planted Aug. 2013 in a completely randomized design at the Fort Pierce U.S. Department of Agriculture (USDA) grove. Plants were assessed visually Aug. 2017, Sept. 2019, and Sept. 2020 for distinctive ACC lesion incidence and severity. Progeny were compared by parent genotypes using nonparametric analysis. Incidence of ACC [percentage of leaves displaying symptoms, verified by quantitative polymerase chain reaction (qPCR) to be associated with Xanthomonas citri pv. citri] across parent genotypes ranged from 8% to 80% (mean, 49%) of leaves affected in 2017, from 4% to 58% (mean, 29%) in 2019, and 8% to 46% (mean, 25%) in 2020. In 2017, of 49 C. trifoliata parent genotypes, only four separated from the two highest ACC-incidence statistical categories [Citrus Research Center (CRC) 3345, 3484, 3888, and 4017]. whereas 29 of the 35 C. trifoliata hybrids displayed lower ACC incidence, which separated from the two highest statistical categories. In 2019, of the C. trifoliata, only six separated from the highest ACC-incidence statistical category (CRC 3330, 3484, 3547, 3549, 3876, and 3888), whereas all 35 C. trifoliata hybrids displayed lower ACC incidence and separated from the highest statistical category, and 26 hybrids separated from 18 of the C. trifoliata. In 2020, only three C. trifoliata separated from the highest ACC-incidence statistical category (CRC 2861, 3549, and 3888) and 20 hybrids separated from 18 of the C. trifoliata. By parent genotype, ACC incidence correlated substantially between each pair of the 3 years, with r 2 values of 0.39, 0.57, and 0.65. Of 34 hybrids validated, similar numbers had C. trifoliata, grapefruit (C. ×aurantium var. racemosa), and sweet orange (C. ×aurantium var. sinensis) chloroplasts. Chloroplast type affected ACC incidence and severity, but not in a consistent manner. Near-isogenic groups within C. trifoliata, as determined by DNA markers, were associated with some statistically different ACC sensitivity. Overall, hybrids of C. trifoliata with other citrus types displayed markedly reduced ACC sensitivity compared with C. trifoliata, indicating that this trait is readily overcome through breeding.
Tyler J. Simons, Christopher J. McNeil, Aubrey D. Pham, Carolyn M. Slupsky, Mikeal L. Roose, and Jean-Xavier Guinard
‘DaisySL’ mandarins (Citrus reticulata Blanco) grafted to Schaub Rough lemon (C. jambhiri Lush; SHRL) Carrizo citrange (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.; CARR), and Rubidoux Trifoliate [Poncirus trifoliata (L.) Raf.; RUBT] rootstocks were measured to determine their liking by adults and children, sensory properties, and targeted metabolomics over the course of 2 consecutive years. Chemical measurements showed differences in sugars, acids, and ethanol content, whereas a descriptive analysis found variations in sweetness, mandarin flavor, juiciness, and peelability. During both years, adults significantly preferred ‘DaisySL’ mandarins grafted to CARR and RUBT over those grafted to SHRL (P ≤ 0.05). Children liked the fruit grafted to CARR and RUBT rootstocks significantly more than fruit grafted to SHRL during the first year, but they did not prefer fruit grafted to any rootstock during the second year. This research found that ‘DaisySL’ mandarins are a well-liked variety of mid-to-late season mandarin capable of filling the seasonal gap between clementine and W. Murcott varieties. We concluded that the rootstock can affect the chemical composition, sensory profile, and consumer preferences for ‘DaisySL’ mandarins.