Inheritance pattern of tetraploids pummelo, mandarin, and their interspecific hybrid sour orange is highly influenced by their phylogenomic structure.

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.

Inheritance and Quantitative Trait Loci Mapping of Aromatic Compounds from Clementine (Citrus × clementina Hort. ex Tan.) and Sweet Orange (C. × sinensis (L.) Osb.) Fruit Essential Oils.

Despite their importance in food processing, perfumery and cosmetics, the inheritance of sweet orange aromatic compounds, as well as their yield in the fruit peel, has been little analyzed. In the present study, the segregation of aromatic compounds was studied in an F1 population of 77 hybrids resulting from crosses between clementine and blood sweet orange. Fruit-peel essential oils (PEOs) extracted by hydrodistillation were analyzed by gas chromatography coupled with flame ionization detection. Genotyping by sequencing was performed on the parents and the hybrids. The resulting "clementine × sweet blood orange" genetic map consists of 710 SNP markers distributed in nine linkage groups (LGs), representing the nine citrus chromosomes, and spanning 1054 centimorgans. Twenty quantitative trait loci (QTLs) were identified, explaining between 20.5 and 55.0% of the variance of the major aromatic compounds and PEO yield. The QTLs for monoterpenes and aliphatic aldehydes predominantly colocalized on LGs 5 and 8, as did the two QTLs for PEO yield. The sesquiterpene QTLs were located on LGs 1, 3, 6 and 8. The detection of major QTLs associated with the synthesis of aliphatic aldehydes, known for their strong aromatic properties, open the way for marker-assisted selection.

Insight into Physiological and Biochemical Determinants of Salt Stress Tolerance in Tetraploid Citrus.

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.

Genotyping by Sequencing for SNP-Based Linkage Analysis and the Development of KASPar Markers for Male Sterility and Polyembryony in Citrus.

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.

Meiotic Behaviors of Allotetraploid Citrus Drive the Interspecific Recombination Landscape, the Genetic Structures, and Traits Inheritance in Tetrazyg Progenies Aiming to Select New Rootstocks.

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.

Better tolerance to Huanglongbing is conferred by tetraploid Swingle citrumelo rootstock and is influenced by the ploidy of the scion.

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.

Insight into resistance to 'Candidatus Liberibacter asiaticus,' associated with Huanglongbing, in Oceanian citrus genotypes.

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.

Specific Physiological and Anatomical Traits Associated With Polyploidy and Better Detoxification Processes Contribute to Improved Huanglongbing Tolerance of the Persian Lime Compared With the Mexican Lime.

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.

Chemical Variability of Peel and Leaf Essential Oils in the Citrus Subgenus Papeda (Swingle) and Few Relatives.

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 ß-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.

Characterization of Odor-Active Compounds of Ichang Lemon (Citrus wilsonii Tan.) and Identification of Its Genetic Interspecific Origin by DNA Genotyping.

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 × 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.

Preferential Disomic Segregation and C. micrantha/C. medica Interspecific Recombination in Tetraploid 'Giant Key' Lime; Outlook for Triploid Lime Breeding.

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.

Resistance to 'Candidatus Liberibacter asiaticus,' the Huanglongbing Associated Bacterium, in Sexually and/or Graft-Compatible Citrus Relatives.

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.

Male and female inheritance patterns in tetraploid 'Moncada' mandarin.

KEY MESSAGE: Tetraploid `Moncada´ mandarin, used as male and female in interploidy hybridizations, displays mainly tetrasomic inheritance for most LGs, with slight variations according to the direction of the crossing. 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.

Phylogenetic Origin of Primary and Secondary Metabolic Pathway Genes Revealed by C. maxima and C. reticulata Diagnostic SNPs.

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.

Genetic, morphological and chemical investigations reveal the genetic origin of Pompia (C. medica tuberosa Risso & Poiteau) - An old endemic Sardinian citrus fruit.

Citrus fruits have been introduced to the Mediterranean area from Asia for centuries and spontaneous crosses have generated several hybrid forms, some of which have had agricultural or industrial success while others have remained niche food or ornamental products, or have disappeared. Pompia (C. medica tuberosa Risso & Poiteau) is an old endemic citrus fruit from Sardinia of unknown genetic origin. Initial phenotypic and molecular characterizations revealed a high degree of similarity with lemon (C. limon (L.) Burm.) and citron (C. medica L.). To identify the ancestors of Pompia, 70 citrus species of the Citrus genus were genotyped with 36 codominant molecular markers (SSR and InDel) of nuclear and cytoplasmic genomes. Diversity analysis and allelic comparisons between each citrus species at each locus indicated that Pompia resembles lemon and limonette of Marrakech, i.e. the result of a cross between sour orange (C. aurantium L.) and citron, where citron was the pollinator. Two Italian citron varieties were identified as potential male parents, i.e. Diamante and Common Poncire. However, we were unable to differentiate varieties of sour oranges because varietal diversification in this horticultural group resulted from DNA sequence variations that SSR or InDel markers could not reveal. Rhob el Arsa and Poncire de Collioure were found to be two synonyms of Pompia. Pompia appeared to be equally distinct from citron, lemon and sour orange based on the overall analysis of the fruit, leaf and seed phenotype, and juice chemical composition. At the leaf level, the Pompia essential oil (EO) composition is close to that of citron whereas the zest is much closer to that of sour orange.

Facing Climate Change: Biotechnology of Iconic Mediterranean Woody Crops.

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.

Genotyping by sequencing can reveal the complex mosaic genomes in gene pools resulting from reticulate evolution: a case study in diploid and polyploid citrus.

BACKGROUND AND AIMS: Reticulate evolution, coupled with reproductive features limiting further interspecific recombinations, results in admixed mosaics of large genomic fragments from the ancestral taxa. Whole-genome sequencing (WGS) data are powerful tools to decipher such complex genomes but still too costly to be used for large populations. The aim of this work was to develop an approach to infer phylogenomic structures in diploid, triploid and tetraploid individuals from sequencing data in reduced genome complexity libraries. The approach was applied to the cultivated Citrus gene pool resulting from reticulate evolution involving four ancestral taxa, C. maxima, C. medica, C. micrantha and C. reticulata. METHODS: A genotyping by sequencing library was established with the restriction enzyme ApeKI applying one base (A) selection. Diagnostic single nucleotide polymorphisms (DSNPs) for the four ancestral taxa were mined in 29 representative varieties. A generic pipeline based on a maximum likelihood analysis of the number of read data was established to infer ancestral contributions along the genome of diploid, triploid and tetraploid individuals. The pipeline was applied to 48 diploid, four triploid and one tetraploid citrus accessions. KEY RESULTS: Among 43 598 mined SNPs, we identified a set of 15 946 DSNPs covering the whole genome with a distribution similar to that of gene sequences. The set efficiently inferred the phylogenomic karyotype of the 53 analysed accessions, providing patterns for common accessions very close to that previously established using WGS data. The complex phylogenomic karyotypes of 21 cultivated citrus, including bergamot, triploid and tetraploid limes, were revealed for the first time. CONCLUSIONS: The pipeline, available online, efficiently inferred the phylogenomic structures of diploid, triploid and tetraploid citrus. It will be useful for any species whose reproductive behaviour resulted in an interspecific mosaic of large genomic fragments. It can also be used for the first generations of interspecific breeding schemes.

Preferential Homologous Chromosome Pairing in a Tetraploid Intergeneric Somatic Hybrid (Citrus reticulata + Poncirus trifoliata) Revealed by Molecular Marker Inheritance.

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.

Molecular Characterization and Stress Tolerance Evaluation of New Allotetraploid Somatic Hybrids Between Carrizo Citrange and Citrus macrophylla W. rootstocks.

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.

Genomics of the origin and evolution of Citrus.

The genus Citrus, comprising some of the most widely cultivated fruit crops worldwide, includes an uncertain number of species. Here we describe ten natural citrus species, using genomic, phylogenetic and biogeographic analyses of 60 accessions representing diverse citrus germ plasms, and propose that citrus diversified during the late Miocene epoch through a rapid southeast Asian radiation that correlates with a marked weakening of the monsoons. A second radiation enabled by migration across the Wallace line gave rise to the Australian limes in the early Pliocene epoch. Further identification and analyses of hybrids and admixed genomes provides insights into the genealogy of major commercial cultivars of citrus. Among mandarins and sweet orange, we find an extensive network of relatedness that illuminates the domestication of these groups. Widespread pummelo admixture among these mandarins and its correlation with fruit size and acidity suggests a plausible role of pummelo introgression in the selection of palatable mandarins. This work provides a new evolutionary framework for the genus Citrus.