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Peter C. Andersen and Brent V. Brodbeck

-sectional area of ‘Brown Select’ (BS) and ‘Owari’ (OW) satsuma scions on Poncirus trifoliata ‘Flying Dragon’ (FD) and ‘Rubidoux’ (RB) rootstocks. Tree canopy areas and canopy volumes were calculated in 2010, 2012, and 2014 to assess yield efficiency on the

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Milton E. Tignor, Frederick S. Davies, Wayne B. Sherman, and John M. Davis

Poncirus trifoliata (L.) Raf. seeds were germinated in perlite under intermittent mist at about 25 °C and natural daylight in a greenhouse. Two-week-old seedlings were then transferred into a growth chamber at 25 °C and 16-hour daylength for 1 week. Tissue samples were collected at 0, 6, 24, 168, and 504 hours after temperature equilibration at 10 °C. Freezing tolerance at –6.7 °C, as determined by electrolyte leakage, and stem (leaves attached) water potential (ψx), measured using a pressure chamber, was recorded for a subset of seedlings for each time interval. Red coloration (apparently anthocyanin) developed at the petiole leaflet junction and buds after 48 hours at 10 °C and gradually occurred throughout the leaves during further exposure. Complementary DNA clones for phenylalanine ammonia lyase (PAL), 4-coumarate: coA ligase (4CL), and chalcone synthase (CHS) were used to probe RNA isolated from the leaves. No increase in steady-state messenger RNA level was detected. Increases in freeze hardiness occurred within 6 hours in the leaves, and continued for up to 1 week. Water potential initially decreased from –0.6 to –2.0 MPa after 6 hours, then returned to –0.6 MPa after 1 week. Thus, Poncirus trifoliata seedlings freeze-acclimate significantly after only 6 hours at 10 °C.

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Frank Suozhan Cheng and Mikeal L. Roose

`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.

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Zhan'ao Deng, Fred G. Gmitter Jr., Shunyuan Xiao, and Shu Huang

Citrus tristiza virus (CTV) is the most-significant viral pathogen of citrus in the world. Rapid decline of trees on sour orange and stem pitting of grapefruit and sweet orange, two diseases induced by CTV, severely jeopardize citrus production worldwide. It is recognized that all future rootstocks should be resistant to this virus, and scion resistance to stem pitting stains is desirable. To facilitate introgression of the CTV resistance gene from Poncirus trifoliata and development of CTV-resistant varieties in citrus, gene mapping projects have been initiated and more than a dozen RAPD markers have been identified with tight linkage to the resistance gene. As part of our efforts to use marker-assisted selection with a large number of crosses, and ultimately to accomplish map-based cloning of the CTV resistance gene, we have been converting the most tightly linked RAPD markers into SCAR (sequence characterized amplified region) markers by cloning, sequencing the marker fragments, and designing locus-specific primers. One codominant and several dominant SCARs have been developed thus far. The updated progress and utilization of these SCARs in marker-assisted selection and possibly in characterization of a BAC library will be presented and discussed.

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Milton E. Tignor Jr. and Courtney A. Weber

Current efforts in the study of citrus freeze hardiness including gene mapping and elucidating early induction processes require large populations of uniform seedlings. Related genera and intergeneric hybrids are often used in these studies and little is known about factors effecting their seedling emergence. We tested a total of 8 genotypes including Poncirus trifoliata `Rubidoux', Citrus grandis, C. sinensis `Pineapple', C. jambhiri `Schaub', C. paradisi `Duncan', C. aurantium (Brazilian), Carrizo citrange (P. trifoliata × C. sinensis), and Troyer citrange. A total of seven pre-planting treatments were used to evaluate seedling emergence rates. Expanding on the work of previous researchers, treatments were seed coat removal, hydrating in water (96 hours) at either 4, 25, or 35°C, acid scarification, or boiling. Generally, seed coat removal resulted in the most uniform emergence as compared to untreated controls. Presoaking at each temperature enhanced emergence in most varieties tested and 25°C was the best hydrating temperature. Acid scarification greatly delayed emergence in all genotypes tested except Troyer citrange and `Pineapple' orange which had enhanced emergence rates as compared to controls. Preplanting treatment with 100°C water was lethal in all varieties. Pretreatment of citrus seeds can enhance uniformity of germination, although optimum treatments for individual genotypes vary.

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Courtney A. Weber, Gloria A. Moore, Zhanao Deng, and Fred G. Gmitter Jr.

Mapping quantitative trait loci (QTL) associated with freeze tolerance was accomplished using a Citrus grandis (L.) Osb. × Poncirus trifoliata (L.) Raf. F1 pseudo-testcross population. A progeny population of 442 plants was acclimated and exposed to temperatures of -9 °C and -15 °C in two separate freeze tests. A subpopulation of 99 progeny was genotyped for random amplified polymorphic DNA (RAPD), cleaved amplified polymorphic sequence (CAPS), sequence characterized amplified region (SCAR), and sequence tagged site (STS) markers to produce a linkage map for each parent. Potential QTL were identified by interval mapping, and their validity was corroborated with results from means comparison (t test), one-way analysis of variance (F test), and bulked segregant analysis (BSA). Multiple analytical methods provided evidence supporting putative QTL and decreased the probability of missing significant QTL associated with freeze tolerance. QTL with a large effect on freeze tolerance were located on both the Citrus and Poncirus linkage maps. In addition, clusters of markers with significantly different means between marker present and absent classes indicating minor QTL that contribute smaller effects on the level of tolerance were found on the linkage maps of both species.

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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

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Ute Albrecht and Kim D. Bowman

were also identified as highly tolerant to HLB by Folimonova et al. (2009) after controlled inoculations with Ca . L. asiaticus. No well-defined disease symptoms have been observed in trifoliate orange ( Poncirus trifoliata ) trees and seedlings

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Milton E. Tignor, John M. Davis, Frederick S. Davies, and Wayne B. Sherman

Poncirus trifoliata is a comparatively hardy, cross compatible, and graft compliant relative of Citrus. The citrus industry in Florida has suffered immense economic losses due to freezes. Although much research has been done in citrus freeze hardiness, little work has been on the early induction of freeze tolerance by low temperature. Poncirus trifoliata `Rubidoux' seedlings were germinated in perlite under intermittent mist at about 25°C and natural daylight conditions in a greenhouse and grown 2 weeks. See dlings were then transferred into a growth chamber at 25°C and 16 hour daylength for 1 week. Temperature was lowered to 10°C and tissue samples were collected at 0, 6, 24, and 168 hours. Freezing tolerance, at –6.7°C as determined by electrolyte leakage, and stem (leaves attached) water potential, measured using a pressure bomb, were also recorded for a subset of seedlings for the above intervals. After exposure to low temperature for 48 hours a red coloration became visible at the petiole leaflet junction an d at the buds, with subsequent exposure to low temperature the coloration spread to the leaves. Clones for phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), and chlorophyll ab binding protein (CAB), and chalcone synthase (CHS) were used to probe RNA isolated from P. trifoliata. PAL and 4CL transcripts increased in response to the low temperature. Significant increases in freeze hardiness occurred within 6 hours in the leaves, and increases continued for up to one week. Water potential increased from –0.6 to –2.0 MPa after 6 hours, then returned to –0.6 MPa after 1 week. These data indicate that increases in freezing tolerance and changes in water potential and gene expression can be detected shortly after low temperature treatments are imposed on P. trifoliata seedlings.

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Courtney A. Weber and Gloria A. Moore

A greater saturation of the previously constructed genetic linkage map of Citrus is important in the long term goal of mapping quantitative trait loci (QTL) such as those controlling cold and salt tolerance. Segregation for cold tolerance appears to be greatly enhanced in the intergeneric F1 population of Citrus grandis × Poncirus trifoliata as compared to the BC1 population previously used for mapping due to the higher percentage of P. trifoliata genes present. This is not unexpected since P. trifoliata is the source of cold tolerance in this cross and is a highly heterozygous species. An integration of the maps of the two populations using about 50 random amplified polymorphic DNA (RAPD) markers common to the two populations is possible using the JoinMap computer program. This will allow the placing of approximately 100 new polymorphic RAPD markers from the F1 population identified by screening from 42 random oligonucleotide primers onto the Citrus map. This saturated map will be used to locate QTL following bulk segregation analysis of cold tolerance in the F1 population.