Search

Citrus Huanglongbing (HLB) is the most devastating disease of citrus, presumably caused by “Candidatus Liberibacter asiaticus” (CaLas). Although transcriptomic profiling of HLB-affected citrus plants has been studied extensively, the initial steps in pathogenesis have not been fully understood. In this study, RNA sequencing (RNA-seq) was used to compare very early transcriptional changes in the response of Valencia sweet orange (VAL) to CaLas after being fed by the vector, Diaphorina citri (Asian citrus psyllid, or ACP). The results suggest the existence of a delayed defense reaction against the infective vector in VAL, while the attack by the healthy vector prompted immediate and substantial transcriptomic changes that led to the rapid erection of active defenses. Moreover, in the presence of CaLas-infected psyllids, several downregulated differentially expressed genes (DEGs) were identified on the pathways, such as signaling, transcription factor, hormone, defense, and photosynthesis-related pathways at 1 day post-infestation (dpi). Surprisingly, a burst of DEGs (6,055) was detected at 5 dpi, including both upregulated and downregulated DEGs on the defense-related and secondary metabolic pathways, and severely downregulated DEGs on the photosynthesis-related pathways. Very interestingly, a significant number of those downregulated DEGs required ATP binding for the activation of phosphate as substrate; meanwhile, abundant highly upregulated DEGs were detected on the ATP biosynthetic and glycolytic pathways. These findings highlight the energy requirement of CaLas virulence processes. The emerging picture is that CaLas not only employs virulence strategies to subvert the host cell immunity, but the fast-replicating CaLas also actively rewires host cellular metabolic pathways to obtain the necessary energy and molecular building blocks to support virulence and the replication process. Taken together, the very early response of citrus to the CaLas, vectored by infective ACP, was evaluated for the first time, thus allowing the changes in gene expression relating to the primary mechanisms of susceptibility and host–pathogen interactions to be studied, and without the secondary effects caused by the development of complex whole plant symptoms.

SummaryHistone acetylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by HATs and HDACs. This study determined the function of 6 histone acetyltransferases (HATs) (Gcn5, RTT109, Elp3, Sas3, Sas2, Nat3) and 6 histone deacetylases (HDACs) (Hos2, Rpd3, Hda1, Hos3, Hst2, Sir2) in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. Our data provide evidence that HATs and HDACs are both required for mycelium growth, cell development and pathogenicity as many gene deletion mutants (ΔGcn5, ΔRTT109, ΔElp3, ΔSas3, ΔNat3, ΔHos2, and ΔRpd3) displayed reduced growth, conidiation or virulence at varying degrees. In addition, HATs and HDACs are involved in the resistance to multiple stresses such as oxidative stress (Sas3, Gcn5, Elp3, RTT109, Hos2), osmotic stress (Sas3, Gcn5, RTT109, Hos2), cell wall-targeting agents (Sas3, Gcn5, Hos2), and fungicide (Gcn5, Hos2). ΔGcn5, ΔSas3 and ΔHos2 displayed severe growth defects on sole carbon source medium suggesting a vital role of HATs and HDACs in carbon source utilization. More SNPs were generated in ΔGcn5 in comparison to wild-type when they were exposed to ultraviolet ray. Moreover, ΔRTT109, ΔGcn5 and ΔHos2 showed severe defects in resistance to DNA-damaging agents, indicating the critical role of HATs and HDACs in DNA damage repair. These phenotypes correlated well with the differentially expressed genes in ΔGcn5 and ΔHos2 that are essential for carbon sources metabolism, DNA damage repair, ROS detoxification, and asexual development. Furthermore, Gcn5 is required for the acetylation of H3K4. Overall, our study provides genetic evidence to define the central role of HATs and HDACs in the pathological and biological functions of A. alternata.ImportanceIn filamentous fungi, HATs and HDACs have been found to be involved in growth, development, virulence, synthesis of secondary metabolites and multi-stress resistance. However, the role of all HATs and HDACs in the same fungal pathogen has not been systematically explored. Our study revealed that HATs and HDACs are required for the vegetative growth, conidiation, pathogenicity, multiple stresses resistance, DNA damage repair, and carbon source utilization in A. alternata. Moreover, HATs and HDACs identified in A. alternata are highly conserved in many notorious pathogenic fungi, such as Magnaporthe grisea and Candida albicans. Therefore, this study not only systematically reveals the biological functions of twelve histone acetylation-required enzymes for the first time, but also provides a foundation for future investigations in A. alternata or other pathogenic fungi.

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen &ldquo, Candidatus Liberibacter asiaticus&rdquo, and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) &times, pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

Pioneering investigations referring to citrus flavor have been intensively conducted. However, the characteristic flavor difference between sweet orange and mandarin has not been defined. In this study, sensory analysis illustrated the crucial role of aroma in the differentiation between orange flavor and mandarin flavor. To study aroma, Valencia orange and LB8-9 mandarin were used. Their most aroma-active compounds were preliminarily identified by aroma extract dilution analysis (AEDA). Quantitation of key volatiles followed by calculation of odor activity values (OAVs) further detected potent components (OAV ≥ 1) impacting the overall aromatic profile of orange/mandarin. Follow-up aroma profile analysis revealed that ethyl butanoate, ethyl 2-methylbutanoate, octanal, decanal, and acetaldehyde were essential for orange-like aroma, whereas linalool, octanal, α-pinene, limonene, and (E,E)-2,4-decadienal were considered key components for mandarin-like aroma. Furthermore, an unreleased mandarin hybrid producing fruit with orange-like flavor was used to validate the identification of characteristic volatiles in orange-like aroma.

Background: Citrus fruit flavor is an important attribute prioritized in variety improvement. The present study compared juice volatiles compositions from 13 selected citrus genotypes, including six mandarins (Citrus reticulata), three sour oranges (Citrus aurantium), one blood orange (Citrus sinensis), one lime (Citrus limonia), one Clementine (Citrus clementina) and one satsuma (Citrus unshiu).; Results: Large differences were observed with respect to volatile compositions among the citrus genotypes. 'Goutou' sour orange contained the greatest number of volatile compounds and the largest volatile production level. 'Ponkan' mandarin had the smallest number of volatiles and 'Owari' satsuma yielded the lowest volatile production level. 'Goutou' sour orange and 'Moro' blood orange were clearly distinguished from other citrus genotypes based on the analysis of volatile compositions, even though they were assigned into one single group with two other sour oranges by the molecular marker profiles.; Conclusions: The clustering analysis based on the aroma volatile compositions was able to differentiate mandarin varieties and natural sub-groups, and was also supported by the molecular marker study. The gas chromatography-mass spectrometry analysis of citrus juice aroma volatiles can be used as a tool to distinguish citrus genotypes and assist in the assessment of future citrus breeding programs. The aroma volatile profiles of the different citrus genotypes and inter-relationships detected among volatile compounds and among citrus genotypes will provide fundamental information on the development of marker-assisted selection in citrus breeding. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

The main purposes of this study were to identity major peel oil odorants and determine which aroma compounds are primarily responsible for the overall aroma profile of a recently developed Huanglongbing (HLB)-tolerant mandarin hybrid. The aroma-active compounds present in the recently developed mandarin hybrid peel oil were extracted by solvent-assisted flavor evaporation (SAFE) and then analyzed by gas chromatography-mass spectrometry/olfactometry (GC-MS/O) and aroma extract dilution analysis (AEDA). Thirty odor-active compounds in the flavor dilution (FD) factor range of 2 to 256 have been identified. On the basis of high FD factors, α-pinene, limonene, β-myrcene, linalool, and β-caryophyllene were characterized as the most essential aroma compounds at a FD factor ≥ 128, followed by γ-terpinene, β-pinene, terpinolene, p-dimethylstyrene, (E)-linalool oxide, decanal, β-cubebene, α-terpineol, and perilla aldehyde at a FD factor ≥ 16. Among all the detected aroma compounds, limonene was the most abundant compound (86.5 ± 2.8%), followed by γ-terpinene (5.3 ± 0.1%), β-myrcene (2.4 ± 0.1%), and α-pinene (1.2 ± 0.0%). The compounds including α-pinene, limonene, β-myrcene, linalool, and β-caryophyllene were characterized as the most essential aromas. The sensory evaluation results indicated that the major attributes (FD ≥ 2) such as floral, lemon, peel-like, green, mint, and sweet were comparable to that of natural mandarin peel oil. Based on the human perception, olfactometry and AEDA were used to determine the aroma-active compounds in the peel oil. Human perception also involved in aroma reconstitution to compare the re-engineering solution containing the essential aroma compounds with the natural mandarin oil.

Huanglongbing (HLB) is a disease that has devastated the Florida citrus industry, threatens the entire U.S. citrus industry, and globally is rapidly spreading. Florida’s citrus production is 90% sweet orange, which is quite sensitive to HLB. The heavy reliance on sweet orange for Florida citrus production makes the industry especially vulnerable to diseases that are damaging to this type of citrus. Furthermore, 90% of Florida oranges are used in producing orange juice that is defined by a federal regulation known as the “orange juice standard”, specifying that at least 90% of “orange juice” must be derived fromCitrus sinensis. Genomic analyses definitively reveal that sweet orange is not a true species, but just one of many introgression hybrids ofC. reticulataandC. maxima, with phenotypic diversity resulting from accumulated mutations in this single hybrid, the “sweet orange”. No other fruit industry is limited by law to such a narrow genetic base. Fortunately, there are new citrus hybrids displaying reduced sensitivity to HLB, and in some cases they produce juice, alone or in blends, that consumers would recognize as “orange juice”. Reconsidering current regulations on orange juice standards may permit use of such hybrids in “orange juice”, providing greater latitude for commercialization of these hybrids, leading to higher-quality orange juice and a more sustainable Florida orange juice industry.

The invasive Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector of the phloem-infecting bacterium, Candidatus Liberibacter asiaticus. Candidatus L. asiaticus is the putative causal agent of Huanglongbing (HLB) disease, a destructive disease of Citrus. While many Citrus species are susceptible to D. citri probing and HLB disease, there are marked behavioral differences in D. citri probing responses and Ca. Liberibacter asiaticus infection severity among Citrus species. Using four mandarin hybrid selections and pummelo plants variably resistant to D. citri probing, oviposition, and survival, we explored probing differences using electropenetrography (EPG), conducted an oviposition and survival study, and determined host plant metabolites using gas-chromatography mass-spectroscopy (GC-MS). We found thirty-seven D. citri probing variables to be significantly different among tested mandarin selections and pummelo, in addition to differential oviposition and survivorship abilities on tested plants. We found sixty-three leaf metabolites with eight being significantly different among tested mandarin selections and pummelo. Detailed analysis of probing behavior, oviposition, survivorship, and host plant metabolite concentrations reveals the complex, layered resistance mechanisms utilized by resistant Citrus against D. citri probing. EPG is a powerful technology for screening Asian citrus psyllid resistant Citrus to elucidate host plant-vector interactions, with an aim to minimize vector probing and eliminate the spread of the bacterial pathogen, Ca. L. asiaticus.

High quality chromosome-scale assemblies from an interspecific hybrid between walnut and a wild relative reveal the persistence of asymmetric fractionation between the sub-genomes and suggest a late-Miocene origin for the genus Juglans.

Background Valencene is a major sesquiterpene in citrus oil and biosynthesized by valencene synthase (Cstps1; EC: 4.2.3.73) from the 15-carbon substrate farnesyl diphosphate. It is abundant in juice of some mandarins (e.g. Citrus reticulata Blanco cv. Fortune), however, it is undetectable in others (e.g. C. reticulata Blanco cv. Murcott), We have discovered that the Murcott mandarin Cstps1 gene expression is severely reduced. A previous genetic mapping study using an F1 population of Fortune × Murcott found that the segregation of valencene production in fruit exhibited a Mendelian inheritance ratio of 1:1. There was only one dominant locus associated with valencene content detected on the mandarin genetic map. The goal of this study was to understand the molecular mechanism underlying the valencene deficiency observed in some citrus hybrids. Results There was a clear relationship between presence or absence of the valencene synthase gene (Cstps1) expression, and presence or absence of valencene among randomly selected mandarin hybrids. Cloning the coding regions of Cstps1 from Fortune and Murcott mandarin, and aligning with previous reported Valencia orange Cstps1 sequence, showed that they both exhibited extremely high similarity with the known Cstps1. By further cloning and analyzing the promoter region of Cstps1 from Valencia, Fortune and Murcott, a 12-nucleotide deletion at approximately − 270 bp from the Cstps1 coding region was only found in Murcott. Three binary vectors, designated as p1380-FortP-GUSin, p1380-MurcP-GUSin and p1380-MurcP(+ 12)-GUSin, were developed for promoter activity analysis. Transient over-expression of Fortune Cstps1 promoter in sweet orange showed notable GUS activity, but the Murcott Cstps1 promoter did not. In addition, by re-inserting the 12-nucleotide fragment, the activity of the Murcott Cstps1 promoter was mostly recovered. Conclusion The deficiency of valencene production in some mandarins is probably due to a 12-nucleotide deletion in the promoter region of the Cstps1, which could be a crucial switch of Cstps1 transcription. Our results further enhanced the understanding of valencene biosynthesis in citrus. Electronic supplementary material The online version of this article (10.1186/s12870-019-1701-6) contains supplementary material, which is available to authorized users.

The Australian finger lime is a unique citrus species that has gained importance due to its unique fruit characteristics and perceived tolerance to Huanglongbing (HLB), an often-fatal disease of citrus trees. In this study, we developed allotetraploid finger lime hybrids and cybrids by utilizing somatic cell fusion techniques to fuse diploid ‘OLL8’ sweet orange or ‘Page’ tangelo callus-derived protoplasts with finger lime (FL) mesophyll-derived protoplasts. Six somatic fusions were regenerated from the ‘OLL8’ + FL fusion, while three putative cybrids were regenerated from the ‘Page’ + FL fusion. Ploidy levels and nuclear-expressed sequence tag derived simple sequence repeat (EST-SSR) markers confirmed the somatic hybrid production, and mitochondrial DNA primer sets confirmed the cybrid nature. Several trees produced by the somatic fusion remained HLB negative even after 6 years of growth in an HLB-endemic environment. Pathogenesis related (PR) and other genes that are often upregulated in HLB-tolerant trees were also upregulated in our somatic fusions. These newly developed somatic fusions and cybrids could potentially be used as breeding parents to develop the next generation of improved HLB-tolerant rootstocks and scions.

Citrus huanglongbing, putatively caused by the associated bacterium ‘Candidatus Liberibacter asiaticus’, is the greatest threat to the world citrus industry today. The bacterium is spread locally and regionally by the citrus psyllid Diaphorina citri, and also can be disseminated by propagation of contaminated scion budwood that is grafted to the appropriate rootstock. The planting of ‘Ca. Liberibacter asiaticus’-free trees is a component of a comprehensive strategy to manage huanglongbing. In contrast to the scion budwood, the rootstocks used to produce these trees are grown from seed. This research was undertaken to provide evidence as to whether or not ‘Ca. L. asiaticus’ can be transmitted through seed. Two groups of 360 or more seedlings each of various citrus species were grown from seed removed from fruit on trees that were symptomatic for huanglongbing and confirmed to be infected with ‘Ca. L. asiaticus’ by polymerase chain reaction (PCR) tests. These seedlings were tested multiple times over periods of up to 3 years. No symptoms typical of huanglongbing, such as blotchy leaf mottle, chlorotic shoots, or dieback of branches, were observed in these seedlings, and none of these 723 seedlings tested positive for the presence of ‘Ca. L. asiaticus’ even after repeated testing by sensitive quantitative PCR assays. Some sour orange seedlings did have quite pronounced and atypical growth, including stunting and mild to severe leaf malformation. These atypical growth habits were limited to seedlings that arose from zygotic embryos as determined by expressed-sequence tag simple-sequence repeat analyses. Thus, no evidence of transmission of ‘Ca. L. asiaticus’ via seed was obtained, and an earlier report of transmission of the pathogen through seed was not confirmed.

Huanglongbing (HLB), or citrus greening, is the most devastating disease in citrus worldwide. Commercial citrus varieties including sweet orange (Citrus sinensis) are highly susceptible to HLB, and trifoliate orange (Poncirus trifoliata, a close Citrus relative) is widely considered resistant or highly tolerant to HLB. In this study, an intergeneric F1 population of sweet orange and trifoliate orange was genotyped by Genotyping-by-Sequencing, and high-density SNP-based genetic maps were constructed separately for trifoliate orange and sweet orange. The two genetic maps exhibited high synteny and high coverage of the citrus genome. Progenies of the F1 population and their parents were planted in a replicated field trial, exposed to intense HLB pressure for 3 years, and then evaluated for susceptibility to HLB over 2 years. The F1 population exhibited a wide range in severity of HLB foliar symptom and canopy damage. Genome-wide QTL analysis based on the phenotypic data of foliar symptom and canopy damage in 2 years identified three clusters of repeatable QTLs in trifoliate orange linkage groups LG-t6, LG-t8 and LG-t9. Co-localization of QTLs for two traits was observed within all three regions. Additionally, one cluster of QTLs in sweet orange (linkage group LG-s7) was also detected. The majority of the identified QTLs each explained 18-30%25 of the phenotypic variation, indicating their major role in determining HLB responses. These results show, for the first time, a quantitative genetic nature yet the presence of major loci for the HLB tolerance in trifoliate orange. The results suggest that sweet orange also contains useful genetic factor(s) for improving HLB tolerance in commercial citrus varieties. Findings from this study should be very valuable and timely to researchers worldwide as they are hastily searching for genetic solutions to the devastating HLB crisis through breeding, genetic engineering, or genome editing.

Long terminal repeat retrotransposons (LTR-RTs) are a large portion of most plant genomes, and can be used as a powerful molecular marker system. The first citrus reference genome (Citrus x clementina) has been publicly available since 2011; however, previous studies in citrus have not utilized the whole genome for LTR-RT marker development. In this study, 3959 full-length LTR-RTs were identified in the C. x clementina genome using structure-based (LTR_FINDER) and homology-based (RepeatMasker) methods. LTR-RTs were first classified by protein domain into Gypsy and Copia superfamilies, and then clustered into 1074 families based on LTR sequence similarity. Three hundred fifty Copia families were grouped into four lineages: Retrofit, Tork, Sire, and Oryco. One hundred seventy-eight Gypsy families were sorted into six lineages: Athila, Tat, Renia, CRM, Galadriel, and Del. Most LTR-RTs (3218 or 81.3%) were anchored to the nine Clementine mandarin linkage groups, accounting for 9.74% of chromosomes currently assembled. Accessions of 25 Rutaceae species were genotyped using 17 inter-retrotransposon amplified polymorphism (IRAP) markers developed from conserved LTR regions. Sequence-specific amplified polymorphism (SSAP) makers were used to distinguish ‘Valencia’ and ‘Pineapple’ sweet oranges (C. x sinensis), and 24 sweet orange clones. LTR-RT markers developed from the Clementine genome can be transferred within the Rutaceae family demonstrating that they are an excellent tool for citrus and Rutaceae genetic analysis.

Citrus tolerance to huanglongbing could result from tolerance to the pathogen Candidatus Liberibacter asiaticus (CLas) and/or to its vector Diaphorina citri. Field observations and greenhouse-controlled studies showed that some citrus cultivars were more tolerant than others. However, the mechanism(s) behind the tolerance has not been determined yet. Using GC-MS, we investigated the volatile organic compounds (VOCs) and the non-volatile metabolite profiles of two tolerant citrus cultivars- Australian finger lime, ‘LB8-9’ Sugar Belle® mandarin hybrid, and a recently released mandarin hybrid ‘Bingo’. The three were grafted onto the rootstock, Carrizo citrange. Our findings showed that the metabolomic profiles of Australian finger lime were different from that of ‘LB8-9’. Finger lime was high in many amino acids and tricarboxylic acid intermediates, whereas ‘LB8-9’ was high in several amino acids, sugars, and sugar alcohols. ‘LB8-9’ was high in thymol, which is known for its strong antimicrobial activity against a panel of pathogenic bacteria. The metabolomic profiles of ‘Bingo’ were intensely different from the other mandarin hybrid, ‘LB8-9’, including a reduced thymol biosynthetic pathway and low amounts of most of the amino acids and sugar alcohols. Remarkably, 1,8-cineole (eucalyptol) was only detected in ‘Bingo’, indicating that eucalyptol could have feeding and ovipositional repellency against D. citri. The metabolite profiles generated for HLB-tolerant citrus species will improve the ability of citrus breeders and will allow them to take more informed decisions. Metabolomic profiling of HLB-tolerant citrus species could identify tolerance specific markers that can be introduced to other commercial citrus cultivars to improve their tolerance to HLB disease.

The genus Citrus, comprising some of the most widely cultivated fruit crops worldwide, includes an uncertain number of species. Here we describe ten natural citrus species, using genomic, phylogenetic and biogeographic analyses of 60 accessions representing diverse citrus germ plasms, and propose that citrus diversified during the late Miocene epoch through a rapid southeast Asian radiation that correlates with a marked weakening of the monsoons. A second radiation enabled by migration across the Wallace line gave rise to the Australian limes in the early Pliocene epoch. Further identification and analyses of hybrids and admixed genomes provides insights into the genealogy of major commercial cultivars of citrus. Among mandarins and sweet orange, we find an extensive network of relatedness that illuminates the domestication of these groups. Widespread pummelo admixture among these mandarins and its correlation with fruit size and acidity suggests a plausible role of pummelo introgression in the selection of palatable mandarins. This work provides a new evolutionary framework for the genus Citrus. The origin, evolution and domestication of Citrus and the genealogy of the most important wild and cultivated citrus varieties. Citrus fruits are one of the most cultivated crops worldwide, yet the evolutionary relationships among citrus species remain uncertain. Daniel Rokhsar, Manuel Talon and colleagues analyse the genomes of 60 accessions that represent a diverse range of citrus species, including 30 newly sequenced citrus genomes. They characterize the diversity and evolution of citrus at the species level and identify interspecific citrus hybrids and admixtures—genetic mixing between previously isolated populations—that could be the result of human activities such as migration and agriculture. The authors identify 10 progenitor species and suggest that citrus originated in southeast Asia, diversifying during the late Miocene epoch through a rapid southeast Asian radiation that correlated with a changing climate, including the weakening of the monsoons. They also find extensive relatedness among mandarins and sweet oranges, showing a complex history of admixture during the domestication of these groups.

Germplasm characterization is now possible and more reliable using improved molecular markers and genomic tools. Previously citrus cultivars were primarily described according to their morphological or horticultural traits, but trueness-to-type can now be confirmed using molecular markers, thereby limiting inadvertent cultivar misidentification. Following routine testing the reported parentage of two mandarin hybrids is now questioned. ‘Orri’ mandarin is derived from ‘Orah’ mandarin by induced mutation, and was reportedly bred by crossing ‘Temple’ tangor and ‘Dancy’ mandarin. ‘Orri’ has excellent organoleptic qualities, and other favorable traits. With ‘Dancy’ as the pollen parent, at least some degree of susceptibility of ‘Orri’ to Alternaria brown spot (ABS) might be expected, but it is more likely to have inherited a high degree of susceptibility; yet ‘Orri’ appears to be immune to ABS. SSR analysis provided evidence that excludes ‘Dancy’ as the pollen parent of ‘Orah’. Further testing revealed the likely pollen parent candidate as ‘Kinnow’ mandarin. ‘Fortune’ mandarin was a popular late-maturing mandarin cultivar until the high incidence of ABS rendered the cultivar uneconomical to produce in certain citrus production regions. This exceptionally late, high quality, attractive cultivar is reported to have originated from a ‘Clementine’ mandarin A— ‘Dancy’ cross. However, two independent molecular marker studies revealed that the pollen parent of ‘Fortune’ is possibly ‘Orlando’ tangelo and not ‘Dancy’. In both cases, the pollen parent of ‘Orri’ and ‘Fortune’ mandarin hybrids appears not to be ‘Dancy’ as published. This information will assist geneticists and breeders to re-interpret heritability studies on ABS susceptibility of mandarins and their hybrids.

A comprehensive citrus breeding and genetics program was established at the UF/CREC during the mid-1980s combining conventional breeding with tools provided by tissue culture, emerging biotechnologies, molecular genetics and genomics. Scion Improvement: The Florida citrus industry is 90% processing sweet oranges. A major goal has been to develop new cultivars for Not From Concentrate (NFC) juice production with emphasis on increasing juice quality and expanding seasonal availability. Improved clones from seedling introductions, budwood irradiation and somaclonal variation will be discussed. The fresh fruit industry in Florida is dominated by grapefruit and seedy mandarins. Primary objectives for fresh fruit improvement include expanding seasonal availability, development of canker tolerant and low furanocoumarin grapefruit, and development of seedless, easy-peel mandarins. The focus of our program has been on parent development, and seedless triploid production via interploid crosses; strategies and successes will be discussed. Rootstock Improvement: Initial focus was on improving soil adaptation, tree-size control to facilitate harvesting and cold-protection, and disease resistance, especially to blight, CTV-induced quick decline and Phytophthora. Breeding objectives have since been expanded to include tolerance of the Diaprepes/Phytophthora complex and Huanglongbing HLB, and development of rootstocks that facilitate advanced citrus production systems (ACPS). Progress from conventional breeding (both 2x and 4x) and somatic hybridization will be discussed. Genetic Engineering: Due to the HLB and canker epidemics, focus has been on disease resistance gene discovery, transformation, and evaluation of transgenic plants; progress will be discussed.

Background Flavor is an important attribute of mandarin (Citrus reticulata Blanco), but flavor improvement via conventional breeding is very challenging largely due to the complexity of the flavor components and traits. Many aroma associated volatiles of citrus fruit have been identified, which are directly related to flavor, but knowledge of genetic linkages and relevant genes for these volatiles, along with applicable markers potentially for expeditious and economical marker-assisted selection (MAS), is very limited. The objective of this project was to identify single nucleotide polymorphism (SNP) markers associated with these volatile traits. Result Aroma volatiles were investigated in two mandarin parents (‘Fortune’ and ‘Murcott’) and their 116 F1 progeny using gas chromatography mass spectrometry in 2012 and 2013. A total of 148 volatiles were detected, including one acid, 12 alcohols, 20 aldehydes, 14 esters, one furan, three aromatic hydrocarbons, 16 ketones, one phenol, 27 sesquiterpenes, 15 monoterpenes, and 38 unknowns. A total of 206 quantitative trait loci (QTLs) were identified for 94 volatile compounds using genotyping data generated from a 1536-SNP Illumina GoldenGate assay. In detail, 25 of the QTLs were consistent over more than two harvest times. Forty-one QTLs were identified for 17 aroma active compounds that included 18 sesquiterpenes and were mapped onto four genomic regions. Fifty QTLs were for 14 monoterpenes and mapped onto five genomic regions. Candidate genes for some QTLs were also identified. A QTL interval for monoterpenes and sesquiterpenes on linkage group 2 contained four genes: geranyl diphosphate synthase 1, terpene synthase 3, terpene synthase 4, and terpene synthase 14. Conclusions Some fruit aroma QTLs were identified and the candidate genes in the terpenoid biosynthetic pathway were found within the QTL intervals. These QTLs could lead to an efficient and feasible MAS approach to mandarin flavor improvement. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-4043-5) contains supplementary material, which is available to authorized users.

A targeted approach using HS-SPME-GC-MS was performed to investigate volatile compounds of ordinary Valencia (VAL) and its more deeply colored mutant Rohde Red Valencia orange (RRV) at different developmental stages. Fifty-six volatile components classified into six chemical groups were quantified. The individual volatile compounds in each group were significantly influenced by the genotype and harvest date. Cluster analysis suggested two principal clusters. Cluster I included fruits of VAL and RRV harvested in July, September, and November and was characterized by the 2-ethyl-1-hexanol acetate and linalool chemotype; cluster II included fruits harvested in January and March, with the valencene/β-myrcene chemotype. Principal component analysis confirmed that fruits of both cultivars harvested at different dates possessed a unique aroma active profile, especially, the odor-active volatile-norisoprenoids degraded from C40 carotenoid were significantly higher in RRV than in VAL at full maturity. These results could provide information for further study of the flavor metabolism pathways in the two closely related cultivars.

Salt tolerant citrus rootstocks are extremely important in the area with brackish water and soil. To identify salt tolerant accessions, two well-recognized tolerant and susceptible cultivars, Cleopatra mandarin and Troyer citrange, and 42 genetically diverse citrus accessions from Iran, were evaluated under three levels of sodium chloride (NaCl) including 0, 40 and 90 mM. Five genotypes named G25, G8, G34 (Cleopatra), G42 (Bergamot) and G44 (Sour orange) were the most salinity tolerant based on stress tolerance score. Salinity tolerance was highly correlated with chlorophyll fluorescence (r = 0.55**), Na+ concentration (r=-0.51**), ascorbate peroxidase (r = 0.56**) and leaf necrosis and loss (r=-0.64**). Iranian rootstocks, G8 and G44, were more tolerant than Cleopatra. These results showed that Iran is an appropriate gene pool for salinity tolerance in citrus. The findings of relative gene expression indicated that G44 had salinity tolerance alleles for SLAH, ABCT2 and ABCT3. Considering genotypic and phenotypic results, G44 was recognized as the most salinity tolerant rootstock.

Genetic traits in Poncirus trifoliata (Rutaceae) are known to confer resistance to colonization by the Asian citrus psyllid, but field surveys indicated these traits were not expressed in any of 24 simple hybrids between P. trifoliata and sweet orange. No resistance to colonization by the psyllid was observed in 14 other types of P. trifoliata hybrids. Three citrumelo hybrids appeared to be resistant in 1 survey, but in another survey, none of 6 citrumelos exhibited resistance. Interestingly, colonization was somewhat reduced on a hybrid with P. trifoliata in the pedigree of both parents, suggesting the possibility that traits suppressing oviposition may be recovered through similar crosses.

An efficient in vitro regeneration system through direct shoot organogenesis was established for Murraya paniculata (L.) Jack (Orange Jessamine). Epicotyls, leaves, roots, and cotyledons from in vitro-germinated seedlings and several plant growth regulators (PGRs) were evaluated for their effects on plant regeneration. Longitudinally cut epicotyl segments were observed to be the optimal explants followed by uncut epicotyls (not longitudinally cut). Roots, leaves, and cotyledons were not suitable as explants as a result of little or no shoot induction. Adventitious shoot induction was enhanced by the addition of 6-benzyladenine (BA). The highest percentage of shoot induction (87%) and the greatest number of shoots per explant (12.7) occurred on Murashige and Skoog (MS) medium supplemented with 15 μM BA from longitudinally cut epicotyls followed by 5.2 shoots per explant from uncut epicotyls. Optimal concentration of gibberellic acid (GA3) for shoot elongation was observed to be 15 μM. Eighty-five percent of the regenerated shoots produced roots with an average of three roots per shoot on MS medium supplemented with 5 μM indole-3-butyric acid (IBA). Our protocol for direct shoot organogenesis can potentially lead to the development of a robust method for production of transgenic plants of M. paniculata through Agrobacterium-mediated genetic transformation.

Two adjacent rootstock trials were conducted in the east coast Indian River region of Florida with ‘Marsh’ grapefruit (Citrus paradisi Macf.) scion. The objective was to find rootstocks to replace sour orange (C. aurantium L.) because of losses to citrus tristeza virus, and to replace Swingle citrumelo [C. paradisi × Poncirus trifoliata (L.) Raf.] because of its limited usefulness in certain poorly drained coastal sites. The trials were conducted in randomized complete blocks with 12 single-tree replicates spaced 4.6 × 6.9 m. The soils were of the Wabasso and Riviera series. The first trial consisted largely of trees on citrange [C. sinensis (L.) Osb. × P. trifoliata] and citrumelo rootstocks, ‘Cipo’ sweet orange (C. sinensis), and various hybrid rootstocks. The second trial involved mandarin rootstocks (C. reticulata Blanco) and sour orange and related rootstocks. Trees were grown for 7 years and yield and juice quality data were collected for the last 4 years of that period. Those rootstocks identified as the most promising, based on combinations of smaller tree size and high productivity and juice quality, were two Sunki mandarin × Swingle trifoliate orange (TF) hybrids (C-54, C-146), a Sunki mandarin × Flying Dragon TF hybrid, C-35 citrange, and a Cleopatra mandarin × Rubidoux TF hybrid (×639). The trees on these five rootstocks cropped well leading to soluble solids (SS) values of 3000 to 4000 kg/ha when they were 7-years old. The trees on C-54 and C-146 were relatively large, somewhat taller than trees on sour orange, whereas those on C-35 and the Sunki × Flying Dragon hybrid were smaller and similar to sour orange in tree height. Fruit quality among the trees on C-35 and the Sunki × Flying Dragon hybrid had relatively high SS concentration (better than sour orange), and the other three rootstocks had relatively lower solids concentration (poorer than sour orange). The trees on C-35 and the Sunki × Flying Dragon hybrid would be good candidates for higher density orchards.

This study examined the effects of plant growth regulators, explant types, and their orientations on in vitro shoot proliferation of Casuarina cunninghamiana Miq. and also the subsequent rooting ability of shoots. Results showed that shoot proliferation occurred only in shoot tip explants cultured vertically on Murashige and Skoog (MS) medium supplemented with 2 or 4 μM thidiazuron (TDZ). Neither 6-benzylaminopurine alone nor in a combination with 1-naphthalene acetic acid (NAA) or gibberellic acid had any effect on shoot proliferation. TDZ at 4 μM resulted in the greatest percentage of axillary bud sprouting (70%) and mean number of sprouts per explant (2.3). Additionally, no shoot proliferation was observed from detipped or single-node explants or from horizontally placed shoot tip explants when cultured on the same TDZ-containing medium. The induced shoots produced adventitious roots on MS medium supplemented with 2.5, 5, or 10 μM indole-3-butyric acid (IBA), not with indole-3-acetic acid and NAA. Although the mean number of roots per explant was not significantly different between 2.5 and 5 μM IBA, the highest rooting percentage (68%) and mean length of roots per explant (0.7 cm) was achieved at 5 μM IBA. The current study provided preliminary information toward commercial in vitro propagation of Casuarina cunninghamiana male plants.

Based on the former constructed ‘Tyr1’ locus genetic map in family 9145, from LB6-2 [Clementine mandarin (C. reticulata) × Hamlin orange (C. sinensis)] × Swingle citrumelo (C. paradise × P. trifoliata), 9 markers were chosen for application in evaluating their effectiveness in marker-assisted selection (MAS) for citrus rootstock breeding program from many F1 progeny of Poncirus trifoliata. As the mapping revealed that these markers were estimated within a range of 12.1 cM in the linkage group, and among them, SCO07 co-segregated with ‘Tyr1’, and 7A4R as the closest to ‘Tyr1’ with a distance of 1.5 cM, these markers were basically fitful to go MAS screening. The results of screening P. trifoliata F1 progeny indicated that all the markers were inherited in codominant fashion and most of them were heterozygous on PT (Pomery of P. trifoliata)., marker 4L17R/CfoI and 7A4(1407)/BfaI were proved to be consistently reliable for accurate scoring of genotypes and the revealed polymorphism was basically coincided with the citrus nematode resistant phenotype within tested populations. The polymorphic genotype with marker 4L17R/CfoI was found completely matched up with the phenotype of individuals that conferred high resistance to citrus nematode when the USDA hybrid rootstocks were screened. Utilization of these markers, especially the highly specific 4L17R/CfoI and 7A4(1407)/BfaI, should result in great benefit to world citrus industry for early selection in rootstock-breeding program.

An efficient protocol for colchicine mediated production of in vitro autotetetraploids from Ponkan mandarin using cell suspension cultures is described. Cells were treated with 1 g l−1 colchicine for 4 or 8 days before transfer into solid EME medium supplemented with 5% maltose. Colchicine treated cells were placed in medium with or without an overlay of 1:2 medium–mixture of liquid 0.6 M BH3 medium and 0.15 M EME + maltose liquid media. It was observed that modifying the immediate cell environment by addition of the liquid overlay played a positive role in cell differentiation and subsequent plant regeneration. Ploidy levels were determined with a flow cytometer and confirmed by chromosome staining using the enzymatic maceration method. A large number of non-chimeric autotetraploids were generated using this method. Such plants have great value in a breeding program for the development of seedless triploid citrus, as very few available tetraploid breeding parents are easy to peel.

Casuarina cunninghamiana Miq. is an introduced species to Florida that has potential as a windbreak plant to help manage canker in citrus groves; however, only Florida sources can be used for that purpose. Local sources of Casuarina are generally adequate seed producers, but germination percentages are frequently poor. Thus, the causes of low seed germination and methods to improve germination were investigated using C. cunninghamiana and a local hybrid (C. equisetifolia L. × C. glauca Sieb. ex Spreng.). Seeds of the hybrid were larger and heavier (88 mg/100 seeds) than those of C. cunninghamiana (mean wt. 67 mg/100 seeds). Shrunken, insect-damaged, and empty seeds, present in all unsorted seed lots, were responsible for poor seed germination of the four seed sources studied. Petroleum ether separation improved germination by dividing seeds into floaters and sinkers. The floater fraction consisted of 47.5% to 93% insect-damaged seeds compared with 9.0% to 43.5% among sinkers. More than 50% of the sinkers were filled seeds and less than 21% in floaters. No empty seeds were sinkers except for one source of C. cunninghamiana. In sorted hybrid seeds, petroleum ether separation eliminated a large proportion of ungerminable seeds (floaters) and seed germination among sinkers was faster with a higher germination percentage than floaters. Cumulative germination of hybrid seeds in a trial involving two temperatures was 23.0% for sunken seeds at 30 °C at the end of 8 weeks compared with 1% of unsorted seeds. Temperature had no significant effect on seed germination. The germination percentage of hybrid seeds with seedcoats removed was 91.0% in the first week of culture compared with only 1.2% in the first week and 12.6% seed germination at the end of 8 weeks' culture of intact seeds.

Experiments were conducted at 3 sites in Florida to determine whether landscape fabric, used as soil mulch, can reduce damage to citrus trees by Diaprepes abbreviates. The mulches were intended to prevent newly hatched weevil larvae from entering the soil to feed on roots and to prevent teneral adult weevils from exiting the soil to initiate egg-laying. The weight of aboveground parts of trees at a site heavily infested by D. abbreviatus on the east coast was 70% larger (P ≤ 0.05) for trees grown for 3 years on mulched compared to unmulched soil. Mulching did not affect the amount of feeding damage to roots at the east coast site, suggesting that mulched trees tolerated the damage better than trees in bare soil. Small plot size and relatively narrow fabric dimensions at the east coast site may have facilitated the entry of neonate larvae into soil. At a site on the Central Ridge with low weevil population density, average trunk cross-sectional area of 5-year-old mulched trees was 31% greater (P

A suitable protocol for micropropagation of Casuarina hybrid, Casuarina equisetifolia L. × Casuarina glauca Sieber ex Spreng (C. e. × C. g.), was developed. When seeds without seed coats were cultured on 4 germination media, the optimal seed germination percentage (91%) was obtained on 0.8% agar solidified water medium. Shoot multiplication was achieved by culturing 2-cm long epicotyls, excised from germinated seedlings, on MS (Murashige and Skoog 1962) basal medium supplemented with BA (6-benzylaminopurine) at 4.4, 8.8, 17.8 and 35.6 μM. The greatest percentage of axillary bud sproutings (87.5%), mean number of sprouts per explant (3.8), and shoot length (3.2 cm) were achieved on MS medium supplemented with 17.8 μM BA. MS medium supplemented with 4 different concentrations of IBA (indole-3-butyric acid) (4.3, 8.7, 13.0 and 17.4 μM) were used for rooting of in vitro grown shoots. The highest rooting percentage (65.6%), mean number of roots per explant (2.5) and mean length of roots per explant (1.6 cm) was achieved at 13.0 μM IBA. Rooted shoots grew well after transfer to a substrate of peat and pinebark (7:3) in the greenhouse.

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.

Seeds of Coreopsis leavenworthii Torr. & Gray (Asteraceae) are being commercially produced but the lack of genetic diversity information has hindered growers and end users from addressing several critical issues affecting wild collection, commercial production, distribution, and the use of seeds. In this study, the genetic diversity and differentiation among natural, production, and introduced populations were analyzed at the molecular level using 320 amplified fragment length polymorphism (AFLP) markers. A high level of diversity [68.6% average polymorphism; total genetic diversity (H t ) = 0.309] and a moderate level of genetic differentiation [total genetic diversity residing among populations (G st ) = 0.226; Φ st = 0.244; Bayesian analog of Nei's G st (G st -B) = 0.197] was detected among six natural populations—two each from northern, central, and southern Florida. Two distance-based clustering analyses, based on an individual's AFLP phenotypes or a population's allele frequencies, grouped natural populations into three clusters, concordant with our previous results from a common garden study of phenotypic variation. Clustering of populations was mostly according to their respective geographical origin within Florida. The correlation between geographical distances and pairwise F st values between populations was very significant (r = 0.855, P < 0.0001). Two central Florida natural populations were divergent and grouped into separate clusters, indicating that the existence of factors other than physical distance alone were contributing to genetic isolation. Three production populations maintained a level of genetic diversity comparable to that in the natural populations and were grouped with the natural populations from which the production populations were derived, suggesting that the genetic identity of the seed origin was maintained under production practices. The genetic diversity of the introduced population was comparable to that of the source populations (central Florida natural populations), but genetic shift seems to have occurred, causing the introduced population to cluster with local (northern Florida) populations where planted. The observed genetic differentiation among natural populations may indicate a need to develop appropriate zones within Florida for preservation of genetic diversity during seed collection, increase, and distribution. This high level of population differentiation also suggests a need to collect and analyze more natural populations across Florida and from Alabama for a better understanding of the species' genetic diversity and population structure across its distribution range.

A total of 293 putative citrange F 1 seedlings, derived from 13 crosses and visually identified by their trifoliate leaf morphology, were investigated using six characterized expressed sequence tag (EST) microsatellite primers. Their origins and ploidy levels were simultaneously and unambiguously determined by these markers. Besides a majority of diploid zygotic hybrids, as high as 24% triploid hybrids were produced, only by Citrus sinensis (sweet orange) × Poncirus trifoliata (trifoliate orange), but not the reciprocal crosses. The results indicated that 2 n eggs originated only from the maternal sweet orange parent, contributing the extra haploid genome in the triploids. It was further inferred that the 2 n eggs in sweet orange result from first division restitution (FDR), not second division restitution (SDR), according to the marker genotypes of these triploids. High percentages of self-cross and nucellar origin seedlings were found only from P. trifoliata × C. sinensis , but not from the reciprocals, simply due to their different reliabilities in the visual leaf identification of the seedlings. Survey of leaves indicated that adult triploid seedlings had predominantly monofoliate leaves, compared to diploid hybrids with mostly trifoliate leaves. The possible genetic mechanism and potential applications were discussed.

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.

The family of aquaporins (AQPs), or major intrinsic proteins (MIPs), includes integral membrane proteins that function as transmembrane channels for water and other small molecules of physiological significance. MIPs are classified into five subfamilies in higher plants, including plasma membrane (PIPs), tonoplast (TIPs), NOD26-like (NIPs), small basic (SIPs) and unclassified X (XIPs) intrinsic proteins. This study reports a genome-wide survey of MIP encoding genes in sweet orange (Citrus sinensis L. Osb.), the most widely cultivated Citrus spp. A total of 34 different genes encoding C. sinensis MIPs (CsMIPs) were identified and assigned into five subfamilies (CsPIPs, CsTIPs, CsNIPs, CsSIPs and CsXIPs) based on sequence analysis and also on their phylogenetic relationships with clearly classified MIPs of Arabidopsis thaliana. Analysis of key amino acid residues allowed the assessment of the substrate specificity of each CsMIP. Gene structure analysis revealed that the CsMIPs possess an exon-intron organization that is highly conserved within each subfamily. CsMIP loci were precisely mapped on every sweet orange chromosome, indicating a wide distribution of the gene family in the sweet orange genome. Investigation of their expression patterns in different tissues and upon drought and salt stress treatments, as well as with 'Candidatus Liberibacter asiaticus' infection, revealed a tissue-specific and coordinated regulation of the different CsMIP isoforms, consistent with the organization of the stress-responsive cis-acting regulatory elements observed in their promoter regions. A special role in regulating the flow of water and nutrients is proposed for CsTIPs and CsXIPs during drought stress, and for most CsMIPs during salt stress and the development of HLB disease. These results provide a valuable reference for further exploration of the CsMIPs functions and applications to the genetic improvement of both abiotic and biotic stress tolerance in citrus.

The inheritance of resistance to a host-specific isolate (Shinn) of Alternaria alternata (Fr.:Fr.) Keissl. from `Minneola' tangelo (a cross between Citrus paradisi Macf. `Duncan' and C. reticulata Blanco `Dancy') was shown to be controlled by a single recessive allele, aaM1, within the citrus genome. A backcross between resistant `Clementine' mandarin (C. reticulata) and susceptible LB#8-10 (a hybrid of `Clementine' mandarin and `Minneola' tangelo) resulted in 61 resistant (R) and 58 susceptible (S) plants (χ2 = 0.0756, P ≥ 0.05), but the reciprocal cross deviated from the expected 1R:1S ratio (87 R and 36 S plants (χ2 = 21.1463, P ≥ 0.05). A dominant allele, AaM1, of this resistance gene was found in a loose coupling phase linkage with two RAPD markers, P12850 (15.3 cM) and AL31250 (36.7 cM), after JOINMAP computer analysis.

SummaryProtoplast fusion has been an effective tool with several applications for citrus cultivar improvement. The evaluation of fruit characteristics of certain previously produced citrus somatic hybrids with ‘Page’ tangelo or ‘Murcott’ tangor as parents, has shown that some allotetraploids produce high quality fruit with direct fresh-fruit cultivar potential. Based on this information, additional somatic hybrids combining these parents with other high quality scions were produced as follows: ‘Murcott’ tangor + ‘Dancy’ tangerine, ‘Murcott’ + LB8-8 (‘Clementine’ ‘Minneola’), ‘Page’ tangelo + ‘Murcott’, ‘Page’ + LB8-9 (‘Clementine’ ‘Minneola’), ‘Page’+ (‘Clementine’ ‘Satsuma’), ‘Page’ + Orie Lee hybrid (‘Clementine’ ‘Murcott’), and ‘Page’ + ‘Ortanique’ tangor, respectively. Their tetraploid level was confirmed by flow cytometry, and their hybridity was verified by random amplified polymorphic DNA (RAPD) or simple sequence repeat (SSR) analysis. Polymerase chain reaction - restriction fragment length polymo...

Epicotyl explants of sweet orange and citrange were infected with Agrobacterium strain EHA101 harboring binary vector pGA482GG, and factors affecting the plant regeneration and transformation efficiency were evaluated. Increasing the wounded area of explants by cutting longitudinally into two halves, and optimization of inoculation density, dramatically enhanced both regeneration and transformation frequency. Inclusion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the explant pretreatment medium and the co-culture medium improved the transformation efficiency by decreasing the escape frequency. More than 90% rooting frequency of transformed citrange shoots was achieved by two-step culture: first on media supplemented with auxins, and then on media without hormones. Inclusion of 20 mg l−1 kanamycin in rooting medium efficiently discriminated transformed shoots from non-transgenic escaped shoots. Shoot grafting in vitro was used to regenerate transformed plants, due to the slow growth of most sweet orange shoots.

A BAC library was constructed from the genomic DNA of an intergeneric Citrus and Poncirus hybrid. The library consists of 24,576 clones with an average insert size of 115 kb, representing approximately seven haploid genome equivalents and is able to give a greater than 99% probability of isolating single-copy citrus DNA sequences from this library. High-density colony hybridization-based library screening was performed using DNA markers linked to the citrus tristeza virus (CTV) resistance gene and citrus disease resistance gene candidate (RGC) sequences. Between four and eight clones were isolated with each of the CTV resistance gene-linked markers, which agrees with the library’s predicted genome coverage. Three hundred and twenty-two clones were identified using 13 previously cloned citrus RGC sequences as probes in library screening. One to four fragments in each BAC were shown to hybridize with RGC sequences. One hundred and nine of the RGC BAC clones were fingerprinted using a sequencing gel-based procedure. From the fingerprints, 25 contigs were assembled, each having a size of 120–250 kb and consisting of 2–11 clones. These results indicate that the library is a useful resource for BAC contig construction and molecular isolation of disease resistance genes.

Numerous disease resistance gene-like DNA sequences were cloned from an intergeneric hybrid of Poncirus and Citrus, using a PCR approach with degenerate primers designed from conserved NBS (nucleotide-binding site) motifs found in a number of plant resistance genes. Most of the cloned genomic sequences could be translated into polypeptides without stop codons, and the sequences contained the characteristic motifs found in the NBS-LRR class of plant disease resistance genes. Pairwise comparisons of these polypeptide sequences indicated that they shared various degrees of amino-acid identity and could be grouped into ten classes (RGC1–RGC10). When the sequences of each class were compared with known resistance-gene sequences, the percentage of amino-acid identity ranged from 18.6% to 48%. To facilitate genetic mapping of these sequences and to assess their potential linkage relationship with disease resistance genes in Poncirus, we developed CAPS markers by designing specific primers based on the cloned DNA sequences and subsequently identifying restriction enzymes that revealed genetic polymorphisms. Three of the amplified DNA fragment markers (designated as 18P33a, Pt9a, and Pt8a) were associated with the citrus tristeza virus resistance gene (Ctv), and one fragment (Pt8a) was associated with the major gene responsible for the citrus nematode resistance (Tyr1); both genes are from Poncirus and of importance to citrus survival and production. These polymorphic fragments were located on two local genetic linkage maps of the chromosome region from Ctv to Tyr1. These results indicate that resistance-gene candidate sequences amplified with the NBS-derived degenerate primers are valuable sources for developing markers in disease resistance-gene tagging, mapping, and cloning.

Eleven RAPD markers linked to a gene region conferring resistance to citrus nematodes in an intergen-eric backcross family were identified. Two sequence- characterized amplified region markers linked to a citrus tristeza virus resistance gene and one selected resistance gene candidate marker were evaluated for their association with citrus nematode resistance. A nematode-susceptible citrus hybrid, LB6-2 [Clementine mandarin (Citrus reticulata)×Hamlin orange (C. sinensis)], was crossed with the citrus nematode-resistant hybrid Swingle citrumelo (C. paradisi×Poncirus trifoliata) to produce 62 hybrids that were reproduced by rooted cuttings. The plants were grown in a greenhouse and inoculated with nematodes isolated from infected field trees. The hybrids segregated widely for this trait in a continuous distribution, suggesting possible polygenic control of the resistance. Bulked segregant analysis was used to identify markers associated with resistance by bulking DNA samples from individuals at the phenotypic distribution extremes. Linkage relationships were established by the inheritance of the markers in the entire population. A single major gene region that contributes to nematode resistance was identified. The resistance was inherited in this backcross family from the grandparent Poncirus trifoliata as a single dominant gene. QTL analysis revealed that 53.6% of the phenotypic variance was explained by this major gene region. The existence of other resistance-associated loci was suggested by the continuous phenotypic distribution and the fact that some moderately susceptible hybrids possessed the resistance-linked markers. The markers may be useful in citrus rootstock breeding programs if it can be demonstrated that they are valid in other genetic backgrounds.

Genetic differentiation among six Florida populations of Diaprepes abbreviatus (L.) was determined using protein and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers. Proteins were separated by electrophoresis and stained with silver stain and for α-naphthylacetate esterase activity. No differentiation was observed among populations when egg proteins were silver stained: α-naphthylacetate esterase activity differentiated five of the six populations. RAPD-PCR data showed significant differentiation among populations, consistent with the hypothesis of three independent introductions of D. abbreviatus into Florida. Our data indicate that D. abbreviatus populations, once introduced, have generally remained in one locality with limited dispersal to new areas.

Restriction fragment length polymorphisms (RFLPs), were used to analyze the organelle composition of two-year-old trees, recovered from two different experiments: protoplasts from embryogenic cell suspensions of `Succari' sweet orange (C. sinensis L. Osbeck) were fused with leaf protoplasts of Citropsis gilletiana Swingle & M. Kell or to leaf protoplasts of Atalantia ceylanica(Arn.) Oliv. The somatic hybrids of both fusion combinations had the mitochondrial genome from the embryogenic partner. In some somatic hybrids, non-parental fragments were observed among the mitochondrial patterns. Somatic hybrids between `Succari' + Atalantia had plastid DNA from the embryogenic parent, while the somatic hybrids of `Succari' + Citropsis all had both parental chloroplast genomes. The relative abundance of organelle DNAs in the donor embryogenic and leaf cells may explain the consistent transmission of the embryogenic parent mitochondrial DNA and the inheritance of the chloroplast genome from either parent.

[EN] Field evaluations have shown that Satsuma mandarin (Citrus unshiu) ‘Okitsu’ is one of the mandarin cultivars that shows substantial resistance to Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus bacterial canker disease. However, the mechanisms underlying this resistance are not well understood. In this study, we have shown that ‘Okitsu’ leaves are nevertheless susceptible to X. citri infection during a period of their development; however, this period is shorter than that seen in the susceptible mandarin ‘Clemenules’ (C. clementina). Under controlled growth conditions, the resistance of ‘Okitsu’ to X. citri was associated with the age of the leaf and was evident in spray-inoculated plants but not in those inoculated by infiltration. Furthermore, X. citri showed reduced attachment and biofilm formation in ‘Okitsu’ leaves compared with ‘Clemenules’. Taken together, our data suggest that structural features of the ‘Okitsu’ leaf surface, such as the physical properties of the cuticle, are involved in the resistance to X. citri., This work was principally supported by the Agencia Nacional de Promocion Cientifica y Tecnologica PICT-2011-1833 to M. R. Marano and by a grant from the Florida Citrus Research and Development Foundation to F. G. Gmitter, Jr. and M. R. Marano. M. A. Chiesa, A. A. Vojnov, A. P. Castagnaro, and M. R. Marano are Career Investigators of CONICET. We thank R Vena for his technical assistance with the confocal microscopy and J. M. Dow and G. Gudesblat for critical review of the manuscript.

Somatic hybridization experiments in Citrus that involve the fusion of protoplasts of one parent isolated from either nucellus-derived embryogenic callus or suspension cultures with leaf-derived protoplasts of a second parent, often result in the regeneration of diploid plants that phenotypically resemble the leaf parent. In this study, plants of this type regenerated following somatic fusions of the following three parental combinations were analyzed to determine their genetic origin (nuclear and organelle): (embryogenic parent listed first, leaf parent second) (1) calamondin (C. microcarpa Bunge) + 'Keen' sour orange (C. aurantium L.), (2) Cleopatra mandarin (C. reticulata Blanco) + sour orange, and (3) 'Valencia' sweet orange (C. sinensis (L.) Osbeck) + 'Femminello' lemon (C. limon (L.) Burm. f.). Isozyme analyses of PGI, PGM, GOT, and IDH zymograms of putative cybrid plants, along with RFLP analyses using a nuclear genome-specific probe showed that these plants contained the nucleus of the leaf parent. RFLP analyses using mtDNA-specific probes showed that these plants contained the mitochondrial genome of the embryogenic callus donor, thereby confirming cybridization. RFLP analyses using cpDNA-specific probes revealed that the cybrid plants contained the chloroplast genome of either one or the other parent. These results support previous reports indicating that acquisition of the mitochondria of embryogenic protoplasts by leaf protoplasts is a prerequisite for recovering plants with the leaf parent phenotype via somatic embryogenesis following somatic fusion.

We examined the relationship between seed size and shape in Citrus and the number and type of seedlings produced by individual seeds for each of three citrus cultivars. Seed size and shape were related to the number of seedlings produced and the likelihood of recovering a zygotic seedling. The relationship between seed size and shape and the likelihood of recovering a zygotic seedling most often was connected with weight and thickness of a seed. This relationship might be of sufficient strength to use in some aspects of cultivar development. However, the relationship did not appear strong enough to be of practical value for application in commercial production of purely nucellar rootstock seedlings.

BACKGROUND: Tangerines have a distinct flavor among citrus fruit. However, information on tangerine volatiles remains limited. Volatile compounds from a breeding population of tangerines were earlier identified by gas chromatography–mass spectrometry. In this study, five hybrids with a distinct volatile profile were analyzed by gas-chromatography–olfactometry (GC-O) and descriptive sensory analysis. RESULTS: Forty-nine aroma active compounds were found in a consensus by GC-O. Aldehydes were the most important group with odor activity, as well as monoterpenes, esters, alcohols and ketones. 1,8-Cineole, β-myrcene, (E,E)-2,4-nonadienal, hexanal, ethyl-2-methylbutanoate, and linalool were perceived with high intensity in most samples. Two ‘Clementine’ × ‘Minneola’ and one ‘Fortune’ × ‘Murcott’ hybrids with tangerine, sulfury and woody/spicy flavors had aroma active compounds with terpeney, fatty/vegetable and metallic/rubber descriptors. A tangerine with ‘Valencia’ orange in its parentage had a characteristic orange flavor, which could be explained by esters and ketones, high in fruity and floral odor intensities. A hybrid of unknown origin had a distinct fruity–non-citrus and pumpkin/fatty flavor; that sample had the lowest amount of aroma-active volatiles, with the least compounds with terpeney odors. CONCLUSION: There was no one compound characteristic of tangerine flavor. Nevertheless, each sample sensory characteristic could be explained by a set of aroma-active volatile compounds. Published 2011 by John Wiley & Sons, Ltd.

Protoplast fusion using polyethylene glycol (PEG) was conducted to combine Citrus sinensis (L.) Osbeck cv. 'Hamlin' sweet orange protoplasts, isolated from nucellus-derived embryogenic callus with Atalantia ceylanica (Arn.) Oliv, leaf protoplasts. Five plants regenerated from independent fusion events following protoplast culture were identified as intergeneric allotetraploid somatic hybrids of 'Hamlin' sweet orange and A. ceylanica, and confirmed by isozyme analysis and chromosome number determination in root tip cells (2n=4x=36). Two different types of leaf morphology were observed among the hybrids (normal and narrow), although no differences in chromosome number nor isozyme banding patterns were observed. This is the first report of the production of hybrid plants between these sexually incompatible genera.

BACKGROUND: With the desirable combination of sugars and acids, volatile compounds contribute to the essential organoleptic attributes of citrus. This study evaluated the aroma volatiles of 20 tangerine hybrids of the University of Florida breeding program. Volatiles were sampled from hand-squeezed juice by headspace solid-phase microextraction (SPME), and analyzed by gas chromatography–mass spectrometry. Principal component analysis (PCA) and cluster analysis (CA) were used to find similarities among samples due to volatile composition with effect of genetic background. RESULTS: In total, 203 volatiles were detected in all samples. Volatiles in lower amounts were widely distributed among samples and were classified mainly as terpene hydrocarbons and oxygenated compounds, such as aldehydes, esters, alcohols and ketones. PCA, based on relative peak areas (content) clearly separated the samples higher in volatile content, mainly those with sweet orange genetic contributions in their background. CA, based on volatile presence/absence, grouped samples into five clusters, each showing distinctive volatile profiles. CONCLUSION: The genetic background of tangerine hybrids affected volatile composition and content of samples. In general, tangerines were characterized by fewer volatiles (in both quality and quantity) and more aldehydes, and hybrids with sweet orange in their background had more sesquiterpenes and esters, which would likely affect their aroma. Published 2010 by John Wiley & Sons, Ltd.

The NBS-LRR class resistance-gene candidate sequences (Pt8a and Pt9a) were used to develop new specific markers to the citrus nematode resistance gene locus Tyr1 . By high-density colony screening, over 200 positive clones were pulled out from the BAC library. A few of the clones were found to be closely linked with the Tyr1 region, because the primers from these clones insert sequence produced polymorphism which matched up with the phenotype after bulked segregant analysis. By primer walking approach, three integrate NBS-LRR class resistance-gene sequences were tagged and identified separately in three clones (7A4, 4L17 and 29F20). More specific markers were developed from these tagged sequences and relatively high-density genetic maps were constructed by incorporating the newly developed markers and previously developed markers in the ‘9145 family’. New markers were applied in ‘9401 family’ trying to estimate roughly the genetic distance between the Ctv and Tyr1 region.