Search

IntroductionUseful germplasm for citrus breeding includes all sexually compatible species of the former genera Citrus, Clymenia, Eremocitrus, Fortunella, Microcitrus, Oxanthera, and Poncirus, now merged in the single Citrus genus. An improved knowledge on the synteny/collinearity between the genome of these different species, and on their recombination landscapes, is essential to optimize interspecific breeding schemes.MethodWe have performed a large comparative genetic mapping study including several main clades of the Citrus genus. It concerns five species (C. maxima, C. medica, C. reticulata, C. trifoliata and C. glauca), two horticultural groups resulting from interspecific admixture (clementine and lemon) and two recent interspecific hybrids (C. australis x C. australasica and C. maxima x C. reticulata). The nine individual genetic maps were established from GBS data of 1,216 hybrids.Results and discussionThe number of SNPs mapped for each parent varies from 760 for C. medica to 4,436 for the C. maxima x C. reticulata hybrid, with an average of 2,162.3 markers by map. Their comparison with C. clementina v1.0 assembly and inter-map comparisons revealed a high synteny and collinearity between the nine genetic maps. Non-Mendelian segregation was frequent and specific for each parental combination. The recombination landscape was similar for the nine mapped parents, and large genomic regions with very low recombination were identified. A consensus genetic map was successfully established. It encompasses 10,756 loci, including 7,915 gene-based markers and 2,841 non-genic SNPs. The anchoring of the consensus map on 15 published citrus chromosome-scale genome assemblies revealed a high synteny and collinearity for the most recent assemblies, whereas discrepancies were observed for some older ones. Large structural variations do not seem to have played a major role in the differentiation of the main species of the Citrus genus. The consensus genetic map is a useful tool to check the accuracy of genome assemblies, identify large structural variation and focus on analyzing potential relationships with phenotypic variations. It should also be a reference framework to integrate the positions of QTLs and useful genes identified in different analyses.

IntroductionCitrus is an important fruit crop for human health. The sensitivity of citrus trees to a wide range of abiotic stresses is a major challenge for their overall growth and productivity. Among these abiotic stresses, salinity results in a significant loss of global citrus yield. In order to find straightforward and sustainable solutions for the future and to ensure citrus productivity, it is of paramount importance to decipher the mechanisms responsible for salinity stress tolerance. Thisstudy aimed to investigate how ploidy levels influence salt stress tolerance in citrus by comparing the transcriptomic responses of diploid and tetraploid genotypes. In a previous article we investigated the physiological and biochemical response of four genotypes with different ploidy levels: diploid trifoliate orange (Poncirus trifoliata [L.] Raf.) (PO2x) and Cleopatra mandarin (Citrus reshni Hort. Ex Tan.) (CL2x) and their respective tetraploids (PO4x, CL4x).MethodsIn this study, we useda multifactorial gene selection and gene clustering approach to finely dissect the influence of ploidy level on the salt stress response of each genotype. Following transcriptome sequencing, differentially expressed genes (DEGs) were identified in response to salt stress in leaves and roots of the different citrus genotypes.Result and discussionGene expression profiles and functional characterization of genes involved in the response to salt stress, as a function of ploidy level and the interaction between stress response and ploidy level, have enabled us to highlight the mechanisms involved in the varieties tested. Saltstress induced overexpression of carbohydrate biosynthesis and cell wall remodelling- related genes specifically in CL4x Ploidy level enhanced oxidative stress response in PO and ion management capacity in both genotypes. Results further highlighted that under stress conditions, only the CL4x genotype up- regulated genes involved in sugar biosynthesis, transport management, cell wall remodelling, hormone signalling, enzyme regulation and antioxidant metabolism. These findings provide crucial insights that could inform breeding strategies for developing salt-tolerant citrus varieties.

Citrus polyploidy is associated with a wide range of morphological, genetic, and physiological changes that are often advantageous for breeding. Citrus triploid hybrids are very interesting as new seedless varieties. However, tetraploid rootstocks promote adaptation to different abiotic stresses and promote resilience. Triploid and tetraploid hybrids can be obtained through sexual hybridizations using tetraploid parents (2x × 4x, 4x × 2x, or 4x × 4x), but more knowledge is needed about the inheritance pattern of tetraploid parents to optimize the efficiency of triploid varieties and tetraploid rootstock breeding strategies. In this work, we have analyzed the inheritance pattern of three tetraploid genotypes: ‘Chandler’ pummelo (Citrus maxima) and ‘Cleopatra’ mandarin (Citrus reticulata), which represent two clear examples of autotetraploid plants constituted by the genome of a single species, and the ‘Sevillano’ sour orange, which is an allotetraploid interspecific hybrid between C. maxima and C. reticulata. Polymorphic simple sequence repeat (SSR) and single-nucleotide polymorphism (SNP) markers were used to estimate parental heterozygosity restitution, and allele frequencies for centromeric loci were used to calculate the preferential pairing rate related to the proportion of disomic and tetrasomic segregation. The tetraploid pummelo and mandarin displayed tetrasomic segregation. Sour orange evidenced a clear intermediate inheritance for five of the nine chromosomes (1, 2, 5, 7, and 8), a slight tendency toward tetrasomic inheritance on chromosome 3, and intermediate inheritance with a tendency toward disomy for chromosomes 4, 6, and 9. These results indicate that the interspecific versus intraspecific phylogenomic origin affects preferential pairing and, therefore, the inheritance patterns. Despite its high level of heterozygosity, the important preferential chromosome pairing observed in sour orange results in a limited diversity of the genotypic variability of its diploid gametes, and consequently, a large part of the genetic value of the original diploid sour orange is transferred to the tetraploid progenies.

Huanglongbing (HLB) is a disease that is responsible for the death of millions of trees worldwide. The bacterial causal agent belongs to Candidatus Liberibacter spp., which is transmitted by psyllids. The bacterium lead most of the time to a reaction of the tree associated with callose synthesis at the phloem sieve plate. Thus, the obstruction of pores providing connections between adjacent sieve elements will limit the symplastic transport of the sugars and starches synthesized through photosynthesis. In the present article, we investigated the impact of the use of tetraploid Swingle citrumelo (Citrus paradisi Macfrad × Poncirus trifoliata [L.] Raf) rootstock on HLB tolerance, compared to its respective diploid. HLB-infected diploid and tetraploid rootstocks were investigated when grafted with Mexican and Persian limes. Secondary roots were anatomically studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to observe callose deposition at the phloem sieve plate and to evaluate the impact of the bacterium’s presence at the cellular level. Voltammetry of immobilized microparticles (VIMP) in roots was applied to determine the oxidative stress status of root samples. In the field, Mexican and Persian lime leaves of trees grafted onto tetraploid rootstock presented less symptoms of HLB. Anatomical analysis showed much stronger secondary root degradation in diploid rootstock, compared to tetraploid rootstock. Analysis of the root sieve plate in control root samples showed that pores were approximately 1.8-fold larger in tetraploid Swingle citrumelo than in its respective diploid. SEM analyses of root samples did not reveal any callose deposition into pores of diploid and tetraploid genotypes. VIMP showed limited oxidative stress in tetraploid samples, compared to diploid ones. These results were even strongly enhanced when rootstocks were grafted with Persian limes, compared to Mexican limes, which was corroborated by stronger polyphenol contents. TEM analysis showed that the bacteria was present in both ploidy root samples with no major impacts detected on cell walls or cell structures. These results reveal that tetraploid Swingle citrumelo rootstock confers better tolerance to HLB than diploid. Additionally, an even stronger tolerance is achieved when the triploid Persian lime scion is associated.

Citrus are classified as salt-sensitive crops. However, a large diversity has been observed regarding the trends of tolerance among citrus. In the present article, physiological and biochemical studies of salt stress tolerance were carried out according to the level of polyploidy of different citrus genotypes. We particularly investigated the impact of tetraploidy in trifoliate orange (Poncirus trifoliata (L.) Raf.) (PO4x) and Cleopatra mandarin (Citrus reshni Hort. Ex Tan.) (CL4x) on the tolerance to salt stress compared to their respective diploids (PO2x and CL2x). Physiological parameters such as gas exchange, ions contents in leaves and roots were analyzed. Roots and leaves samples were collected to measure polyphenol, malondialdehyde (MDA), ascorbate and H2O2 contents but also to measure the activities of enzymes involved in the detoxification of active oxygen species (ROS). Under control conditions, the interaction between genotype and ploidy allowed to discriminate different behavior in terms of photosynthetic and antioxidant capacities. These results were significantly altered when salt stress was applied when salt stress was applied. Contrary to the most sensitive genotype, that is to say the diploid trifoliate orange PO2x, PO4x was able to maintain photosynthetic activity under salt stress and had better antioxidant capacities. The same observation was made regarding the CL4x genotype known to be more tolerant to salt stress. Our results showed that tetraploidy may be a factor that could enhance salt stress tolerance in citrus.

Huanglongbing (HLB), the most destructive citrus disease, is associated with unculturable, phloem-limited Candidatus Liberibacter species, mainly Ca. L. asiaticus (Las). Las is transmitted naturally by the insect Diaphorina citri. In a previous study, we determined that the Oceanian citrus relatives Eremocitrus glauca, Microcitrus warburgiana, Microcitrus papuana, and Microcitrus australis and three hybrids among them and Citrus were full-resistant to Las. After 2 years of evaluations, leaves of those seven genotypes remained Las-free even with their susceptible rootstock being infected. However, Las was detected in their stem bark above the scion-rootstock graft union. Aiming to gain an understanding of the full-resistance phenotype, new experiments were carried out with the challenge-inoculated Oceanian citrus genotypes through which we evaluated: (1) Las acquisition by D. citri fed onto them; (2) Las infection in sweet orange plants grafted with bark or budwood from them; (3) Las infection in sweet orange plants top-grafted onto them; (4) Las infection in new shoots from rooted plants of them; and (5) Las infection in new shoots of them after drastic back-pruning. Overall, results showed that insects that fed on plants from the Oceanian citrus genotypes, their canopies, new flushes, and leaves from rooted cuttings evaluated remained quantitative real-time polymerase chain reaction (qPCR)-negative. Moreover, their budwood pieces were unable to infect sweet orange through grafting. Furthermore, sweet orange control leaves resulted infected when insects fed onto them and graft-receptor susceptible plants. Genomic and morphological analysis of the Oceanian genotypes corroborated that E. glauca and M. warburgiana are pure species while our M. australis accession is an M. australis × M. inodora hybrid and M. papuana is probably a M. papuana × M. warburgiana hybrid. E. glauca × C. sinensis hybrid was found coming from a cross between E. glauca and mandarin or tangor. Eremocitrus × Microcitrus hybrid is a complex admixture of M. australasica, M. australis, and E. glauca while the last hybrid is an M. australasica × M. australis admixture. Confirmation of consistent full resistance in these genotypes with proper validation of their genomic parentages is essential to map properly genomic regions for breeding programs aimed to generate new Citrus-like cultivars yielding immunity to HLB.

Sexual breeding at the tetraploid level is a promising strategy for rootstock breeding in citrus. Due to the interspecific origin of most of the conventional diploid citrus rootstocks that produced the tetraploid germplasm, the optimization of this strategy requires better knowledge of the meiotic behavior of the tetraploid parents. This work used Genotyping By Sequencing (GBS) data from 103 tetraploid hybrids to study the meiotic behavior and generate a high-density recombination landscape for their tetraploid intergenic Swingle citrumelo and interspecific Volkamer lemon progenitors. A genetic association study was performed with root architecture traits. For citrumelo, high preferential chromosome pairing was revealed and led to an intermediate inheritance with a disomic tendency. Meiosis in Volkamer lemon was more complex than that of citrumelo, with mixed segregation patterns from disomy to tetrasomy. The preferential pairing resulted in low interspecific recombination levels and high interspecific heterozygosity transmission by the diploid gametes. This meiotic behavior affected the efficiency of Quantitative Trait Loci (QTL) detection. Nevertheless, it enabled a high transmission of disease and pest resistance candidate genes from P. trifoliata that are heterozygous in the citrumelo progenitor. The tetrazyg strategy, using doubled diploids of interspecific origin as parents, appears to be efficient in transferring the dominant traits selected at the parental level to the tetraploid progenies.

Polyembryony and male sterility (MS) are essential characters for citrus breeding. MS, coupled with parthenocarpy, allows for addressing the diversification of diploid seedless mandarin varieties, and nucleocytoplasmic MS is the most prevalent system. Polyembryony limits the use of seed parents in scion breeding programs, and the recovery of monoembryonic hybrids to be used as female parents is a crucial pre-breeding component. The objectives of this work were the identification of SNPs closely linked with the genes implied in these traits for marker-assisted selection. Genotyping by sequencing was used to genotype 61 diploid hybrids from an F1 progeny recovered from crossing ‘Kiyomi’ and ‘Murcott’ tangors. A total of 6444 segregating markers were identified and used to establish the two parental genetic maps. They consisted of 1374 and 697 markers encompassing 1416.287 and 1339.735 cM for ‘Kiyomi’ and ‘Murcott’, respectively. Phenotyping for MS and polyembryony was performed. The genotype–trait association study identified a genomic region on LG8 which was significantly associated with MS, and a genomic region on LG1 which was significantly associated with polyembryony. Annotation of the identified region for MS revealed 19 candidate genes. One SNP KASPar marker was developed and fully validated for each trait.

Huanglongbing (HLB) is presently a major threat to the citrus industry. Because of this disease, millions of trees are currently dying worldwide. The putative causal agent is a motile bacteria belonging to Candidatus Liberibacter spp., which is transmitted by psyllids. The bacteria is responsible for the synthesis of callose at the phloem sieve plate, leading to the obstruction of the pores that provide connections between adjacent sieve elements, thus limiting the symplastic transport of the sugars and starches synthesized in leaves to the other plant organs. The Persian triploid lime (Citrus latifolia) is one of the most HLB-tolerant citrus varieties, but the determinants associated with the tolerance are still unknown. HLB-infected diploid Mexican lime (Citrus aurantiifolia) and Persian lime were investigated. The leaf petiole was analyzed using scanning electron microscopy (SEM) to observe callose deposition at the phloem sieve plate. Leaf starch contents and detoxification enzyme activities were investigated. In the field, Persian lime leaves present more limited symptoms due to HLB than the Mexican lime leaves do. Photosynthesis, stomatal conductance, and transpiration decreased compared with control plants, but values remained greater in the Persian than in the Mexican lime. Analysis of the petiole sieve plate in control petiole samples showed that pores were approximately 1.8-fold larger in the Persian than in the Mexican lime. SEM analyses of petiole samples of symptomatic leaves showed the important deposition of callose into pores of Mexican and Persian limes, whereas biochemical analyses revealed better detoxification in Persian limes than in Mexican limes. Moreover, SEM analyses of infected petiole samples of asymptomatic leaves showed much larger callose depositions into the Mexican lime pores than in the Persian lime pores, whereas biochemical traits revealed much better behavior in Persian limes than in Mexican limes. Our results reveal that polyploids present specific behaviors associated with important physiological and biochemical determinants that may explain the better tolerance of the Persian lime against HLB compared with the Mexican lime.

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.

The triploid ‘Tahiti’ lime (C. x latifolia (Yu. Tanaka) Tanaka) naturally originated from a merger between a haploid ovule of lemon (C. x limon (L.) Burm) and a diploid pollen from a ‘Mexican’ lime (C. x aurantiifolia (Christm.) Swing). The very limited natural inter-varietal diversity and gametic sterility of C. latifolia requires a phylogenomic based reconstruction breeding strategy to insure its diversification. We developed a strategy based on interploid hybridization between diploid lemon and the doubled diploid ‘Giant Key’ lime. This lime is a doubled diploid of ‘Mexican’ lime, itself a natural interspecific F1 hybrid between C. medica L. and C. micrantha Wester. For an optimized breeding program, we analyzed the meiotic behavior of the allotetraploid lime, the genetic structure of its diploid gametes, the interspecific recombination between C. medica and C. micrantha, and constructed its genetic map. A population of 272 triploid hybrids was generated using ‘Giant Key’ lime as pollinator. One hundred fifty-eight SNPs diagnostic of C. micrantha, regularly distributed throughout the citrus genome were successfully developed and applied. The genetic structure of the diploid gametes was examined based on C. micrantha doses along the genome. The diploid gametes transmitted in average 91.17% of the parental interspecific C. medica/C. micrantha heterozygosity. Three chromosomes (2, 8, and 9) showed disomic segregation with high preferential pairing values, while the remaining chromosomes showed an intermediate inheritance with a preferential disomic trend. A total of 131 SNPs were assigned to nine linkage groups to construct the genetic map. It spanned 272.8 cM with a low average recombination rate (0.99 cM Mb-1) and high synteny and colinearity with the reference clementine genome. Our results confirmed that an efficient reconstruction breeding strategy for ‘Tahiti’ lime is possible, based on interploid hybridization using a doubled diploid of C. aurantiifolia. The tetraploid parent should be selected for favorable agronomic traits and its genetic value should be efficiently inherited by the progeny thanks to transmission of the high level of parental heterozygosity. However, it would require developing numerous progeny to overcome the linkage drag caused by the limited interspecific recombination associated with the predominant disomic inheritance.

Rootstock is widely used for the cultivation of citrus fruits because it brings resistance or tolerance to diseases or environmental constrains and modulates the fruit quality. Polyploidization is a widespread improvement strategy in citrus. The objective was to evaluate the effect of rootstock and ploidy level on the composition of essential oils. Two trials were conducted, one displaying a ‘Navelina’ orange grafted on three rootstocks and a second combining two ploidy levels (di and tetraploid) of scion (‘Pineapple’ orange) and rootstock (‘Carrizo’ citrange). The composition of peel essential oil (PEO) was analyzed by gas chromatography coupled with mass spectrometry, and a panel of experts analyzed its flavor variation with a triangle test approach. The rootstock influenced the yield and composition of the orange PEO, with a low impact on flavor. Neither the rootstock nor the scion ploidy level affected the PEO yield. Only the tetraploid level of the scion significantly modified the PEO composition, reducing the oxygenated compound fraction. Sensitive significant differences were detected between the reference sample (diploid scion–diploid rootstock) and the three other combinations. These results suggest that for the profiling of an aromatic flavor, the rootstock is a key element as is the ploidy level of the scion.

Self-incompatibility (SI) is present in around half of all species of flowering plants. SI limits endogamy and contributes to increased genetic diversity. SI is a very important trait in citrus because, when coupled with parthenocarpy, it allows seedless fruit production. Otherwise, SI is an impediment to genetic studies and breeding programs. Temperature stress, bud pollination and polyploidization can induce the breakdown of the SI mechanism in several species. In this work, we investigated how the SI mechanism can be broken down in two self-incompatible diploid citrus genotypes: ‘Fortune’ mandarin and ‘Clemenules’ clementine. The influence of temperature stress on the SI mechanism was assessed in self-pollinated flowers of ‘Fortune’ mandarins subjected to 2 temperature regimes (10 °C and 30 °C), whereas the bud pollination effect was investigated in the same genotype and in ‘Clemenules’ clementines cultivated under field conditions. The tetraploid ‘Clemenules’ clementine cultivated under field conditions was used to study if tetraploidization can bypass the SI reaction. Histological observations of pollen tube growth and seed production in self-pollinated flowers were used to evaluate the breakdown of SI, while the genetic analysis with SSR and SNP markers confirmed that all recovered plants were zygotic and had been originated by selfing. Our results confirm that the SI reaction can be surpassed by temperature stress, bud pollination and tetraploidy. To our knowledge, this is the first report in citrus in which the SI reaction breakdown by these three different strategies is demonstrated by molecular markers.

Rootstocks are an important component for citrus adaptation to increasing biotic and abiotic stresses resulting from global climate change. There is a strong complementarity between Citrus species, which adapt to abiotic stresses, and Poncirus trifoliata and its intergeneric hybrids, which exhibit resistances or tolerances to major diseases and pests. Thus, symmetrical somatic hybridization between complementary diploid rootstocks of these two genera appears to be an efficient way to develop new tetraploid rootstocks in order to address the new challenges of the citrus industry. New intergeneric somatic hybrids were obtained by electrofusion between protoplasts of Citrus and P. trifoliata hybrids. Extensive characterization of the nuclear and cytoplasmic genomes was performed by genotyping-by-sequencing (GBS) analysis. This revealed diploid cybrids and nuclear somatic hybrids. Mitochondrial genomes were mostly inherited from the callus parent, but homologous recombination events were observed for one parental combination. Chloroplasts exhibited random uniparental inheritance. GBS revealed local chromosomal instabilities for all nuclear somatic hybrids and whole chromosome eliminations for two hybrids. However, at the whole genome level, symmetrical addition of the nuclear genomes of both parents was predominant and all somatic hybrids displayed at least one trifoliate orange haplotype throughout the genome.

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.

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.

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.

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 (>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.

The Papeda 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.

Self-incompatibility is an important evolutionary feature in angiosperms and has major implications for breeding strategies in horticultural crops. In citrus, when coupled with parthenocarpy, it enables the production of seedless fruits in a mono-varietal orchard. A gametophytic incompatibility system with one S locus was proposed for citrus, but its molecular mechanisms remain the subject of debate. The objective of this work was to locate the S locus by the analyzing segregation distortion in reciprocal crosses of two self-incompatible citrus sharing one self-incompatible allele and to compare this location with previously published models. High density genetic maps of ‘Fortune’ mandarin and ‘Ellendale tangor’ with, respectively, 2164 SNP and 1467 SNP markers, were constructed using genotyping by sequencing data. They are highly syntenic and collinear with the clementine genome. Complete rejection of one allele was only observed in male segregation in the two parents and in only one genomic area, at the beginning of chromosome 7 of the clementine reference genome. Haplotype data in the area surrounding the theoretical S locus were in agreement with previously proposed S genotypes. Overall, our results are in full agreement with the recently proposed gametophytic S-RNase system with the S locus at the beginning of chromosome 7 of the clementine reference genome.

Rootstocks are crucial for the sustainability of the citrus industry worldwide. Diploid intergeneric Citrus × 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. × paradisi Macfad. × P. trifoliata) and to analyze the meiotic behavior of tetraploid citrumelo and citrandarin (C. reticulata Blanco × 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.

The cosmetic and fragrance industry largely exploits citrus essential oils (EOs) because of their aromatic properties. EO compositions are complex and differ between fruit pericarp (PEO) and leaf (LEO). Citrus fruit grow in many countries under very different climates. Seventeen citrus cultivars were selected and their similarities between the two collections were verified by SSR (Single Sequence Repeat) and InDel (Insertion and Deletion) markers to assess the effects of the environment and cultivation practices on the EO yield and composition. LEOs and PEOs were extracted by water distillation and analyzed by GC-MS. PEO yields were generally higher in Corsica than in Bahia, especially in the citron family. PEOs in this family were richer in limonene in Bahia than in Corsica while, conversely, neral, geranial and derivatives were present in a higher proportion in Corsican varieties. A few minor components were site-specific, such as nookaton, a pummelo-specific compound that was not present in grapefruit cultivated in Bahia. If climate change over the last 20 years has not affected the PEO composition in Corsica, the contrasted environmental conditions and cultural practices between Bahia and Corsica could possibly explain the EO variations.

The creation of intergeneric somatic hybrids between Citrus and Poncirus is an efficient approach for citrus rootstock breeding, offering the possibility of combining beneficial traits from both genera into novel rootstock lineages. These somatic hybrids are also used as parents for further tetraploid sexual breeding. In order to optimize these latter breeding schemes, it is essential to develop knowledge on the mode of inheritance in the intergeneric tetraploid hybrids. We assessed the meiotic behavior of an intergeneric tetraploid somatic hybrid resulting from symmetric protoplast fusion of diploid Citrus reticulata and diploid Poncirus trifoliata. The analysis was based on the segregation patterns of 16 SSR markers and 9 newly developed centromeric/pericentromeric SNP markers, representing all nine linkage groups of the Citrus genetic map. We found strong but incomplete preferential pairing between homologues of the same ancestral genome. The proportion of gametes that can be explained by random meiotic chromosome associations (τ) varied significantly between chromosomes, from 0.09 ± 0.02 to 0.47 ± 0.09, respectively, in chromosome 2 and 1. This intermediate inheritance between strict disomy and tetrasomy, with global preferential disomic tendency, resulted in a high level of intergeneric heterozygosity of the diploid gametes. Although limited, intergeneric recombinations occurred, whose observed rates, ranging from 0.09 to 0.29, respectively, in chromosome 2 and 1, were significantly correlated with τ. Such inheritance is of particular interest for rootstock breeding because a large part of the multi-trait value selected at the teraploid parent level is transmitted to the progeny, while the potential for some intergeneric recombination offers opportunities for generating plants with novel allelic combinations that can be targeted by selection.

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.

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.

The mandarin horticultural group is an important component of world citrus production for the fresh fruit market. This group formerly classified as C. reticulata is highly polymorphic and recent molecular studies have suggested that numerous cultivated mandarins were introgressed by C. maxima (the pummelos). C. maxima and C. reticulata are also the ancestors of sweet and sour oranges, grapefruit, and therefore of all the "small citrus" modern varieties (mandarins, tangors, tangelos) derived from sexual hybridization between these horticultural groups. Recently, NGS technologies have greatly modified how plant evolution and genomic structure are analyzed, moving from phylogenetics to phylogenomics. The objective of this work was to develop a workflow for phylogenomic inference from Genotyping By Sequencing (GBS) data and to analyze the interspecific admixture along the nine citrus chromosomes for horticultural groups and recent varieties resulting from the combination of the C. reticulata and C. maxima gene pools. A GBS library was established from 55 citrus varieties, using the ApekI restriction enzyme and selective PCR to improve the read depth. Diagnostic polymorphisms (DPs) of C. reticulata/C. maxima differentiation were identified and used to decipher the phylogenomic structure of the 55 varieties. The GBS approach was powerful and revealed 30,289 SNPs and 8,794 Indels with 12.6% of missing data. 11,133 DPs were selected covering the nine chromosomes with a higher density in genic regions. GBS combined with the detection of DPs was powerful for deciphering the "phylogenomic karyotypes" of cultivars derived from admixture of the two ancestral species after a limited number of interspecific recombinations. All the mandarins, mandarin hybrids, tangelos and tangors analyzed displayed introgression of C. maxima in different parts of the genome. C. reticulata/C. maxima admixture should be a major component of the high phenotypic variability of this germplasm opening up the way for association studies based on phylogenomics.

Most cultivated Citrus species originated from interspecific hybridisation between four ancestral taxa (C. reticulata, C. maxima, C. medica, and C. micrantha) with limited further interspecific recombination due to vegetative propagation. This evolution resulted in admixture genomes with frequent interspecific heterozygosity. Moreover, a major part of the phenotypic diversity of edible citrus results from the initial differentiation between these taxa. Deciphering the phylogenomic structure of citrus germplasm is therefore essential for an efficient utilization of citrus biodiversity in breeding schemes. The objective of this work was to develop a set of species-diagnostic single nucleotide polymorphism (SNP) markers for the four Citrus ancestral taxa covering the nine chromosomes, and to use these markers to infer the phylogenomic structure of secondary species and modern cultivars. Species-diagnostic SNPs were mined from 454 amplicon sequencing of 57 gene fragments from 26 genotypes of the four basic taxa. Of the 1,053 SNPs mined from 28,507 kb sequence, 273 were found to be highly diagnostic for a single basic taxon. Species-diagnostic SNP markers (105) were used to analyse the admixture structure of varieties and rootstocks. This revealed C. maxima introgressions in most of the old and in all recent selections of mandarins, and suggested that C. reticulata × C. maxima reticulation and introgression processes were important in edible mandarin domestication. The large range of phylogenomic constitutions between C. reticulata and C. maxima revealed in mandarins, tangelos, tangors, sweet oranges, sour oranges, grapefruits, and orangelos is favourable for genetic association studies based on phylogenomic structures of the germplasm. Inferred admixture structures were in agreement with previous hypotheses regarding the origin of several secondary species and also revealed the probable origin of several acid citrus varieties. The developed species-diagnostic SNP marker set will be useful for systematic estimation of admixture structure of citrus germplasm and for diverse genetic studies.

Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the "grapefruit juice effect". Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas) and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus varieties for use in creating species devoid of these toxic compounds in future breeding programs.

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.

Despite potential benefits granted by genetically modified (GM) fruit trees, their release and commercialization raises concerns about their potential environmental impact, and the transfer via pollen of transgenes to cross-compatible cultivars is deemed to be the greatest source for environmental exposure. Information compiled from field trials on GM trees is essential to propose measures to minimize the transgene dispersal. We have conducted a field trial of seven consecutive years to investigate the maximum frequency of pollen-mediated crop-to-crop transgene flow in a citrus orchard, and its relation to the genetic, phenological and environmental factors involved.Three different citrus genotypes carrying the uidA (GUS) tracer marker gene (pollen donors) and a non-GM self-incompatible contiguous citrus genotype (recipient) were used in conditions allowing natural entomophilous pollination to occur. The examination of 603 to 2990 seeds per year showed unexpectedly low frequencies (0.17-2.86%) of transgene flow. Paternity analyses of the progeny of subsets of recipient plants using 10 microsatellite (SSR) loci demonstrated a higher mating competence of trees from another non-GM pollen source population that greatly limited the mating chance of the contiguous cross-compatible and flowering-synchronized transgenic pollen source. This mating superiority could be explained by a much higher pollen competition capacity of the non-GM genotypes, as was confirmed through mixed-hand pollinations.Pollen competition strongly contributed to transgene confinement. Based on this finding, suitable isolation measures are proposed for the first time to prevent transgene outflow between contiguous plantings of citrus types that may be extendible to other entomophilous transgenic fruit tree species.

Premise of the study: Single nucleotide polymorphism (SNP) markers based on Competitive Allele-Specific PCR (KASPar) were developed from sequences of three Citrus species. Their transferability was tested in 63 Citrus genotypes and 19 relative genera of the subfamily Aurantioideae to estimate the potential of SNP markers, selected from a limited intrageneric discovery panel, for ongoing broader diversity analysis at the intra- and intergeneric levels and systematic germplasm bank characterization. Methods and Results: Forty-two SNP markers were developed using KASPar technology. Forty-one were successfully genotyped in all of the Citrus germplasm, where intra- and interspecific polymorphisms were observed. The transferability and diversity decreased with increasing taxonomic distance. Conclusions: SNP markers based on the KASPar method developed from sequence data of a limited intrageneric discovery panel provide a valuable molecular resource for genetic diversity analysis of germplasm within a genus and should be useful for germplasm fingerprinting at a much broader diversity level.