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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.

[EN] Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of genetic resistance to HLB-associated 'Candidatus Liberibacter asiaticus' (Las) becomes strategic to warrant crop sustainability, but no resistant Citrus genotypes exist. Some Citrus relatives of the family Rutaceae, subfamily Aurantioideae, were described as full-resistant to Las, but they are phylogenetically far, thus incompatible with Citrus. Partial resistance was indicated for certain cross-compatible types. Moreover, other genotypes from subtribe Citrinae, sexually incompatible but graft-compatible with Citrus, may provide new rootstocks able to restrict bacterial titer in the canopy. Use of seedlings from monoembryonic species and inconsistencies in previous reports likely due to Las recalcitrance encouraged us to evaluate more accurately these Citrus relatives. We tested for Las resistance a diverse collection of graft-compatible Citrinae species using an aggressive and consistent challenge-inoculation and evaluation procedure. Most Citrinae species examined were either susceptible or partially resistant to Las. However, Eremocitrus glauca and Papua/New Guinea Microcitrus species as well as their hybrids and those with Citrus arose here for the first time as full-resistant, opening the way for using these underutilized genotypes as Las resistance sources in breeding programs or attempting using them directly as possible new Las-resistant Citrus rootstocks or interstocks., This work was funded by Fundecitrus, grant no. 817526 from the European Union H2020 Innovation Action Program and project PID2019-104569RB-I00 from the AEI-Spain.

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.

Citrus reproductive biology is complex. One of its characteristic features is parthenocarpy that enables seedless fruit production. Citrus parthenocarpy and self-incompatibility knowledge is only partial and sometimes discrepant. Increasing such knowledge is relevant for better managing cultivated varieties and improving the selection of parents in breeding strategies to recover seedless varieties such as mandarins. This work develops an efficient protocol to characterize self-incompatibility and different parthenocarpy types based on emasculation, hand self-pollination, and hand cross-pollination. It analyzes fruit setting and seed production coupled with histological pollen performance observations. We analyzed the reproductive behavior of nine mandarin varieties with relevant characteristics as parents for seedless mandarin breeding. ‘Clemenules’ clementine and ‘Moncada’ mandarins were strictly self-incompatible with facultative and vegetative parthenocarpy, ‘Imperial’ mandarin and ‘Ellendale’ tangor displayed no strict self-incompatibility associated with facultative and vegetative parthenocarpy, ‘Fortune’ mandarin was self-incompatible with facultative and stimulative parthenocarpy, ‘Campeona’ and ‘Salteñita’ mandarins were self-compatible with vegetative parthenocarpy, ‘Serafines’ satsuma was associated with male sterility together with facultative and vegetative parthenocarpy, and ‘Monreal’ clementine was self-compatible and nonparthenocarpic. Our protocol can be applied for screening of mandarin germplasm and to characterize new parents. Reproductive behavior knowledge is important for optimizing seedless mandarin breeding programs based on diploidy, triploidy, or induced mutagenesis.

International audience; Modern cultivated Citrus species and varieties result from interspecific hybridization between four ancestral taxa. Among them, Citrus maxima and Citrus reticulata, closely associated with the pummelo and mandarin horticultural groups, respectively, were particularly important as the progenitors of sour and sweet oranges (Citrus aurantium and Citrus sinensis), grapefruits (Citrus paradisi), and hybrid types resulting from modern breeding programs (tangors, tangelos, and orangelos). The differentiation between the four ancestral taxa and the phylogenomic structure of modern varieties widely drive the phenotypic diversity’s organization. In particular, strong phenotypic differences exist in the coloration and sweetness and represent important criteria for breeders. In this context, focusing on the genes of the sugar, carotenoid, and chlorophyll biosynthesis pathways, the aim of this work was to develop a set of diagnostic single-nucleotide polymorphism (SNP) markers to distinguish the ancestral haplotypes of C. maxima and C. reticulata and to provide information at the intraspecific diversity level (within C. reticulata or C. maxima). In silico analysis allowed the identification of 3,347 SNPs from selected genes. Among them, 1,024 were detected as potential differentiation markers between C. reticulata and C. maxima. A total of 115 SNPs were successfully developed using a competitive PCR technology. Their transferability among all Citrus species and the true citrus genera was very good, with only 0.87% of missing data. The ancestral alleles of the SNPs were identified, and we validated the usefulness of the developed markers for tracing the ancestral haplotype in large germplasm collections and sexually recombined progeny issued from the C. reticulata/C. maxima admixture gene pool. These markers will pave the way for targeted association studies based on ancestral haplotypes.

Rootstocks are crucial for the sustainability of the citrus industry worldwide. Diploid intergeneric Citrus x Poncirus hybrids have contributed considerably to citrus rootstock improvement and their tetraploid (doubled-diploid) forms are important resources for the creation of a new generation of tetraploid rootstocks. To optimize the efficiency of tetraploid rootstock breeding strategies, more knowledge is required on inheritance in the allotetraploid genitors. A set of 159 new SNP markers that fully distinguish Poncirus trifoliata (L.) Raf. from Citrus species was developed from polymorphisms mined in GBS data and used to establish a genetic map of tetraploid citrumelo (C. x paradisi Macfad. x P. trifoliata) and to analyze the meiotic behavior of tetraploid citrumelo and citrandarin (C. reticulata Blanco x P. trifoliata). The tetraploid citrumelo genetic map was highly syntenic and collinear with the clementine reference genome. The apparent intergeneric recombination rate was strongly limited by high preferential chromosome pairing, resulting in intermediate inheritance with disomic tendency. Such inheritance, also observed in tetraploid citrandarin, results in the transmission by the diploid gametes of a high rate of intergeneric heterozygosity. It is therefore expected that a large part of the genetic value selected in the original diploid intergeneric rootstock is transmitted to the tetraploid sexual progenies.

Citrus classification remains an unresolved problem. In the first part of this chapter, after a short description of their morphological characters, the justification for the separation of the “true citrus” group into six genera is discussed, based on their reproductive biology and plastome phylogenetics. It is concluded that all true citrus plus the genus Oxanthera should be joined within the genus Citrus. Moreover, despite full sexual compatibility between the Citrus species sensu Swingle, the phenotypic and molecular differentiation between the ancestral taxa (C. maxima, C. medica, C. micrantha, C. reticulata) of the cultivated forms fit with phylogenetic species concepts based on diagnosability and monophyly. In the second part, after an historical perspective, the species rank of the genus Citrus is discussed based on the recent phylogenomic data and a new concept is proposed for citrus classification. Finally, in the third part, the phenotypical diversity of the Asian ancestors of edible citrus and of the most important horticultural groups of interspecific origins is presented after a discussion of the implication of the reticulate evolution coupled with apomixis on the phenotypical diversity structure of edible Citrus.

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)

Triploid-breeding programs in citrus are key tool to develop seedless cultivars. Obtaining triploid citrus hybrids may be achieved through different strategies, such as the exploitation of female unreduced gamete in crosses between diploid parents and diploid by tetraploid sexual hybridizations, in which tetraploid genotypes can be used as male or female parents. Genetic configuration of triploid populations from interploid crosses greatly depends on the chromosomic segregation mode of the tetraploid parent used. Here, we have analyzed the inheritance of the tetraploid ‘Moncada’ mandarin and compared the genetic structures of the resulting gametes when used as male and as female parent. The preferential chromosome pairing rate is calculated from the parental heterozygosity restitution (PHR) of codominant molecular markers, indicating the proportion between disomic and tetrasomic segregation. Tetraploid ‘Moncada’ both as female and male parent largely exhibited tetrasomic segregation. However, as female parent, one linkage group (LG8) showed intermediate segregation with tendency towards tetrasomic inheritance, while another linkage group (LG4) evidenced a clear intermediate segregation. On the other hand, when used as male parent two linkage groups (LG5 and LG6) showed values that fit an intermediate inheritance model with tetrasomic tendency. Significant doubled reduction (DR) rates were observed in five linkage groups as female parent, and in six linkage groups as male parent. The new knowledge generated here will serve to define crossing strategies in citrus improvement programs to efficiently obtain new varieties of interest in the global fresh consumption market. EEA Concordia Fil: Garavello, Miguel Fernando. Instituto Valenciano de Investigaciones Agrarias. Centro de Citricultura y Producción Vegetal; España. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Concordia; Argentina Fil: Cuenca, José. Instituto Valenciano de Investigaciones Agrarias. Centro de Citricultura y Producción Vegetal; España. Fil: Garcia-Lor, Andrés. Instituto Valenciano de Investigaciones Agrarias. Centro de Citricultura y Producción Vegetal; España. Fil: Ortega, Neus. Instituto Valenciano de Investigaciones Agrarias. Centro de Citricultura y Producción Vegetal; España. Fil: Navarro, Luis. Instituto Valenciano de Investigaciones Agrarias. Centro de Citricultura y Producción Vegetal; España. Fil: Ollitrault, Patrick. Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD). Amélioration Génétique et Adaptation des Plantes (UMR Agap). Unité Mixte de Recherche; Francia Fil: Aleza, Pablo. Instituto Valenciano de Investigaciones Agrarias. Centro de Citricultura y Producción Vegetal; España.

The Mediterranean basin is especially sensitive to the adverse outcomes of climate change and especially to variations in rainfall patterns and the incidence of extremely high temperatures. These two concurring adverse environmental conditions will surely have a detrimental effect on crop performance and productivity that will be particularly severe on woody crops such as citrus, olive and grapevine that define the backbone of traditional Mediterranean agriculture. These woody species have been traditionally selected for traits such as improved fruit yield and quality or alteration in harvesting periods, leaving out traits related to plant field performance. This is currently a crucial aspect due to the progressive and imminent effects of global climate change. Although complete genome sequence exists for sweet orange (Citrus sinensis) and clementine (Citrus clementina), olive tree (Olea europaea) and grapevine (Vitis vinifera), the development of biotechnological tools to improve stress tolerance still relies on the study of the available genetic resources including interspecific hybrids, naturally occurring (or induced) polyploids and wild relatives under field conditions. To this respect, post-genomic era studies including transcriptomics, metabolomics and proteomics provide a wide and unbiased view of plant physiology and biochemistry under adverse environmental conditions that, along with high-throughput phenotyping, could contribute to the characterization of plant genotypes exhibiting physiological and/or genetic traits that are correlated to abiotic stress tolerance. The ultimate goal of precision agriculture is to improve crop productivity, in terms of yield and quality, making a sustainable use of land and water resources under adverse environmental conditions using all available biotechnological tools and high-throughput phenotyping. This review focuses on the current state-of-the-art of biotechnological tools such as high throughput –omics and phenotyping on grapevine, citrus and olive and their contribution to plant breeding programs.

Temperature in the progamic phase is critical for the success of plant sexual reproduction, and new knowledge is needed to optimise breeding programmes to obtain new varieties that adapt to a climate change scenario. Using three male donors and one female recipient in the genus Citrus, we evaluated the effect of four temperature regimes on each process in the progamic phase. An innovative method based on microscopic observations of cross sections from pollinated pistils collected daily allowed a comprehensive analysis of pollen tube growth (dynamics and kinetics) along the pistil. Pollen grain germination and stigmatic receptivity were evaluated directly on the stigma, which offers more accurate information than previously reported in vitro experiments. Our results showed that warm temperatures reduce the time needed by pollen tubes to reach ovules and accelerate pistil degeneration, while cold temperatures produce the opposite effects. Interestingly, we observed both pollen grain germination and pollen tube growth at 10 °C, which have not been observed in previous studies in citrus. At this temperature, the differences observed in both pollen grain germination and pollen tube growth for different genotypes reflect the adaptation of their sporophytic generation to low temperatures which would enable gametophytic screening to be used as a tool to select better adapted genotypes to different temperature conditions. The differences observed in the growth rates between pollen tubes in each genotype-temperature combination provide an opportunity to explore additional gametophytic selection in this reproductive phase. The capacity to respond to temperature changes in the progamic phase to ensure mating can be useful for breeding programs that focus on obtaining better adapted populations to different temperature conditions.

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.

Tetraploid clementine displays mainly tetrasomic inheritance. Genetic structures of 2n SDR and 2 × gametes from DD clementine are complementary and will guides triploids citrus breeding strategies. Triploid breeding is developed worldwide to create new seedless cultivars. Citrus triploid hybrids can be recovered from 2x × 2x sexual hybridizations as a consequence of the formation of unreduced gametes (2n), or from 4x × 2x interploid hybridizations in which tetraploid parents used are most often doubled-diploid (DD). Here we have analyzed the inheritance in doubled-diploid clementine and compared the genetic structures of gametes of DD clementine with SDR unreduced gametes of diploid clementine. Parental heterozygosity restitution (PHR) with DD parents depends on the rate of preferential chromosome pairing and thus the proportion of disomic versus tetrasomic segregations. Doubled-diploid clementine largely exhibited tetrasomic segregation. However, three linkage groups had intermediate segregation and one had a tendency for disomy. Significant doubled reduction rates (DR) rates were observed in six of the nine LGs. Differences of PHR between 2n SDR and 2x DD gametes were highest in the centromeric region and progressively decreased toward the distal regions where they were not significant. Over all markers, PHR was lower (two-thirds) in SDR 2n gametes than in DD-derived diploid gametes. The two strategies appear complementary in terms of genotypic variability. Interploid 4x × 2x hybridization is potentially more efficient for developing new cultivars that are phenotypically closer to the diploid parent of the DD than sexual hybridization through SDR 2n gametes. Conversely, 2x × 2x triploidisation has the potential to produce novel products with characteristics for market segmentation strategies.

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)

Polyploidy is one of the main forces that drives the evolution of plants and provides great advantages for breeding. Somatic hybridization by protoplast fusion is used in citrus breeding programs. This method allows combining the whole parental genomes in a single genotype, adding complementary dominant characters, regardless of parental heterozygosity. It also contributes to surpass limitations imposed by reproductive biology and quickly generates progenies that combine the required traits. Two allotetraploid somatic hybrids recovered from the citrus rootstocks—Citrus macrophylla (CM) and Carrizo citrange (CC)—were characterized for morphology, genome composition using molecular markers (SNP, SSR, and InDel), and their tolerance to iron chlorosis, salinity, and Citrus tristeza virus (CTV). Both hybrids combine the whole parental genomes even though the loss of parental alleles was detected in most linkage groups. Mitochondrial genome was inherited from CM in both the hybrids, whereas recombination was observed for chloroplastic genome. Thus, somatic hybrids differ from each other in their genome composition, indicating that losses and rearrangements occurred during the fusion process. Both inherited the tolerance to stem pitting caused by CTV from CC, are tolerant to iron chlorosis such as CM, and have a higher tolerance to salinity than the sensitive CC. These hybrids have potential as improved rootstocks to grow citrus in areas with calcareous and saline soils where CTV is present, such as the Mediterranean region. The provided knowledge on the effects of somatic hybridization on the genome composition, anatomy, and physiology of citrus rootstocks will be key for breeding programs that aim to address current and future needs of the citrus industry.

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.

Background and Aims Two main types of triploid limes are produced worldwide. The 'Tahiti' lime type (Citrus latifolia) is predominant, while the 'Tanepao' type (C. aurantiifolia) is produced to a lesser extent. Both types result from natural interspecific hybridization involving a diploid gamete of C. aurantiifolia 'Mexican' lime type (itself a direct interspecific C. micrantha × C. medica hybrid). The meiotic behaviour of a doubled-diploid 'Mexican' lime, the interspecific micrantha/medica recombination and the resulting diploid gamete structures were analysed to investigate the possibility that 'Tahiti' and 'Tanepao' varieties are derived from natural interploid hybridization. Methods A population of 85 tetraploid hybrids was established between a doubled-diploid clementine and a doubled-diploid 'Mexican' lime and used to infer the genotypes of 'Mexican' lime diploid gametes. Meiotic behaviour was studied through combined segregation analysis of 35 simple sequenbce repeat (SSR) and single nucleotide polymorphismn (SNP) markers covering the nine citrus chromosomes and cytogenetic studies. It was supplemented by pollen viability assessment. Key Results Pollen viability of the doubled-diploid Mexican lime (64 %) was much higher than that of the diploid. On average, 65 % of the chromosomes paired as bivalents and 31.4 % as tetravalents. Parental heterozygosity restitution ranged from 83 to 99 %. Disomic inheritance with high preferential pairing values was deduced for three chromosomes. Intermediate inheritances, with disomic trend, were found for five chromosomes, and an intermediate inheritance was observed for one chromosome. The average effective interspecific recombination rate was low (1.2 cM Mb–1). Conclusion The doubled-diploid 'Mexican' lime had predominantly disomic segregation, producing interspecific diploid gamete structures with high C. medica/C. micrantha heterozygosity, compatible with the phylogenomic structures of triploid C. latifolia and C. aurantiifolia varieties. This disomic trend limits effective interspecific recombination and diversity of the diploid gamete population. Interploid reconstruction breeding using doubled-diploid lime as one parent is a promising approach for triploid lime diversification. (Resume d'auteur)

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.

International audience; SSRs have been considered as almost ideal markers for genetic diversity analysis. With the increasing availability of sequencing data, SNPs and InDels become major classes of codominant markers with genome wide coverage. We have analyzed the respective values of SSRs, InDels, and SNPs for intra and interspecific Citrus genetic diversity analysis. Moreover, we have compared the diversity structure revealed by markers mined in a single heterozygous genotype (clementine) and markers mined from a large interspecific survey. A random set of markers was selected for each marker class to genotype 48 citrus accessions. SSRs were the most polymorphic markers at the intraspecific level allowing complete varietal differentiation within basic taxa (C. reticulata, C. maxima, C. medica). However, SSRs gave the lowest values for interspecific differentiation, followed by SNPs and InDels, that in average displayed low intraspecific variability but high interspecific differentiation. A clear effect of the discovery panel was observed for SNPs and InDels. The ascertainment biases associated with the clementine heterozygosity mining resulted mainly in an over estimation of within C. reticulata diversity and an underestimation of the interspecific differentiation. In conclusion pluri-allelic SSRs are very useful for intraspecific structure analysis but have limitations for interspecific and phylogenetic studies. SNPs and InDels mined in large discovery panels appear to allow efficient selection of more specialized markers with high potential for phylogenetic diagnosis and to decipher the interspecific mosaic structure of secondary cultivated species or to analyze intraspecific diversity. Several SNP genotyping methods are available for different analysis scales, from high throughput genotyping to small set of data production. With the ongoing resequencing projects, SNPs markers will soon be the most important class of markers for citrus genetics.

Seedlessness is a major criterion for the citrus fresh fruit market. Therefore, triploid breeding appears very promising for the selection of new citrus cultivars. CIRAD has developed a method to produce triploid hybrids by somatic hybridization between diploid and haploid lines, and demonstrates the potential for haploid citrus production by induced gynogenesis in clementine. In this work we analysed the efficiency of induced gynogenesis by irradiated pollen in 'Wilking' mandarin (Citrus reticulata). Five doses of gamma irradiation of 'Meyer' lemon pollen were tested (0, 150, 200, 250, 300 Grays). Embryos were extracted from small seeds, obtained in immature fruits, and cultivated in vitro. The ploidy level of plantlets was determined by flow cytometry analysis. Irradiated pollen appeared to be efficient for gynogenesis induction in 'Wilking'. A relatively high number of haploids (7 plantlets) were obtained, essentially after hybridisation with 'Meyer' lemon pollen irradiated at 250 Grays. The ploidy level of all haploid plantlets was confirmed by chromosome counts, and molecular markers demonstrated their gynogenetic origin. These haploid lines should be useful for triploid breeding by somatic hybridisation, and also constitute interesting resources for genomic studies. (Resume d'auteur)

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.

Unreduced (2n) gametes have played a pivotal role in polyploid plant evolution and are useful for sexual polyploid breeding in various species, particularly for developing new seedless citrus varieties. The underlying mechanisms of 2n gamete formation were recently revealed for Citrus reticulata but remain poorly understood for other citrus species, including lemon (C. limon [L.] Burm. f.). Here, we investigated the frequency and causal meiotic mechanisms of 2n megagametophyte production in lemon. We genotyped 48progeny plants of two lemon genotypes, “Eureka Frost” and “Fino”, using 16 Simple Sequence Repeat (SSR) and 18 Single Nucleotide Polymorphism (SNP) markers to determine the genetic origin of the progenies and the underlying mechanisms for 2n gamete formation. We utilized a maximum-likelihood method based on parental heterozygosity restitution (PHR) of centromeric markers and analysis of PHR patterns along the chromosome. The frequency of 2n gamete production was 4.9% for “Eureka Frost” and 8.3% for “Fino”, with three meiotic mechanisms leading to 2n gamete formation. We performed the maximum-likelihood method at the individual level via centromeric marker analysis, finding that 88% of the hybrids arose from second-division restitution (SDR), 7% from first-division restitution (FDR) or pre-meiotic doubling (PRD), and 5% from post-meiotic genome doubling (PMD). The pattern of PHR along LG1 confirmed that SDR is the main mechanism for 2n gamete production. Recombination analysis between markers in this LG revealed partial chiasma interference on both arms. We discuss the implications of these restitution mechanisms for citrus breeding and lemon genetics.

Polyploid citrus arise by somatic and sexual polyploidization, and both have been used in triploid breeding programs. Sexual polyploidization is mainly achieved by first-division restitution (FDR) or second-division restitution (SDR) meiotic restitution mechanisms. In citrus, mostly SDR producing 2n ovule has been described. However, we obtained 72 tetraploid hybrids from 4x X 2x sexual hybridizations using two doubled-diploid mandarins as female parents ('Moncada' mandarin and 'Fina' clementine) and a diploid hybrid tangor as male parent (clementine X sweet orange—'CSO') suggesting 2n pollen formation. This material was used to confirm the existence of 2n pollen in Citrus and to analyze its origin. SSR and SNP molecular marker analyses revealed that 64 out of the 72 recovered tetraploid plants resulted from the fertilization of a reduced diploid female gamete by unreduced (diploid) pollen from 'CSO', whereas eight tetraploid plants arose from self-pollination of the tetraploid parent. The maximum-likelihood method based on parental heterozygosity restitution (PHR) of centromeric loci identified both FDR and SDR as the mechanisms leading to unreduced male gamete formation. From the 64 unreduced gametes produced by diploid 'CSO' tangor, 41 (64.1%) were originated by FDR, whereas 12 (18.8%) were significant for SDR. Non-conclusive results were obtained for 11gametes (17.2%). The pattern of PHR variation of markers located along the linkage group 2 confirmed our results at population level. To our knowledge, this is the first report of tetraploid citrus progenies arising from unreduced pollen and the first description of the coexistence of two meiotic restitution mechanisms (SDR and FDR) producing unreduced pollen in citrus.

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.

Alternaria brown spot (ABS) is a serious disease affecting susceptible citrus genotypes, which is a strong concern regarding citrus breeding programs. Resistance is conferred by a recessive locus (ABSr) previously located by our group within a 3.3 Mb genome region near the centromere in chromosome III. This work addresses fine-linkage mapping of this region for identifying candidate resistance genes and develops new molecular markers for ABS-resistance effective marker-assisted selection (MAS). Markers closely linked to ABSr locus were used for fine mapping using a 268-segregating diploid progeny derived from a heterozygous susceptible × resistant cross. Fine mapping limited the genomic region containing the ABSr resistance gene to 366 kb, flanked by markers at 0.4 and 0.7 cM. This region contains nine genes related to pathogen resistance. Among them, eight are resistance (R) gene homologs, with two of them harboring a serine/threonine protein kinase domain. These two genes along with a gene encoding a S-adenosyl-L-methionine-dependent-methyltransferase protein, should be considered as strong candidates for ABS-resistance. Moreover, the closest SNP was genotyped in 40 citrus varieties, revealing very high association with the resistant/susceptible phenotype. This new marker is currently used in our citrus breeding program for ABS-resistant parent and cultivar selection, at diploid, triploid and tetraploid level.

International audience; Genome expression in polyploid is usually considered to be quite different depending on the genetic context. While it is admitted that autotetraploids resulting from polyploidization events within species do not present major changes of genes expression, allotetraploid which result from hybridization between divergent species or lines were showed to present very important changes of expression compared to the diploid (2x) parental lines (genomic shock). In this work, we investigated the root citrus genome expression using custom Agilent Citrus microarray (4x44K format) of different couples of 2x and doubled diploid (4x) in absence of stress: Poncirus, Cleopatra Mandarin as well in the Cleopatra Mandarin × Poncirus intergeneric hybrid. We also evaluated the plant hydraulic architecture of this plant material. Our goal was to evaluate if any change in root genome expression related to polyploidy could lead to plant hydraulic architecture changes.

International audience; For grafted plants, salt stress tolerance of the aerial plant part is poorly documented. Thus, we developed a simple, fast and inexpensive method to identify tolerant genotypes. Twigs of 14 mandarin accessions that we previously analyzed as seedlings were cut in solution to prevent embolism and were then evaluated in salt stress condition for a week. Physiological parameters such as gas exchanges, leaf Cl- and Na+, as well as the presence of H2O2 and the activity of enzymes involved in ROS synthesis and detoxification processes were analyzed. One accession known to be tolerant as rootstock was shown to be sensitive with limited Cl- translocation from the solution to the shoot while sensitive accessions when grown as seedlings presented limited wilting symptoms and accumulated large leaf Cl- content. A model is proposed to explain the different strategies of the plant to cope with high toxic ion content. This method allows separation of the root compartment, where ion exclusion mechanisms may exist and have an impact on the salt stress tolerance of the whole plant. (C) 2015 Elsevier GmbH. All rights reserved.

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.

Background Chloroplast DNA is a primary source of molecular variations for phylogenetic analysis of photosynthetic eukaryotes. However, the sequencing and analysis of multiple chloroplastic regions is difficult to apply to large collections or large samples of natural populations. The objective of our work was to demonstrate that a molecular taxonomic key based on easy, scalable and low-cost genotyping method should be developed from a set of Single Nucleotide Polymorphisms (SNPs) diagnostic of well-established clades. It was applied to the Aurantioideae subfamily, the largest group of the Rutaceae family that includes the cultivated citrus species. Results The publicly available nucleotide sequences of eight plastid genomic regions were compared for 79 accessions of the Aurantioideae subfamily to search for SNPs revealing taxonomic differentiation at the inter-tribe, inter-subtribe, inter-genus and interspecific levels. Diagnostic SNPs (DSNPs) were found for 46 of the 54 clade levels analysed. Forty DSNPs were selected to develop KASPar markers and their taxonomic value was tested by genotyping 108 accessions of the Aurantioideae subfamily. Twenty-seven markers diagnostic of 24 clades were validated and they displayed a very high rate of transferability in the Aurantioideae subfamily (only 1.2 % of missing data on average). The UPGMA from the validated markers produced a cladistic organisation that was highly coherent with the previous phylogenetic analysis based on the sequence data of the eight plasmid regions. In particular, the monophyletic origin of the “true citrus” genera plus Oxanthera was validated. However, some clarification remains necessary regarding the organisation of the other wild species of the Citreae tribe. Conclusions We validated the concept that with well-established clades, DSNPs can be selected and efficiently transformed into competitive allele-specific PCR markers (KASPar method) allowing cost-effective highly efficient cladistic analysis in large collections at subfamily level. The robustness of this genotyping method is an additional decisive advantage for network collaborative research. The availability of WGS data for the main “true citrus” species should soon make it possible to develop a set of DSNP markers allowing very fine resolution of this very important horticultural group. Electronic supplementary material The online version of this article (doi:10.1186/s12863-016-0426-x) contains supplementary material, which is available to authorized users.

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.

Polyploidisation is a key source of diversification and speciation in plants. Most researchers consider sexual polyploidisation leading to unreduced gamete as its main origin. Unreduced gametes are useful in several crop breeding schemes. Their formation mechanism, i.e., First-Division Restitution (FDR) or Second-Division Restitution (SDR), greatly impacts the gametic and population structures and, therefore, the breeding efficiency. Previous methods to identify the underlying mechanism required the analysis of a large set of markers over large progeny. This work develops a new maximum-likelihood method to identify the unreduced gamete formation mechanism both at the population and individual levels using independent centromeric markers. Knowledge of marker-centromere distances greatly improves the statistical power of the comparison between the SDR and FDR hypotheses. Simulating data demonstrated the importance of selecting markers very close to the centromere to obtain significant conclusions at individual level. This new method was used to identify the meiotic restitution mechanism in nineteen mandarin genotypes used as female parents in triploid citrus breeding. SDR was identified for 85.3% of 543 triploid hybrids and FDR for 0.6%. No significant conclusions were obtained for 14.1% of the hybrids. At population level SDR was the predominant mechanisms for the 19 parental mandarins.

International audience; The mandarin horticultural varietal group (a basic taxon of the cultivated citrus (Citrus reticulata)) is highly polymorphic. It includes also genotypes introgressed by other species. The precise contribution of ancestral species to the mandarin group is not known. The goals of this work were (1) to characterise the mandarin germplasm using nuclear (simple sequence repeat (SSR), indel, single nucleotide polymorphism (SNP)), chloroplastic and mitochondrial markers; (2) to evaluate genetic diversity and detect redundancies; (3) to determine the possible presence of citrus ancestral genome introgressions into the mandarin genome; and (4) to determine the genetic structure within the mandarin group. Fifty microsatellites (SSRs), 24 insertion-deletions (indels), 67 SNPs, 8 chloroplastic SSRs (cpDNA) and 4 mitochondrial (mtDNA) indel markers were analysed for 191 genotypes, including the 4 main citrus ancestral species (C. reticulata, C. maxima, C. medica and C. micrantha) and Fortunella. C. maxima was the main genome introgressed in the mandarin germplasm. Seven clusters were revealed by Structure analysis at the nuclear level (N) within the mandarin germplasm. At least four of these clusters showed a clear introgression from other ancestral species. Moreover, most of the mandarins appeared to be complex mixtures of these groups. The maternal indel analysis (mtDNA and cpDNA) revealed ten cytotypes in which mandarins were represented in seven of them. This work provides new insights into the organisation of the mandarin germplasm and its structure at the nuclear and cytoplasmic levels and will be useful to design more efficient breeding programmes and management of citrus germplasm collections.

It is believed that Citrus medica, C. maxima, C. reticulate and C. micrantha have generated all cultivated citrus species. Depending on the classification, lemons and limes are classified either into two species, C. limon and C. aurantifolia (Swingle and Reece, 1967) or into more than 30 (Tanaka, 1977). In order to study the molecular phylogeny of this citrus group, we analyzed 23 targeted sequenced nuclear genes and used three mitochondrial and five chloroplastic markers for 15 lemons and limes compared with representatives of the four basic taxa. We observed three main groups, each one derived from direct interspecific hybridizations: (1) the Mexican lime group (C. aurantifolia), including C. macrophylla, arising from hybridizations between papeda (C. micrantha) and citron (C. medica); (2) the yellow lemon group (C. limon) that are hybrids between sour orange (C. aurantium, which is believed to be a hybrid between C. maxima and C. reticulata) and citron; and (3) a rootstock lemon/lime group (Rough lemon and Rangpur lime) that are hybrids between an acid mandarin and a citron. We also identified different probable backcrosses and genotypes with more complex origin. None of the analyzed limes and lemons shared the C. medica cytoplasm, while this taxon is the common nuclear contributor of all limes and lemons. Limes and lemons appear to be a very complex citrus varietal group with the contribution of the 4 basic taxa. Neither the Swingle and Reece classification nor the Tanaka fit with the genetic evidence. (Resume d'auteur)

The volatile compounds from leaves and peels of an interspecific citrus somatic hybrid, Citrus aurantifolia (Christm.) Swing. + Citrus paradisi Macfayden, obtained by fusion of protoplasts from lime, Citrus aurantifolia (cv. Mexican Lime) and grapefruit, Citrus paradisi (cv. Star Ruby), were extracted by pentane: ether (1 : 1) from liquid nitrogen ball-milled leaves and flavedo and examined by GC-MS in comparison to those of its parents. The hybrid quantitatively retained the ability of the lime parent to synthesize in its leaves the major monoterpene aldehydes (neral, geranial) the monoterpene alcohols (nerol, geraniol), and their acetates, and also the capacity of the grapefruit parent to produce a sesquiterpene aldehyde ([beta]-sinensal) in its leaves and nootkatone in its peel. Conversely, synthesis of most sesquiterpene hydrocarbons and long chain aliphatic aldehydes, which are present in the lime parent leaves and peel, was strongly inhibited in the hybrid, as in the grapefruit parent. In comparison to its parents, the hybrid overproduced citronellal in its leaves and [alpha]-sinensal and [beta]-sinensal in its peel. Based on these results, the future prospects for a better understanding of the inheritance mechanisms with regards to aroma biosynthesis in citrus leaves and peels are discussed. (Resume d'auteur)

International audience; Genetic analysis of phenotypical traits and marker-trait association in polyploid species is generally considered as a challenge. In the present work, different approaches were combined taking advantage of the particular genetic structures of 2n gametes resulting from second division restitution (SDR) to map a genome region linked to Alternaria brown spot (ABS) resistance in triploid citrus progeny. ABS in citrus is a serious disease caused by the tangerine pathotype of the fungus Alternaria alternata. This pathogen produces ACT-toxin, which induces necrotic lesions on fruit and young leaves, defoliation and fruit drop in susceptible genotypes. It is a strong concern for triploid breeding programs aiming to produce seedless mandarin cultivars. The monolocus dominant inheritance of susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. Bulk segregant analysis coupled with genome scan using a large set of genetically mapped SNP markers and targeted genetic mapping by half tetrad analysis, using SSR and SNP markers, allowed locating a 3.3 Mb genomic region linked to ABS resistance near the centromere of chromosome III. Clusters of resistance genes were identified by gene ontology analysis of this genomic region. Some of these genes are good candidates to control the dominant susceptibility to the ACT-toxin. SSR and SNP markers were developed for efficient early marker-assisted selection of ABS resistant hybrids.

Background Three gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules, designated ESP, FRA, and ITA (derived from three labs in Spain, France, and Italy, respectively), were selected for cytological and molecular characterization in order to elucidate genomic rearrangements provoked by haploidization. The study included comparisons of their ploidy, homozygosity, genome integrity, and gene dosage, using chromosome counting, flow cytometry, SSR marker genotyping, and array-Comparative Genomic Hybridization (array-CGH). Results Chromosome counting and flow cytometry revealed that ESP and FRA were haploid, but ITA was tri-haploid. Homozygous patterns, represented by a single peak (allele), were observed among the three plants at almost all SSR loci distributed across the entire diploid donor genome. Those few loci with extra peaks visualized as output from automated sequencing runs, generally low or ambiguous, might result from amplicons of paralogous members at the locus, non-specific sites, or unexpected recombinant alleles. No new alleles were found, suggesting the genomes remained stable and intact during gametogenesis and regeneration. The integrity of the haploid genome also was supported by array-CGH studies, in which genomic profiles were comparable to the diploid control. Conclusions The presence of few gene hybridization abnormalities, corroborated by gene dosage measurements, were hypothetically due to the segregation of hemizygous alleles and minor genomic rearrangements occurring during the haploidization procedure. In conclusion, these plants that are valuable genetic and breeding materials contain completely homozygous and essentially intact genomes.

Background and Aims: Despite differences in morphology, the genera representing ‘true citrus fruit trees’ are sexually compatible, and their phylogenetic relationships remain unclear. Most of the important commercial ‘species’ of Citrus are believed to be of interspecific origin. By studying polymorphisms of 27 nuclear genes, the average molecular differentiation between species was estimated and some phylogenetic relationships between ‘true citrus fruit trees’ were clarified. Methods: Sanger sequencing of PCR-amplified fragments from 18 genes involved in metabolite biosynthesis pathways and nine putative genes for salt tolerance was performed for 45 genotypes of Citrus and relatives of Citrus to mine single nucleotide polymorphisms (SNPs) and indel polymorphisms. Fifty nuclear simple sequence repeats (SSRs) were also analysed. Key Results: A total of 16 238 kb of DNA was sequenced for each genotype, and 1097 single nucleotide polymorphisms (SNPs) and 50 indels were identified. These polymorphisms were more valuable than SSRs for inter-taxon differentiation. Nuclear phylogenetic analysis revealed that Citrus reticulata and Fortunella form a cluster that is differentiated from the clade that includes three other basic taxa of cultivated citrus (C. maxima, C. medica and C. micrantha). These results confirm the taxonomic subdivision between the subgenera Metacitrus and Archicitrus. A few genes displayed positive selection patterns within or between species, but most of them displayed neutral patterns. The phylogenetic inheritance patterns of the analysed genes were inferred for commercial Citrus spp. Conclusions: Numerous molecular polymorphisms (SNPs and indels), which are potentially useful for the analysis of interspecific genetic structures, have been identified. The nuclear phylogenetic network for Citrus and its sexually compatible relatives was consistent with the geographical origins of these genera. The positive selection observed for a few genes will help further works to analyse the molecular basis of the variability of the associated traits. This study presents new insights into the origin of C. sinensis.

International audience; Citrus diversity is known to be essentially supported by three essential basic species, mandarin, pummelo and citron, which are at the origin of quite all the cultivated citrus. Based on studies of phenotype, the citron group (Citrus medica) is supposedly low in genetic diversity compared to mandarins and pummelos. To assess the diversity of the citron group we analyzed the molecular polymorphism of nuclear and cytoplasmic genetic markers and the variation in composition of essential oils from leaves and peels of 24 varieties of citron or apparent hybrids of citron. Our results demonstrate that the citron group has a relatively high allelic diversity, probably due to multiple introductions of different varieties. Self-fertilization was the mode of reproduction which most frequently led to the development of commercial varieties with specific aromas and flavors, such as the cultivar 'Corsican', a putative 'Common Poncire' self-crossed hybrid. However, some varieties appeared to be interspecific hybrids related to citron but only by the male parent. This varietal diversity was probably favored by spreading seeds to extend the cultivation of citron in Mediterranean countries. The chemical diversity does not always correspond to genetic diversity but nevertheless reveals specific profiles associated with some genotypes.

Rootstock provides better adaptation to biotic and abiotic constraints. Poncirus and its hybrids are widely used since they are tolerant to Citrus Tristeza Virus. However, when grafted, most of them are considered to be sensitive to salinity and water deficit. Diversity does exist within the Poncirus trifoliata specie but little is known regarding the behaviour variability of genotypes to abiotic constraints. The citrus diversity of 72 P. trifoliata genotypes was investigated using SSR markers. Two mayor genetic groups were clearly identified. Five genotypes, belonging to each genetic group, were then selected to investigate their properties of tolerance to water deficit. Water deficit was applied by withdrawing irrigation for 4 weeks. Physiological parameters such leaf stomatal conductance and quantum yield of PSII electron transport, soil water potential, leaf osmotic pressure and transpiration were estimated. Among genotypes, we observed that some genotypes such as' Rubidoux' were clearly more tolerant to water deficit than others such as 'Pomeroy'. Interestingly, the genotypes that were sensitive belonged to one genetic group and the tolerant ones belonged to the other group. Therefore, it sounds that among the Poncirus genus, it is possible to select genotypes as parent in breeding programs, which are more tolerant to water deficit. (Resume d'auteur)

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Background: With the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers. However, SNP chip data generally suffers from ascertainment biases caused by the SNP discovery and selection process in which a small number of individuals are used as discovery panels. The ongoing International Citrus Genome Consortium sequencing project of the highly heterozygous Clementine and sweet orange genomes will soon result in the release of several hundred thousand SNPs. The primary goals of this study were: (i) to estimate the transferability within the genus Citrus of SNPs discovered from Clementine BACend sequencing (BES), (ii) to estimate bias associated with the very narrow discovery panel, and (iii) to evaluate the usefulness of the Clementine-derived SNP markers for diversity analysis and comparative mapping studies between the different cultivated Citrus species. Results: Fifty-four accessions covering the main Citrus species and 52 interspecific hybrids between pummelo and Clementine were genotyped on a GoldenGate array platform using 1,457 SNPs mined from Clementine BES and 37 SNPs identified between and within C. maxima, C. medica, C. reticulata and C. micrantha. Consistent results were obtained from 622 SNP loci. Of these markers, 116 displayed incomplete transferability primarily in C. medica, C. maxima and wild Citrus species. The two primary biases associated with the SNP mining in Clementine were an overestimation of the C. reticulata diversity and an underestimation of the interspecific differentiation. However, the genetic stratification of the gene pool was high, with very frequent significant linkage disequilibrium. Furthermore, the shared intraspecific polymorphism and accession heterozygosity were generally enough to perform interspecific comparative genetic mapping. Conclusions: A set of 622 SNP markers providing consistent results was selected. Of the markers mined from Clementine, 80.5% were successfully transferred to the whole Citrus gene pool. Despite the ascertainment biases in relation to the Clementine origin, the SNP data confirm the important stratification of the gene pools around C. maxima, C. medica and C. reticulata as well as previous hypothesis on the origin of secondary species. The implemented SNP marker set will be very useful for comparative genetic mapping in Citrus and genetic association in C. reticulata.

Phytochemistry Luro, Francois Venturini, Nicolas Costantino, Gilles Paolini, Julien Ollitrault, Patrick Costa, Jean Research Support, Non-U.S. Gov't England Phytochemistry. 2012 May;77:186-96. doi: 10.1016/j.phytochem.2011.12.013. Epub 2012 Jan 19.; International audience; Native to southeast Asia, the citron (Citrus medica L.) was the first citrus fruit to be introduced to the Mediterranean area, in the third century BC, and remained its only citrus representative until the tenth century. The citron was used for its aroma - stemming from its essential oils in leaves and fruit peels - and as symbols in the Jewish religion. Subsequently, the cultivation of citron was extended significantly, peaking in the nineteenth century, when its fruits were used in cosmetics and confectioneries. The objec- tive of this study was to examine the genetic diversity of the Mediterranean citron with regard to the multiplication and dissemination practices that were related to its uses. We studied the polymorphisms of 27 nuclear and cytoplasmic genetic markers of 24 citron varieties, preserved in the citrus germplasm of INRA-CIRAD, San Giuliano, France. The composition of leaf essential oils was determined to establish varieties and phylogenic relationships between accessions. Other major citrus species were included in the molecular analysis, which demonstrated the existence of 13 geneti- cally linked citrons, differing from other citrus species, based on low heterozygosity and specific alleles; these citrons were considered true-type citrons, confirmed by their convergent chemical profiles.We also detected a polymorphism in the chloroplastic genome in these 13 citrons, which, when combined with allelic diversity of 2.4 alleles per locus, suggests that multiple citrons were introduced to the Mediterra- nean area in last 2 millennia. We determined the genetic origin and relationships of several varieties, such as Corsican, which could have arisen from the selfing of Poncire Commun. We noted a higher-than-expected polymorphism rate among Mediterranean citron varieties, likely due to crossfecundation. The chemical leaf oil composition of several economical varieties, such as Corsican, is distinct and can increase the quality of specific agriculture products for the cosmetics and candy industries.

International audience; Despite the economic importance of citrus, insights on the genetic response to stress are scarce. The aim of the present study was to compare fundamental citrus species for their response to photooxidative stress. The experiment was conducted under orchard conditions on three fundamental citrus species C. medica L., C. reticulata Blanco and C. maxima (Burm.) Merr., and on Fortunella japonica (Thunb.) Swing. We examined their respective net photosynthesis (Pnet), stomatal conductance (Gs) and chlorophyll fluorescence (Fv/Fm) on sun-acclimated leaves and shade-acclimated leaves returned under natural sunlight irradiance. To compare the respective response mechanism, we analyzed changes in oxidative status (hydrogen peroxide (H2O2) and malondialdehyde (MDA)), reactive oxygen species (ROS)-scavenging enzymes (superoxide dismutase (SOD), catalase, ascorbate peroxidase), recycling enzymes (monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase) and antioxidant metabolites (ascorbate and glutathione). Kumquat and pummelo exposed lower down-regulation and full recovery of photosynthetic parameters, lower accumulation of oxidized compounds associated with greater production of reduced glutathione (Gsh) and enhanced activity of the three ROS scavenging enzymes, especially SOD. Citron and mandarin showed a marked decrease and incomplete recovery in photosynthetic performance, mainly in Pnet and Fv/Fm, larger accumulation of oxidative parameters, slighter induction of antioxidant enzymes and down-regulation of reduced ascorbate (Asa) and Gsh synthesis. These results suggest that kumquat and pummelo have a greater tolerance to photooxidative stress than citron and mandarin.

Background Most modern citrus cultivars have an interspecific origin. As a foundational step towards deciphering the interspecific genome structures, a reference whole genome sequence was produced by the International Citrus Genome Consortium from a haploid derived from Clementine mandarin. The availability of a saturated genetic map of Clementine was identified as an essential prerequisite to assist the whole genome sequence assembly. Clementine is believed to be a ‘Mediterranean’ mandarin × sweet orange hybrid, and sweet orange likely arose from interspecific hybridizations between mandarin and pummelo gene pools. The primary goals of the present study were to establish a Clementine reference map using codominant markers, and to perform comparative mapping of pummelo, sweet orange, and Clementine. Results Five parental genetic maps were established from three segregating populations, which were genotyped with Single Nucleotide Polymorphism (SNP), Simple Sequence Repeats (SSR) and Insertion-Deletion (Indel) markers. An initial medium density reference map (961 markers for 1084.1 cM) of the Clementine was established by combining male and female Clementine segregation data. This Clementine map was compared with two pummelo maps and a sweet orange map. The linear order of markers was highly conserved in the different species. However, significant differences in map size were observed, which suggests a variation in the recombination rates. Skewed segregations were much higher in the male than female Clementine mapping data. The mapping data confirmed that Clementine arose from hybridization between ‘Mediterranean’ mandarin and sweet orange. The results identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. Conclusions A reference genetic map of citrus, used to facilitate the chromosome assembly of the first citrus reference genome sequence, was established. The high conservation of marker order observed at the interspecific level should allow reasonable inferences of most citrus genome sequences by mapping next-generation sequencing (NGS) data in the reference genome sequence. The genome of the haploid Clementine used to establish the citrus reference genome sequence appears to have been inherited primarily from the ‘Mediterranean’ mandarin. The high frequency of skewed allelic segregations in the male Clementine data underline the probable extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in male parents.

Two autotetraploid (4x) trifoliate orange (Poncirus trifoliata (L) Rat.) rootstocks and the corresponding diploid (2 x) rootstocks were compared for their impact on clementine "Commune" (Citrus clementina Hort. ex Tan) production and on rootstock/scion physiology. Cumulative fruit yield, fruit quality (sugars, organic acids, carotenoids, hesperidin) and scion physiology (stem growth, leaf starch, sugars, chlorophyll content, phenolic content and gas exchange parameters) were investigated. The cumulative fruit yield over 11 years was lower in 4x rootstock/clementine associations and correlated with reduced vigor of the clementine scion. Fruit quality criteria were not affected by the ploidy level of the rootstocks, with the exception of hesperidin content, which was higher with 4x than 2x rootstock/clementine associations. Leaf chlorophyll and phenolic contents were higher with 4x than 2x rootstock/clementine associations. In leaves, the rate of photosynthesis and stomatal conductance was much higher with 2x rootstocks; however, the maximum electron transport rate was lower when compared to the 4x rootstocks. Similarly, leaf starch and sugar contents were much higher with 4x than 2x. Taken together, these results demonstrate that the use of 4x rootstocks dramatically changes both tree physiology and fruit yield, without promoting large changes in fruit quality criteria. (C) 2011 Elsevier B.V. All rights reserved.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Genetic stratification associated with domestication history is a key parameter for estimating the pertinence of genetic association study within a gene pool. Previous molecular and phenotypic studies have shown that most of the diversity of cultivated citrus results from recombination between three main species: C. medica (citron), C. reticulata (mandarin) and C. maxima (pummelo). However, the precise contribution of each of these basic species to the genomes of secondary cultivated species, such as C. sinensis (sweet orange), C. limon (lemon), C. aurantium (sour orange), C. paradisi (grapefruit) and recent hybrids is unknown. Our study focused on: (1) the development of insertion-deletion (InDel) markers and their comparison with SSR markers for use in genetic diversity and phylogenetic studies; (2) the analysis of the contributions of basic taxa to the genomes of secondary species and modern cultivars and (3) the description of the organisation of the Citrus gene pool, to evaluate how genetic association studies should be done at the cultivated Citrus gene pool level. InDel markers appear to be better phylogenetic markers for tracing the contributions of the three ancestral species, whereas SSR markers are more useful for intraspecific diversity analysis. Most of the genetic organisation of the Citrus gene pool is related to the differentiation between C. reticulata, C. maxima and C. medica. High and generalised LD was observed, probably due to the initial differentiation between the basic species and a limited number of interspecific recombinations. This structure precludes association genetic studies at the genus level without developing additional recombinant populations from interspecific hybrids. Association genetic studies should also be affordable at intraspecific level in a less structured pool such as C. reticulata.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Tolerant citrus rootstocks are defined as Cl- excluders. However, little is known about the salt tolerance of cultivars used as scions, particularly the tolerance of monoembryonic citrus genotypes. To enhance the genetic resources for generating improved hybrid rootstocks, the evaluation of large samples of citrus species, including both monoembryonic and polyembryonic genotypes, is necessary. In this study, 12 citrus genotypes representing the major Citrus species and all the three genera of the Rutaceae family were subjected to moderate salt stress (75 mM) for 12 weeks to characterise their physiological response to salt stress. Various symptoms and physiological parameters were evaluated to characterise their salt sensitivity. These included plant growth (stem diameter), leaf chlorophyll content, leaf flavonoid content, maximum quantum yield of PSII [(F-m-F-0)/F-m)], net photosynthesis, stomatal conductance and leaf Na+ and Cl- contents. The results clearly demonstrated that the most salt sensitive genotypes accumulated high concentrations of Na+ and Cl- and maintained a fair growth and photosynthetic rate. By contrast, salt-tolerant genotypes accumulated less Na+ and Cl- and decreased their growth and gas exchange. Poncire commun citron and Marumi kumquat were the most sensitive species, while mandarins, pummelo and Australian sour orange were the most tolerant species. Among the genotypes, Engedi pummel presented a specific trait for salt tolerance that has not been previously reported. Taken together, the results suggest that low leaf chloride content can be used as an indicator of salt stress tolerance in citrus genotypes. Exploitation of this indicator will enable the improved evaluation of citrus genetic resources and should lead to the identification of new sources of tolerance for rootstock breeding.

International audience; A dinucleotide-enriched genomic library was obtained from mandarin orange (Citrus reticulata Blanco). A subset of 101 positive clones was sequenced and primers were designed. The loci were screened for levels of variation using 26-29 wild mandarin oranges collected in Vietnam. Forty-three loci were polymorphic with the number of alleles ranging from two to 18. The observed heterozygosity (H-O) and expected heterozygosity (H-E) were from 0.03 to 0.96 and from 0.03 to 0.92, respectively.

¿ Background and Aims: Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation. ¿ Methods: Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat (SSR) markers to analyse their genetic origin. Embryo rescue was used to grow all embryos contained in polyembryonic seeds of 'Tardivo di Ciaculli' mandarin, followed by characterization of the plantlets obtained by flow cytometry and SSR markers to accurately establish the rate of tetraploidization events and their potential tissue location. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Variation in tetraploid plantlet rates was analysed between different seedlings of the same genotype ('Carrizo' citrange; Citrus sinensis ¿ Poncirus trifoliata) from seeds collected in different tropical, subtropical and Mediterranean countries. ¿ Key Results: Tetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric. Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates. The higher frequencies (20 %) of tetraploids were observed for citranges cultivated in the Mediterranean area. ¿ Conclusions: Tetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression of tetraploidization. Tetraploid genotypes arising from chromosome doubling of apomictic citrus are extensively being used as parents in breeding programmes to develop seedless triploid cultivars and have potential direct use as new rootstocks. © The Author 2011. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved., This work was jointly financed by the AGL2008-00596-MCI and Prometeo 2008/121 Generalidad Valenciana projects and supported by the European Commission, under the FP6-2003-INCO-DEV-2 project CIBEWU (no. 015453). We thank J. A. Pina for growing plants in the greenhouse and in the field.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; All commercial citrus rootstocks are polyembryonic and propagated by seeds. Although these seeds produce uniform plant material most of the time, zygotic or polyploid citrus seedlings may arise. The aim of this study was to understand how zygotic or tetraploid rootstock could affect a citrus rootstock selection field trial. A trifoliate orange selection field trial, which was planted in 1974 and grafted with clementine, was re-investigated with respect to the presence of rootstocks that were not true-to-type. Among the 288 trees investigated, flow cytometry identified 2.4% of rootstocks as tetraploid and SSR markers indicated that 6.6% were zygotic. Yield data showed that the presence of tetraploid rootstock dramatically decreased (by about 45%) clementine fruit production. However, zygosity did not always affect fruit production and a range of effects were observed, from a slight increase in production to a 24% decrease. Exclusion of non-true-to-type genotypes from the production analysis indicated that the best candidate for rootstock was a clone previously ranked in the middle of the 32 rootstocks under evaluation. However, the presence of zygotic rootstock did not appear to cause any significant differences in fruit quality during the first 5 years of the investigation, which suggests that non-true-to-type plants cannot be identified by fruit quality parameters. This study indicates that tetraploid and zygotic rootstocks have a strong impact on citrus fruit production in orchards, and removal of off-type seedlings is required prior to planting in any agronomic trial.