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Citrus fruit quality is defined as the combination of physical and chemical traits; some of which may change during the ripening phase, e.g., acidity and sugar content. A clear understanding of their genetic control would be very helpful for marker-assisted breeding programs especially with regard to the juvenile phase and some reproductive features that hamper the selection of improved hybrids. A genetic study was thus performed on the heredity of quality traits and QTL detection based on segregation in a progeny generated from a cross between clementine cv “Commun” (Citrus × reticulata cv clementine) and mandarin cv “Willow leaf” (C. reticulata Blanco). Parental and consensus genetic linkage maps were constructed using 645 SNP and SSR markers. These maps were represented by 10 linkage groups in clementine and 12 linkage groups in mandarin, representing 75% and 58% respectively of the previously published clementine reference map. A total of 16 traits, including fruit mass, equatorial diameter, juice percentage, total soluble solids, acidity, pH, glucose, fructose, sucrose, and citric and malic acid concentrations were evaluated at three maturation dates. High variations indicating transgressive segregation were found for all traits, with normal or close to normal distributions. QTL analysis performed using the multiple QTL model allowed the detection of 34 QTLs on the three maps. QTLs were distributed in different linkage groups and generally detected at only one date of the ripening phase. The percentage of total variation explained ranged from 12 to 37% per QTL. Major QTLs (R2 ≥ 30%) were detected for equatorial diameter, glucose, and fructose (expressed in percentage dry matter) on linkage groups 8 and 9. Co-localization of QTLs controlling correlated and uncorrelated traits were mainly found on linkage groups 2, 4, 8, and 9, particularly between fruit mass and acidity.

International audience; Ichang lemon is a citrus fruit whose rind gives off a delicious and much appreciated fragrance and flavor. The volatile components of the fruit peel of Ichang lemon were investigated by GC-MS and GC-O (AEDA method). Simultaneously, its genetic origin was identified by using diagnostic SNP markers specific to ancestral species and multiallelic SSR and InDel markers. Ichang lemon combines three ancestral genomes (Citrus maxima, Citrus ichangensis, and Citrus reticulata) and may be a pummelo x Yuzu hybrid. Although the major compounds of the Ichang lemon aromatic profile were present in Citrus junos, a few pummelo-specific compounds were also detected, such as indole and nootkatone, in agreement with its maternal lineage. 3-Methyl-3-sulfanylbutyl acetate, reported to occur in passion fruit and brewed coffee, was identified by GC-MS, GC-QTOF-MS, and GC-FTIR for the first time in citrus. This odor-active compound has a sulfurous, tropical fruity, green note.

Citrus phylogeny is currently based on genome analysis using molecular markers and sequencing. The 7 pure genetic groups that gave rise to all cultivated citrus underlie the diversity of citrus accessions originating from Asia. However, there are wild citrus forms whose phylogenetic position is unknown, such as mountain citron (Citrus halimii B.C. Stone) that was discovered in Malaysia in the early 1970s. We sought to elucidate its status by determining its genetic profile with 30 SSR and InDel markers distributed on the 9 chromosomes of the citrus reference genome as compared to those of the 7 pure genetic groups represented by 4 or 5 varieties each. The genetic study was supplemented by a comparison of the composition of essential oils obtained by fruit peel and leaf hydrodistillation to those of the citrus fruits used for genotyping. The genetic study demonstrated that C. halimii is not an interspecific hybrid (low heterozygosity) but rather a true species that shares a common ancestor with kumquats (Fortunella sp.), which would have evolved separately. The fruit aromatic profiles confirmed this kumquat/mountain citron relationship but also highlighted the uniqueness of C. halimii due to the presence of high proportions of compounds that have never been observed in other citrus fruits, such as germacrene d-8-one (accounting for 8.7% of the leaf essential oil).

The Papeda&nbsp, Citrus subgenus includes several species belonging to two genetically distinct groups, containing mostly little-exploited wild forms of citrus. However, little is known about the potentially large and novel aromatic diversity contained in these wild citruses. In this study, we characterized and compared the essential oils obtained from peels and leaves from representatives of both Papeda groups, and three related hybrids. Using a combination of GC, GC-MS, and 13C-NMR spectrometry, we identified a total of 60 compounds in peel oils (PO), and 76 compounds in leaf oils (LO). Limonene was the major component in almost all citrus PO, except for C. micrantha and C. hystrix, where β-pinene dominated (around 35%). LO composition was more variable, with different major compounds among almost all samples, except for two citrus pairs: C. micrantha/C. hystrix and two accessions of C. ichangensis. In hybrid relatives, the profiles were largely consistent with their Citrus/Papeda parental lineage. This high chemical diversity, not only among the sections of the subgenus Papeda, but also between species and even at the intraspecific level, suggests that Papeda may be an important source of aroma diversity for future experimental crosses with field crop species.

Orange fruit abscission usually occurs at the final stage of fruit maturation but in some areas of citrus production, in advance of the usual harvest period, and sometimes suddenly and intensely. The reasons for this precocious citrus fruit abscission remains unclear. Therefore, the aim of this study was to try to clarify what the determinants of this phenomenon are. A multi-site experimentation was carried out on six orange cultivars, in Corsica, Spain and Tunisia where the phenomenon of early massive fruit drop varies. Climatic parameters, fruit maturity parameters and fruit detachment force (FDF) were recorded along the fruit maturation period. Respectively to the fruit drop, the FDF decreased in Tunisia and in Spain until the fruit falls, whereas in Corsica, it remained relatively constant throughout fruit maturation. Although data on fruit maturity parameters (rind color, acidity and total soluble solids) differed at the three sites, their evolution was similar during the period of maturation. FDF was not related to changes in any fruit maturity parameters, and more likely depended on changes in temperatures on days when the mean temperature was above 13 &deg, C. Massive fruit abscission could be linked to the earlier more rapid restart of vegetative growth in Tunisia and Spain than in Corsica.

Gene banks have been established to conserve the genetic diversity of crop species. Large germplasm collections lead to management problems (space, maintenance costs, etc.), especially in collections involving species with recalcitrant seeds that must be maintained as growing plants. Core collections (CCs) are thus developed to reduce the size of large germplasm collections while keeping the maximum variability. This also facilitates fine phenotypic evaluation. In this study, several software packages (DARwin, PowerMarker and MSTRAT) and methods (Max length subtree, M strategy, simulated annealing and MinSD) were compared to define a mandarin (Citrus reticulata) CC. One hundred and sixty-seven accessions were sampled from two germplasm collections, which were genotyped with 50 SSR, 24 InDel and 68 single nucleotide polymorphism markers. All the CC obtained were tested for the maintenance of the genetic variability parameters (Ho and He) of the initial collection, the level of linkage disequilibrium (LD) and the phenotypic diversity retention. The Max length subtree function from DARWin seemed to be the most appropriate method for establishing a CC in C. reticulata. It maintained 96.82% of the allelic richness and 17.96% of the size of the initial collection with only 30 accessions. Besides it did not increase the LD (r2 value) of the initial collection and retained the vast majority of the phenotypic variability. However, a CC with 70 accessions would be more helpful for genetic association studies. (Resume d'auteur)

International audience; Plants require essential minerals for their growth and development that are mainly acquired from soil by their roots. Nutrient deficiency is an environmental stress that can seriously affect fruit production and quality. In citrus crops, rootstock/scion combinations are frequently employed to enhance tolerance to various abiotic stresses. These tolerances can be improved in doubled diploid genotypes. The aim of this work was to compare the impact of nutrient deficiency on the physiological and biochemical response of diploid (2x) and doubled diploid (4x) citrus seedlings: Volkamer lemon, Trifoliate orange ´ Cleopatra mandarin hybrid, Carrizo citrange, Citrumelo 4475. Flhorag1 (Poncirus trifoliata + and willow leaf mandarin), an allotetraploid somatic hybrid, was also included in this study. Our results showed that depending on the genotype, macronutrient and micronutrient deficiency affected certain physiological traits and oxidative metabolism differently. Tetraploid genotypes, mainly Flhorag1 and Citrumelo 4475, appeared resistant compared to the other genotypes as indicated by the lesser decrease in photosynthetic parameters (Pnet, Fv/Fm and Gs) and the lower accumulation of oxidative markers (MDA and H202) in roots and leaves, especially after long-term nutrient deficiency. Their higher tolerance to nutrient deficiency could be explained by better activation of their antioxidant system. For the other genotypes, tetraploidization did not induce greater tolerance to nutrient deficiency.

The nutraceutical properties of extra-virgin olive oil (EVOO) can be further improved by the addition of olive leaves during olive pressing. However, while Citrus leaves are rich sources of bioactive substances, no data are available in the literature about the effect of Citrus leaf addition on the nutraceutical and sensorial profiles of olive oil. This study aimed at comparing the chemical and sensorial qualities of olive oils obtained from ripe olives pressed together with either Olea or Citrus spp. (lemon or orange) cryomacerated leaves. General composition parameters as well as major antioxidants and antioxidant activity were measured. A panel test evaluation, as well as headspace volatile characterization (headspace solid phase microextraction, HS-SPME), were also performed. All data were compared with an EVOO extracted from the same olive batch used as control. It was possible to obtain Leaf Olive Oils (LOOs) characterized by a higher (p &lt, 0.05) content of antioxidants, compared to the control sample, and the highest oleuropein concentration was detected in the olive oil extracted in presence of olive leaf (+50% in comparison with the control). All the LOOs showed a higher smell complexity and the scent of ripe fruit was generally mitigated. Lemon and olive LOOs showed the best smell profile.

International audience; Nutrient deficiency has economic and ecological repercussions for citrus fruit crops worldwide. Citrus crops rely on fertilization to maintain good fruit output and quality, whereas new crop management policy aims to reduce fertilizers input. New rootstocks are needed to meet to this constraint, and the use of new tetraploid rootstocks better adapted to lower nutrient intake could offer a promising way forward. Here we compared physiological, biochemical and anatomic traits of leaves in diploid (2x) and doubled-diploid (4x) Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) and Volkamer lemon (Citrus limonia Osb.) seedlings over 7 months of nutrient deficiency. Photosynthetic parameters (Pnet, Gs and Fv/Fm) decreased, but to a lesser extent in 4x genotypes than 2x. Degradation of the ultrastructural organelles (chloroplasts and mitochondria) and compound cells (thylakoids and starches) was also lower in 4x genotypes, suggesting that tetraploidy may enhance tolerance to nutrient deficiency. However, leaf surface (stomata, stomatal density and epithelial cells) showed no nutrient deficiency-induced change. In 4x Citrumelo 4475, the higher tolerance to nutrient deficiency was associated with a lower MDA and H2O2 accumulation than in the 2x, suggesting a more efficient antioxidant system in the 4x genotype. However, few differences in antioxidant system and oxidative status were observed between 2x and 4x Volkamer lemons.

Sour oranges (Citrus aurantium L.) are well known in the processing and cosmetics industries for the aromatic properties of their essential oils. Intercultivar genetic and aromatic diversity is not well documented. The objective of this study was to evaluate the impact of morphological selection and genetic mechanisms of varietal diversification (mutation or hybridization) on the aromatic and odor variability of sour orange essential oils. Forty-five sour orange accessions from INRAE-CIRAD citrus Biological Resources Center (France) were assessed for ten simple sequence repeat (SSR) and 54 single nucleotide polymorphism (SNP) markers, nine morphochemical fruit traits and with the aromatic components of leaf and peel essential oils. Thirty-nine sour oranges displayed no intercultivar molecular polymorphism and six genotypes originated from interspecific hybridizations involving sour orange, citron, pummelo or mandarin. The peel essential oil (PEO) diversity was low, in accordance with the genetic diversity. The predominance of limonene (&gt, 90%) prevents any possible correlation to be made between the composition and the variation in sensory profiles detected by panelists. Few compounds in the leaf essential oil (LEO), such as linalool, linalyl acetate, α-terpineol and geraniol were significantly different across sour oranges varieties. The morphological fruit attributes mainly used in varietal selection differed highly across the main genetically identical group of sour orange accessions. These results confirm that mutation can generate variability in aromatic compounds and aromas and that their exploitation requires an improvement in characterization processes.

Background and Aims The origin of limes and lemons has been a source of conflicting taxonomic opinions. Biochemical studies, numerical taxonomy and recent molecular studies suggested that cultivated Citrus species result from interspecific hybridization between four basic taxa (C. reticulata, C. maxima, C. medica and C. micrantha). However, the origin of most lemons and limes remains controversial or unknown. The aim of this study was to perform extended analyses of the diversity, genetic structure and origin of limes and lemons. Methods The study was based on 133 Citrus accessions. It combined maternal phylogeny studies based on mitochondrial and chloroplastic markers, and nuclear structure analysis based on the evaluation of ploidy level and the use of 123 markers, including 73 basic taxa diagnostic single nucleotide polymorphism (SNP) and indel markers. Key Results The lime and lemon horticultural group appears to be highly polymorphic, with diploid, triploid and tetraploid varieties, and to result from many independent reticulation events which defined the sub-groups. Maternal phylogeny involves four cytoplasmic types out of the six encountered in the Citrus genus. All lime and lemon accessions were highly heterozygous, with interspecific admixture of two, three and even the four ancestral taxa genomes. Molecular polymorphism between varieties of the same sub-group was very low. Conclusions Citrus medica contributed to all limes and lemons and was the direct male parent for the main sub-groups in combination with C. micrantha or close papeda species (for C. aurata, C. excelsa, C. macrophylla and C. aurantifolia – 'Mexican' lime types of Tanaka's taxa), C. reticulata (for C. limonia, C. karna and C. jambhiri varieties of Tanaka's taxa, including popular citrus rootstocks such as 'Rangpur' lime, 'Volkamer' and 'Rough' lemons), C. aurantium (for C. limetta and C. limon – yellow lemon types – varieties of Tanaka's taxa) or the C. maxima × C. reticulata hybrid (for C. limettioides – 'Palestine sweet' lime types – and C. meyeri). Among triploid limes, C. latifolia accessions ('Tahiti' and 'Persian' lime types) result from the fertilization of a haploid ovule of C. limon by a diploid gamete of C. aurantifolia, while C. aurantifolia triploid accessions ('Tanepao' lime types and 'Madagascar' lemon) probably result from an interspecific backcross (a diploid ovule of C. aurantifolia fertilized by C. medica). As limes and lemons were vegetatively propagated (apomixis, horticultural practices) the intra-sub-group phenotypic diversity results from asexual variations. (Resume d'auteur)

BACKGROUND The physiological roles of organic acids in fruit cells are not fully understood, especially in citrus, whereas the decline in titratable acidity during ripening shown by many citrus fruits is due to the utilization of citric acid. We induced carbohydrate depletion by removing source leaves at two key periods in mandarin development (early and full citric acid accumulation). Then, we assessed the resulting changes in the short term (within 48 h) and long term (several weeks until ripening). RESULTS Control mature fruits were characterized by elevated fresh weight, large diameters and high quantities of malic acid, citric acid and sucrose. At the same stage, fruits subjected to early or late defoliation had higher glucose, fructose, citric acid concentrations and lower sucrose concentrations. They differed only in their malic acid concentrations, which were higher in early defoliation fruits and similar in late defoliation fruits when compared to control fruits. Finally, fruits subjected to late defoliation were characterized by high proline and γ-aminobutyric acid concentrations, and low fructose and glucose concentrations. CONCLUSION We have shown that short- and long-term carbohydrate limitation modifies sugar and organic acid metabolism during mandarin fruit growth. © 2015 Society of Chemical Industry

The chemical composition of the essential oil (EO), microwave extract (ME) and hydrolate extract (HE) from the same batch of leaves of Fortunella japonica, was investigated: by combination of chromatographic (GC, CC) and spectroscopic techniques (GC-MS, 13C NMR). F. japonica essential oil and extracts are complex mixtures of 28-60 compounds being mainly oxygenated sesquiterpenes. The EO composition was dominated by germacrene D (14.9%), β-elemol (9.1%), cis-guai-6-en-10β-ol (6.3%), β-eudesmol (5.5%), and δ-elemene (5.2%). Limonene was the unique monoterpene identified at appreciable amount (7.1%). The extract obtained by microwave assisted hydrodistillation contained as main components: β-elemol (12.4%), germacrene D (9.9%), cis-guai-6-en- 10β-ol (9.0%), β-eudesmol (8.2%), germacra-l(l0),5-dien-4α-ol (7.1%) and α-eudesmol (6.4%). Finally, the highest content of oxygenated sesquiterpenes (near 92%) was found in the hydrolate extract displaying cryptomeridiol (23.3%, but totally absent in the EO and ME), β-eudesmol (20.6%) and α-eudesmol (10.7%). Combined analysis by chromatographic and spectroscopic techniques appeared useful for identification of various sesquiterpenols bearing a tertiary alcohol function.

The genome doubling of the allotetraploid somatic hybrid can confer greater tolerance to cold and light stress than the diploid parents and their respective tetraploid. Allopolyploids are generally known to display broader adaptation to abiotic stresses than their parental diploid species. In the Mediterranean area, Citrus species are subjected to abiotic constraints such as low temperature and high radiation. Tetraploids are known to resist these environmental constraints better, and so the use of new tetraploid rootstocks offers an alternative to overcome these threats to crop productivity. The objective of this study was to determine whether the use of an allotetraploid hybrid could provide greater tolerance to cold and light stresses than its diploid parents or respective doubled-diploid parents. We compared cold and light stress responses of the allotetraploid hybrid FlhorAG1 (FL-4x) with those of its diploid parents, the willow leaf mandarin (Citrus deliciosa Ten) (WLM-2x) and the Poncirus Pomeroy (Poncirus trifoliata (L.) Raf.) (POP-2x), and their respective doubled-diploids (WLM-4x and POP-4x, respectively) by measuring physiological and biochemical parameters. When subjected to cold and light stress, FL-4x showed lower photoinhibition (Fv/Fm) and less accumulation of oxidative markers (MDA and H2O2) than diploid and doubled-diploid WLM and POP genotypes. This was correlated with a greater increase for FL-4x in some antioxidant activities during cold stress (SOD, APX and GR) and light stress (SOD, APX and MDHAR mainly). Overall, our results suggest that greater antioxidant capability in FL-4x should make this allotetraploid hybrid more tolerant to low temperatures than the two WLM genotypes, and more tolerant to light stress than the two WLM and POP genotypes.

International audience; During the last decade, the worldwide market for fresh citrus fruits, mainly those that are seedless with easy-peeling and nice orange-colored skin, greatly increased. The elaboration of organoleptic quality is strongly defined by genetic and environmental components and their interaction. While several mandarin breeding programs have been developed worldwide, very few focus on the issues of tropical areas, and few data are available for citrus germplasm evaluation in a tropical environment. To optimize the mandarin production system, research related to the exploration of existing citrus germplasm, the expansion of the genetic base of orchards and the evaluation of the agronomic potential of new genotypes must be conducted. The objective of this study was to characterize and evaluate 25 mandarin varieties from typical Brazilian tropical citrus germplasm according to genetic, morphological and physicochemical traits over two consecutive years. Simple correlation coefficient analysis showed significant positive and negative correlations among characteristics (e.g., fruit diameter and total soluble solids). Principal component analysis showed that fruit diameter, fruit weight, total soluble solids, total titratable acidity, fruit surface texture and adherence of albedo to pulp were the variables that most contributed to phenotypic variability. Genetic diversity analysis grouped accessions in four main clusters corresponding to traditional edible Chinese mandarins, Mediterranean mandarins, tangelos and tango hybrids and nonedible mandarins. A multiple factorial analysis showed that phenotypic diversity was associated with genetic diversity mainly for taste, shape and color traits. The results of this study will be used for the conservation of the indigenous germplasm, identification of varieties with valuable traits related to fruit quality and subsequent exploitation in citrus breeding programs.

The composition of an oil sample isolated from leaves of Fortunella japonica (Thunb.) Swingle was investigated by combination of chromatographic and spectroscopic techniques. Among the compounds present at appreciable amounts, a special attention was brought to sesquiterpenes bearing a tertiary alcohol function and particularly to germacra-1(10),5-dien-4α-ol. Indeed, identification of diastereoisomers α and β of germacra-1(10),5-dien-4-ol has been the subject of controversy in the literature. We report here for the first time 13C NMR data of the α isomer. The criteria for differentiation of both diastereoisomers are discussed. In parallel, leaf oils from four other Fortunella species (F. crassifolia, F. hindsii, F. margarita and F. obovata) have been analyzed by GC(RI) and 13C NMR. The main components of the investigated Fortunella leaf oils were β-elemol (8.7-30.9%), germacrene D (2.1–16.3%), β-eudesmol (4.6–13.1%) and Δ9-10-eremophilen-11-ol (1.4–11.2%). The occurrence of germacra-1(10),5-dien-4α-ol (1.1–7.7%) was observed in the five Fortunella leaf oils. Copyright © 2015 John Wiley & Sons, Ltd.

International audience; Citrus reticulata is considered one of the four main ancestral taxa of cultivated citrus and is widely associated with the mandarin horticultural group. However, several studies suggest introgressions of other taxa and the precise structure of the mandarin group is not known. To analyse the structuration of mandarin germplasm and the potential interspecific introgressions, 208 accessions were genotyped by the KASPar method, with sixty-seven single nucleotide polymorphism (SNP) markers mined in 27 nuclear genes and in clementine BAC-ends. These SNP markers were employed for the study of 208 genotypes comprised of 169 ` mandarin-like' genotypes and 39 genotypes belonging to ancestral taxa. Structure software was used to identify groups within the mandarin germplasm. Six mandarin groups were identified, some of them in agreement with the species defined by Tanaka. Many mandarins were located between these mandarin groups, indicating a high level of admixture between the identified parental groups. New insights in the organization of mandarin germplasm and its structure are presented in this work, which will be useful for better management of citrus germplasm collections.

International audience; Despite considerable differences in morphology, the genera representing 'true citrus fruit trees' are sexually compatible, but their phylogenetic relationships remain unclear. Most of the important commercial species of Citrus are believed to be of interspecific origin. By studying SNP and indel polymorphisms of 27 nuclear genes on 32 genotypes of Citrus and relatives of Citrus, the phylogenetic relationship between 'true citrus fruit trees' was clarified. Additionally, four mitochondrial (mtDNA) and five chloroplastic (cpDNA) SSRs markers were analysed for the citrus species to specify the maternal phylogeny of Citrus secondary species. A total of 16238 kb of DNA was sequenced for each genotype, and 1156 SNPs and 58 indels were identified. Nuclear phylogenetic analysis revealed that C. reticulata, Poncirus, and Fortunella form a clade that is clearly differentiated from the clade that includes three other basic taxa of cultivated citrus (C. maxima, C. medica and C. micrantha). Clymenia is included in a third strong clade with Eremocitrus and Microcitrus. The nuclear phylogeny of Citrus and its sexually compatible relatives was consistent with the geographic origins of these genera. The origin of Citrus secondary species was analysed both at nuclear and cytoplasmic level and the conclusions mostly agree with previous hypotheses. The SNPs and indels identified at the nuclear level from a relatively large discovery panel will be useful for systematic characterisation of citrus germplasm, both at intra-and inter-specific level.

The availability of a saturated genetic map of Clementine was identified by the International Citrus Genome Consortium as an essential prerequisite to assist the assembly of the reference whole genome sequence based on a Clementine derived haploid. The primary goals of the present study were to establish a Clementine reference map, and to perform comparative mapping with pummelo and sweet orange. Five parental genetic maps were established with SNPs, SSRs and InDels. A medium density reference map (961 markers for 1084.1 cM) of Clementine was established and used by the ICGC to facilitate the chromosome assembly of the haploid genome sequence. Comparative mapping with pummelo and sweet orange revealed that the linear order of markers was highly conserved. Reasonable inferences of most citrus genomes should be obtained by mapping next-generation sequencing data against the haploid reference genome sequence. Skewed segregations were frequent and higher in the male than female Clementine potentially leading to false interpretation of the genetic determinism of phenotypic traits. The mapping data confirmed that Clementine arose from hybridization between 'Mediterranean' mandarin and sweet orange and identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. Introgression of pummelo genome fragments were identified in heterozygosity in each chromosome. Moreover, it appeared that the genome of the haploid Clementine used to establish the citrus reference genome sequence was inherited primarily from the 'Mediterranean' mandarin. The usefulness of this genetic map, anchored in the reference whole genome sequence, is discussed. Citrus Research and Development Foundation 108O568 Ministerio de Ciencia e Innovación: AGL2008-00596-MCI, 2008/121, IPT-010000-2010-43, PSE-060000-2009-8, AGL2007-65437-C04-01/AGR European Commission Citrus Research and Development Foundation itl-bio-03-10122 015453 This work was principally funded by the French ANR CITRUSSEQ project. The European Commission, under the FP6-2003-INCO-DEV-2 project CIBEWU (n°015453), the Spanish Ministerio de Ciencia e Innovación grants, AGL2007-65437-C04-01/AGR and AGL2008-00596-MCI, the Spanish PSE-060000-2009-8 and IPT-010000-2010-43 projects, the Prometeo project 2008/121 Generalidad Valenciana, the Turkish TUBITAK Project No: 108O568; the California Citrus Research Board and UC Discovery grant itl-bio-03-10122 and the Florida Citrus Research and Development Foundation (CRDF), grants #67 and 71 also contributed to the work.

International audience; Combination of biotic and abiotic stress is a major challenge for crop and fruit production. Thus, identification of genes involved in cross-response to abiotic and biotic stress is of great importance for breeding superior genotypes. Lectins are glycan-binding proteins with a functions in the developmental processes as well as in the response to biotic and abiotic stress. In this work, a lectin like gene, namely ClLectin1, was characterized in Volkamer lemon and its expression was studied in plants exposed to either water stress, hormonal elicitors (JA, SA, ABA) or wounding to understand whether this gene may have a function in the response to multiple stress combination. Results showed that ClLectin1 has 100% homology with a L-type lectin gene from C. sinensis and the in silico study of the 5'UTR region showed the presence of cis-responsive elements to SA, DRE2 and ABA. ClLectin1 was rapidly induced by hormonal treatments and wounding, at local and systemic levels, suggesting an involvement in defence signalling pathways and a possible role as fast detection biomarker of biotic stress. On the other hand, the induction of ClLectin1 by water stress pointed out a role of the gene in the response to drought. The simultaneous response of ClLectin1 expression to water stress and SA treatment could be further investigated to assess whether a moderate drought stress may be useful to improve citrus performance by stimulating the SA-dependent response to biotic stress.

Pompia is a citrus fruit endemic of Sardinia, Italy, with an essential oil profile showing outstanding anti-inflammatory and anti-microbic properties. Despite its remarkable pharmaceutical potential, little taxonomic and genetic information is available for this species. We applied flow cytometry and classical cytogenetic techniques to assess the DNA content and to reconstruct the karyotype of several Pompia accessions. Molecular data from plastid DNA barcoding and nuclear DNA sequencing were used to study the genetic distance between Pompia and other citrus species. Flow cytometric estimates of DNA content and somatic chromosome counts suggest that Pompia is a regular diploid Citrus species. DNA polymorphisms of nuclear and chloroplast markers allowed us to investigate the genetic relationships between Pompia accessions and other Citrus species. Based on DNA polymorphism data we propose that Pompia is a very recent interspecific hybrid generated by a cross between C. aurantium (as seed bearer) and C. medica (as pollen donor). Our findings pave the way for further and more specific investigations of local Pompia germplasm resources that may help the preservation and valorisation of this valuable citrus fruit tree.

International audience; To understand the genotypic variation of citrus to mild salt stress, a proteomic approach has been carried out in parallel on two citrus genotypes ('Cleopatra' and 'Willow leaf' mandarins), which differ for Na+ and Cl- accumulation, and their cognate autotetraploids (4x). Using two-dimensional electrophoresis approximately 910 protein spots were reproducibly detected in control and salt-stressed leaves of all genotypes. Among them, 44 protein spots showing significant variations at least in one genotype were subjected to mass spectrometry analysis for identification. Salt-responsive proteins were involved in several functions, including photosynthetic processes, ROS scavenging, stress defence, and signalling. Genotype factors affect the salt-responsive pattern, especially that of carbon metabolism. The no ion accumulator 'Cleopatra' mandarin genotype showed the highest number of salt-responsive proteins, and up-regulation of Calvin cycle-related proteins. Conversely the ion accumulator 'Willow leaf' mandarin showed high levels of several photorespiration-related enzymes. A common set of proteins (twelve spots) displayed higher levels in salt-stressed leaves of 2x and 4x 'Cleopatra' and 4x 'Willow leaf' mandarin. Interestingly, antioxidant enzymes and heat shock proteins showed higher constitutive levels in 4x 'Cleopatra' mandarin and 4x 'Willow leaf' mandarin compared with the cognate 2x genotype. This work provides for the first time information on the effect of 8 weeks of salt stress on citrus genotypes contrasting for ion accumulation and their cognate autotetraploids. Results underline that genetic factors have a predominant effect on the salt response, although a common stress response independent from genotype was also found. (C) 2013 Elsevier GmbH. All rights reserved.

International audience; The composition of twenty-four samples of essential oil isolated from leaves of Citrus paradisi Macf. growing in a northern area of citrus cultivation in Mediterranean climate (Corsica) was investigated by GC (RI), GC-MS and C-13 NMR. Thirty-six compounds accounting for 94.9-99.3% of the oil composition were identified. The oils contained mainly monoterpene hydrocarbons and specially sabinene (up to 60.2%),(E)-beta-ocimene (up to 15.0%) and punctually gamma-terpinene (up to 56.1%), beta-pinene (up to 30.9%) and p-cymene (up to 12.5%). The twenty-four compositions were submitted to k-means partitioning and principal component analysis, which allowed the distinction of one homogeneous group of twenty-two grapefruits (main compound sabinene, mean = 47.1%). This group included eighteen cultivars derived by mutation from the ancestral grapefruit tree (Duncan) and four cultivars known to be interspecific hybrids. Two out group cultivars were clearly differentiated from others by original chemical compositions, in agreement with their hybrid origin.

In Citrus species chromosome doubling naturally occurs in somatic embryos and doubled diploid plants often show better adaptation to adverse environmental condition. To understand the molecular determinants of stress acclimation, we examined the response to water deficit in diploid (2×VL) and doubled diploid (4×VL) seedlings of Volkamer lemon (Citrus limonia Osb.) assessing the profile of constitutive volatile organic compound (VOC) in control and stressed conditions. Physiological parameters and leaf volatile compound profiles were measured during water deficit and 24 h after rehydration of plants to field capacity. Net photosynthesis and stomatal conductance were reduced in water stressed leaves, with no significant differences between 2×VL and 4×VL plants. Malondialdehyde concentration, a marker of lipid peroxidation of cellular membranes, was significantly more higher in stressed 2×VL leaves than in 4×VL. The blend of constitutive VOC was different in control leaves being oxygenated monoterpenoids more abundant in 2×VL leaves, and monoterpenoids more abundant in 4×VL leaves. Water deficit did not stimulate biosynthesis of terpenoids, whereas accumulation of trans-2 hexenal, a green leaf volatile (GLV) synthesized after membrane denaturation, was observed in stressed leaves of 2×VL leaves, but not in 4×VL leaves. Semiquantitative PCR showed an increase of the expression of HPL, the gene encoding for hydroperoxidase lyase which catalyzes 2-hexenal formation, only in 2×VL plants. The expression of the putative dehydration transcription factor DREB2A was also observed only in 2×VL water stressed plants. This work shows that level of ploidy may alter constitutive content of GLV by Citrus, therefore likely affecting plants capacity of protection and interaction with other organisms. Whereas diploid and double diploid plants showed similar physiological responses to water deficit, a biochemical marker indicated that membranes of double diploid leaves were more resistant to the stress. These results provide intriguing insights into the regulation of terpenoids and oxylipins pathways as a function of polyploidization in a non-model plant species.

SUMMARYTrifoliate orange (Poncirus trifoliata(L.) Raf.) is a very useful taxon for the citrus industry since this rootstock is immune to the Citrus Tristeza virus and confers cold tolerance. Numerous trifoliate orange varieties exist but little is known regarding their behavioural variability when subjected to abiotic constraints. The diversity of 74P. trifoliataaccessions maintained in the INRA-CIRAD Citrus Germplasm Collection was investigated using simple sequence repeat markers. Two major genetic groups were clearly identified as a few homonyms, intergroup or intra-group hybrids and doubled-chromosome tetraploid forms. The Group 1 phenotype was characterized by larger flowers and leaves and smaller seeds than Group 2. Tetraploid accessions showed larger leaves and heavier seeds than all other diploid accessions, regardless of genetic classification. Eight genotypes belonging to both genetic groups, as well as two hybrids between the two groups, were selected to investigate their water deficit tolerance. Stress was applied by withdrawing irrigation for 4 weeks. Physiological parameters such as leaf stomatal conductance, quantum yield of photosystem II electron transport, soil water potential, leaf osmotic potential and transpiration rate were estimated. Some varieties, such as Rubidoux 0101033, were clearly more tolerant to water deficit than others, such as Pomeroy 0101040 and Pomeroy 0110081. Interestingly, accessions that had the highest soil water potential and were the least affected by stress belonged to genetic Group 2. Conversely, trifoliate oranges of genetic Group 1 were the least tolerant.

Trees; International audience; Citrus plants originate from southeastern Asia, in a large area with various climates characterized by a broad range of temperatures. Some species have been diversified in temperate climates, others in subtropical climates. Temperature is assumed to be a key factor in citrus species adaptation and diversification of basic cellular functions. In a field experiment, the tolerance of the three fundamental Citrus species C. medica L., C. reticulata Blanco and C. maxima (Burm.) Merr., and Fortunella japonica (Thunb.) Swing. to photooxidative stress caused by seasonal climatic changes was evaluated on adult trees by measuring net photosynthesis (Pnet), stomatal conductance (Gs), maximum photosynthesis (Pmax) and chlorophyll fluorescence (Fv/Fm). In addition, seasonal changes in oxidative status, antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase) and antioxidant metabolites (ascorbate and glutathione) were monitored. Mandarin and pummelo appeared to be the most tolerant, showing the lowest down-regulation of photosynthetic parameters, and the lowest accumulation of oxidized compounds associated with efficiency of their antioxidant system. Kumquat showed intermediate behaviour, with a large diminution of photosynthetic parameters and marked accumulation of hydrogen peroxide, whereas the malondialdehyde content remained low, with a strong induction of glutathione synthesis. Finally, citron appeared to be the most sensitive genotype with a marked decrease in photosynthetic performance, the largest accumulation of oxidative parameters, insufficient induction of antioxidant enzymes and down-regulation of ascorbate and glutathione synthesis.

Resume Apparu lors d'un croisement naturel entre un mandarinier et un oranger a la fin du XIXe siecle, la clementine s'est ensuite diversifiee par selection clonale de mutants somatiques. Distinguer les differentes varietes clonales etant pratiquement impossible, le developpement d'outils moleculaires d'identification varietale s'est donc avere necessaire. Nous avons etudie trois cultivars, representant des groupes de maturite des fruits tres distincts. Nous avons recherche des criteres d'identification au niveau phenotypique (caracteristiques organoleptiques, morphologie des feuilles) et au niveau de l'ADN (isozymes, RAPD et ISSR). La diversite phenotypique observee est relativement elevee et contraste avec le faible polymorphisme moleculaire. En effet, seul le cultivar « Guerdane » presente des profils d'empreintes genetiques differents de ceux des deux autres cultivars. La frequence des modifications genetiques serait donc variable d'un cultivar a un autre. Les marqueurs moleculaires specifiques du cultivar « Guerdane » s'ajoutent aux marqueurs phenotypiques et etendent les possibilites d'identification aux jeunes plants en pepiniere. Pour citer cet article : B. Chahidi et al., C. R. Biologies 331 (2008).

Essential oils of 113 Citrus clementina (Commun) × Citrus deliciosa (Willow Leaf) hybrids were obtained from leaves collected on trees located in the same orchard. Their chemical composition was investigated by capillary GC, GC–MS and 13C-NMR and the results were submitted to statistical analysis. Three chemical compositions were characterized. The crossing between clementine and mandarin produces mainly leaf oils characterized by a composition similar to those of the parents (sabinene/linalool or methyl N-methylanthranylate). However, several samples exhibited an original composition (γ-terpinene/linalool). Copyright © 2008 John Wiley & Sons, Ltd.

International audience; Fruits are consumed not just for their taste but also their nutritional value. The major primary metabolites in fruit are sugars and acids, whose contents change during fruit growth and determine ultimate fruit quality. Fruits are also a source of antioxidant metabolites, which are important to human health due to their role in reducing risk of cancer and cardiovascular diseases. Antioxidants are equally important in the plant as they help fight against oxidative stress. Here we investigated the consequences of changes in the primary metabolism in acidic and acidless citrus genotypes during the major stages of fruit growth on the expression of antioxidant enzymes and the markers of cellular oxidation (hydrogen peroxide, malondialdehyde) in acidless (Iaffaoui orange and sweet lemon) and acidic (Salustiana orange and Villafranca lemon) citrus fruits. Glucose and fructose were the major sugars in the acidless lemon. Sucrose was the major sugar in the acidic lemon. Oranges shared a balance of glucose, fructose and sucrose. Malic and citric acid concentrations were higher in acidic lemons than acidless fruits. Acidic genotypes had higher hydrogen peroxide concentrations than acidless genotypes, whereas MDA concentrations were higher in oranges than in lemons. Specific activities of ascorbate peroxidase, catalase, superoxide dismutase and dehydroascorbate reductase were on the whole higher in acidic than acidless fruits. Principal component analysis revealed between-genotype divergence in antioxidant system, giving three groups: acidic lemons, acidless lemons, and oranges.

International audience; Mandarin is an important genetic group in citrus largely used for variety breeding programs and also to generate new rootstocks better adapted to abiotic stress. In this study, 14 mandarin accessions representative of the diversity in this group were analyzed for their salt stress tolerance properties. Physiological parameters such as gas exchange, osmotic pressure, as well as leaf and root chloride contents were measured. Samples were harvested to characterize the presence of hydrogen peroxide and to monitor the activity of enzymes involved in reactive oxygen species synthesis and detoxification processes. Very different physiological behaviors were noted among accessions. Most cultivars that are used as rootstocks such as Citrus reshni Hort. ex Tan. ('Cleopatra') or Citrus depressa Hayata ('Shekwasha'), were found to be tolerant, with limited root-to-shoot chloride translocation. Also, some accessions used as varieties showed similar tolerance traits. Interestingly some accessions that presented high leaf chloride contents were not always associated with sensitivity traits. Also accessions such as Willowlear showed quite high leaf chloride contents that were correlated with better detoxification processes. Tolerance traits that we identified are discussed in relation to the known genetic structure in mandarins. Finally, some of the salt stress tolerant accessions that we identified could potentially be used as parents in rootstock breeding programs and others in scion variety breeding programs. (c) 2015 Published by Elsevier B.V.

International audience; Citrus is an economically important world tree fruit crop with production in more than 146 countries. The center of origin for citrus is considered to be Southeastern Asia including southern China, northeastern India, and Malaysia, with secondary centers in surrounding areas. Novel and commercially significant scion and rootstock cultivars originating by natural mutation or directed hybridization were introduced during the past century. Significant genetic resource collections exist in many countries. A global citrus germplasm network was developed in 1997. Now that the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGR) has recognized Citrus as an Annex 1 crop, a global conservation strategy needs to be established. The objective of this workshop was to plan the development of this strategy. More than 60 delegates attended with representation from Argentina, Australia, Bhutan, Brazil, China, Columbia, France, Iran, Israel, Italy, Japan, Mexico, Morocco, Portugal, Spain, Turkey, UAE, US, and Vietnam. Nine oral presentations were given describing multiple aspects of genebank operations, technologies and gaps in Brazil, China, France, Australia, Spain, and the US. A draft genebank survey questionnaire was presented to the group and comments and suggested changes for improvement were received. Citrus curators and genebank managers throughout the world will be surveyed for the status and health of their collections. The surveys will be received and compiled by collaborators at the University of California, Riverside, and at the USDA ARS Riverside. This information will be compiled and reported. The strategy will include background and history of the development of Citrus as a cultivated crop, conservation protocols, vulnerabilities, and recommendations for safeguarding Citrus germplasm. This strategy will be implemented through the Global Crop Diversity Trust with collaboration from the International Society for Horticultural Science and the international Citrus research community.