Metabolite Variations during the First Weeks of Growth of Immature Citrus sinensis and Citrus reticulata by Untargeted Liquid Chromatography-Mass Spectrometry/Mass Spectrometry Metabolomics.

Immature citruses are an important resource for the pharmaceutical industry due to their high levels of metabolites with health benefits. In this study, we used untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics to investigate the changes associated with fruit size in immature citrus fruits in the first weeks of growth. Three orange cultivars (Citrus sinensis 'Navel', Citrus sinensis 'Valencia', and Citrus sinensis 'Valencia Late') and a mandarin (Citrus reticulata Blanco 'Fremont') were separated into eight fruit sizes, extracted, and analyzed. Statistical analyses revealed a distinct separation between the mandarin and the oranges based on 56 metabolites, with an additional separation between the 'Navel' orange and the 'Valencia' and 'Valencia Late' oranges based on 21 metabolites. Then, metabolites that evolved significantly with fruit size growth were identified, including 40 up-regulated and 31 down-regulated metabolites. This study provides new insights into the metabolite modifications of immature Citrus sinensis and Citrus reticulata in the first weeks of growth and emphasizes the significance of including early sampled fruits in citrus maturation studies.

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.

Changes in Anthocyanin Production during Domestication of Citrus.

Mandarin (Citrus reticulata), citron (Citrus medica), and pummelo (Citrus maxima) are important species of the genus Citrus and parents of the interspecific hybrids that constitute the most familiar commercial varieties of Citrus: sweet orange, sour orange, clementine, lemon, lime, and grapefruit. Citron produces anthocyanins in its young leaves and flowers, as do species in genera closely related to Citrus, but mandarins do not, and pummelo varieties that produce anthocyanins have not been reported. We investigated the activity of the Ruby gene, which encodes a MYB transcription factor controlling anthocyanin biosynthesis, in different accessions of a range of Citrus species and in domesticated cultivars. A white mutant of lemon lacks functional alleles of Ruby, demonstrating that Ruby plays an essential role in anthocyanin production in Citrus Almost all the natural variation in pigmentation by anthocyanins in Citrus species can be explained by differences in activity of the Ruby gene, caused by point mutations and deletions and insertions of transposable elements. Comparison of the allelic constitution of Ruby in different species and cultivars also helps to clarify many of the taxonomic relationships in different species of Citrus, confirms the derivation of commercial varieties during domestication, elucidates the relationships within the subgenus Papeda, and allows a new genetic classification of mandarins.

Molecular cloning and characterization of a geranyl diphosphate-specific aromatic prenyltransferase from lemon.

Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species.

Mapping the genetic and tissular diversity of 64 phenolic compounds in Citrus species using a UPLC-MS approach.

BACKGROUND AND AIMS: Phenolic compounds contribute to food quality and have potential health benefits. Consequently, they are an important target of selection for Citrus species. Numerous studies on this subject have revealed new molecules, potential biosynthetic pathways and linkage between species. Although polyphenol profiles are correlated with gene expression, which is responsive to developmental and environmental cues, these factors are not monitored in most studies. A better understanding of the biosynthetic pathway and its regulation requires more information about environmental conditions, tissue specificity and connections between competing sub-pathways. This study proposes a rapid method, from sampling to analysis, that allows the quantitation of multiclass phenolic compounds across contrasting tissues and cultivars. METHODS: Leaves and fruits of 11 cultivated citrus of commercial interest were collected from adult trees grown in an experimental orchard. Sixty-four phenolic compounds were simultaneously quantified by ultra-high-performance liquid chromatography coupled with mass spectrometry. KEY RESULTS: Combining data from vegetative tissues with data from fruit tissues improved cultivar classification based on polyphenols. The analysis of metabolite distribution highlighted the massive accumulation of specific phenolic compounds in leaves and the external part of the fruit pericarp, which reflects their involvement in plant defence. The overview of the biosynthetic pathway obtained confirmed some regulatory steps, for example those catalysed by rhamnosyltransferases. The results suggest that three other steps are responsible for the different metabolite profiles in 'Clementine' and 'Star Ruby' grapefruit. CONCLUSIONS: The method described provides a high-throughput method to study the distribution of phenolic compounds across contrasting tissues and cultivars in Citrus, and offers the opportunity to investigate their regulation and physiological roles. The method was validated in four different tissues and allowed the identification and quantitation of 64 phenolic compounds in 20 min, which represents an improvement over existing methods of analysing multiclass polyphenols.

Cytological and molecular characterization of three gametoclones of Citrus clementina.

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

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.

SNP mining in C. clementina BAC end sequences; transferability in the Citrus genus (Rutaceae), phylogenetic inferences and perspectives for genetic mapping.

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

A reference genetic map of C. clementina hort. ex Tan.; citrus evolution inferences from comparative mapping.

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

Assessment of the genetic diversity of the Tunisian citrus rootstock germplasm.

BACKGROUND: Citrus represents a substantial income for farmers in the Mediterranean Basin. However, the Mediterranean citrus industry faces increasing biotic and abiotic constraints. Therefore the breeding and selection of new rootstocks are now of the utmost importance. In Tunisia, in addition to sour orange, the most widespread traditional rootstock of the Mediterranean area, other citrus rootstocks and well adapted to local environmental conditions, are traditionally used and should be important genetic resources for breeding. To characterize the diversity of Tunisian citrus rootstocks, two hundred and one local accessions belonging to four facultative apomictic species (C. aurantium, sour orange; C. sinensis, orange; C. limon, lemon; and C. aurantifolia, lime) were collected and genotyped using 20 nuclear SSR markers and four indel mitochondrial markers. Multi-locus genotypes (MLGs) were compared to references from French and Spanish collections. RESULTS: The differentiation of the four varietal groups was well-marked. The groups displayed a relatively high allelic diversity, primarily due to very high heterozygosity. Sixteen distinct MLGs were identified. Ten of these were noted in sour oranges. However, the majority of the analysed sour orange accessions corresponded with only two MLGs, differentiated by a single allele, likely due to a mutation. The most frequent MLG is shared with the reference sour oranges. No polymorphism was found within the sweet orange group. Two MLGs, differentiated by a single locus, were noted in lemon. The predominant MLG was shared with the reference lemons. Limes were represented by three genotypes. Two corresponded to the 'Mexican lime' and 'limonette de Marrakech' references. The MLG of 'Chiiri' lime was unique. CONCLUSIONS: The Tunisian citrus rootstock genetic diversity is predominantly due to high heterozygosity and differentiation between the four varietal groups. The phenotypic diversity within the varietal groups has resulted from multiple introductions, somatic mutations and rare sexual recombination events. Finally, this diversity study enabled the identification of a core sample of accessions for further physiological and agronomical evaluations. These core accessions will be integrated into citrus rootstock breeding programs for the Mediterranean Basin.

Enhanced Photosynthetic Capacity, Osmotic Adjustment and Antioxidant Defenses Contribute to Improve Tolerance to Moderate Water Deficit and Recovery of Triploid Citrus Genotypes.

Currently, drought stress is a major issue for crop productivity, and future climate models predict a rise in frequency and severity of drought episodes. Polyploidy has been related to improved tolerance of plants to environmental stresses. In Citrus breeding programs, the use of triploidy is an effective way to produce seedless fruits, one of the greatest consumer expectations. The current study used physiological and biochemical parameters to assess the differential responses to moderate water deficit of 3x genotypes compared to 2x genotypes belonging to the same hybridization. Both parents, the mandarin Fortune and Ellendale tangor, were also included in the experimental design, while the 2x common clementine tree was used as reference. Water deficit affects leaf water status, as well as physiological and detoxification processes. Triploid genotypes showed a better ability to maintain water status through increased proline content and photosynthetic capacity. Moreover, less oxidative damage was associated with stronger antioxidant defenses in triploid genotypes. We also found that triploidy improved the recovery capacity after a water deficit episode.

Large changes in anatomy and physiology between diploid Rangpur lime (Citrus limonia) and its autotetraploid are not associated with large changes in leaf gene expression.

Very little is known about the molecular origin of the large phenotypic differentiation between genotypes arising from somatic chromosome set doubling and their diploid parents. In this study, the anatomy and physiology of diploid (2x) and autotetraploid (4x) Rangpur lime (Citrus limonia Osbeck) seedlings has been characterized. Growth of 2x was more vigorous than 4x although leaves, stems, and roots of 4x plants were thicker and contained larger cells than 2x that may have a large impact on cell-to-cell water exchanges. Leaf water content was higher in 4x than in 2x. Leaf transcriptome expression using a citrus microarray containing 21 081 genes revealed that the number of genes differentially expressed in both genotypes was less than 1% and the maximum rate of gene expression change within a 2-fold range. Six up-regulated genes in 4x were targeted to validate microarray results by real-time reverse transcription-PCR. Five of these genes were apparently involved in the response to water deficit, suggesting that, in control conditions, the genome expression of citrus autotetraploids may act in a similar way to diploids under water-deficit stress condition. The sixth up-regulated gene which codes for a histone may also play an important role in regulating the transcription of growth processes. These results show that the large phenotypic differentiation in 4x Rangpur lime compared with 2x is not associated with large changes in genome expression. This suggests that, in 4x Rangpur lime, subtle changes in gene expression may be at the origin of the phenotypic differentiation of 4x citrus when compared with 2x.

Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment.

BACKGROUND AND AIMS: Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation. METHODS: Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat (SSR) markers to analyse their genetic origin. Embryo rescue was used to grow all embryos contained in polyembryonic seeds of 'Tardivo di Ciaculli' mandarin, followed by characterization of the plantlets obtained by flow cytometry and SSR markers to accurately establish the rate of tetraploidization events and their potential tissue location. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Variation in tetraploid plantlet rates was analysed between different seedlings of the same genotype ('Carrizo' citrange; Citrus sinensis × Poncirus trifoliata) from seeds collected in different tropical, subtropical and Mediterranean countries. KEY RESULTS: Tetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric. Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates. The higher frequencies (20 %) of tetraploids were observed for citranges cultivated in the Mediterranean area. CONCLUSIONS: Tetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression of tetraploidization. Tetraploid genotypes arising from chromosome doubling of apomictic citrus are extensively being used as parents in breeding programmes to develop seedless triploid cultivars and have potential direct use as new rootstocks.

Influence of mitochondria on gene expression in a citrus cybrid.

The production of cybrids, combining nucleus of a species with alien cytoplasmic organelles, is a valuable method used for improvement of various crops. Several citrus cybrids have been created by somatic hybridization. These genotypes are interesting models to analyze the impact of cytoplasmic genome change on nuclear genome expression. Herein, we report genome-wide gene expression analysis in leaves of a citrus cybrid between C. reticulata cv 'Willowleaf mandarin' and C. limon cv 'Eureka lemon' compared with its lemon parent, using a Citrus 20K cDNA microarray. Molecular analysis showed that this cybrid possesses nuclear and chloroplast genomes of Eureka lemon plus mitochondria from Willowleaf mandarin and, therefore, can be considered as a lemon bearing foreign mitochondria. Mandarin mitochondria influenced the expression of a large set of lemon nuclear genes causing an over-expression of 480 of them and repression of 39 genes. Quantitative real-time RT-PCR further confirmed the credibility of microarray data. Genes over-expressed in cybrid leaves are predominantly attributed to the functional category "cellular protein metabolism" whereas in the down-regulated none functional category was enriched. Overall, mitochondria replacement affected different nuclear genes including particularly genes predicted to be involved in mitochondrial retrograde signaling. Mitochondria regulate all cell structures even chloroplast status. These results suggest that nuclear gene expression is modulated with respect to new information received from the foreign organelle, with the final objective to suit specific needs to ensure better cell physiological balance.

Evidence for non-disomic inheritance in a Citrus interspecific tetraploid somatic hybrid between C. reticulata and C. limon using SSR markers and cytogenetic analysis.

Artificial tetraploid somatic hybrids have been developed for sterile triploid citrus breeding by sexual hybridization between diploid and tetraploid somatic hybrids. The genetic structure of diploid gametes produced by tetraploid genotypes depends on the mode of chromosome association at meiosis. In order to evaluate tetraploid inheritance in a tetraploid interspecific somatic hybrid between mandarin and lemon, we performed segregation studies using cytogenetic and single sequence repeat molecular markers. Cytogenetic analysis of meiosis in the somatic hybrid revealed 11% tetravalents and 76% bivalents. Inheritance of the tetraploid hybrid was analyzed by genotyping the triploid progeny derived from a cross between a diploid pummelo and the tetraploid somatic hybrid, in order to derive genotypes of the meiospores produced by the tetraploid. A likelihood-based approach was used to distinguish between disomic, tetrasomic, and intermediate inheritance models and to estimate the double reduction rate. In agreement with expectations based the cytogenetic data, marker segregation was largely compatible with tetrasomic and inheritance intermediate between disomic and tetrasomic, with some evidence for preferential pairing of homoeologous chromosomes. This has important implications for the design of breeding programs that involve tetraploid hybrids, and underscores the need to consider inheritance models that are intermediate between disomic and tetrasomic.

Somatic hybridization for citrus rootstock breeding: an effective tool to solve some important issues of the Mediterranean citrus industry.

The prevalence of sour orange rootstock in the southern and eastern part of the Mediterranean Basin is presently threatened by the spread of Citrus Tristeza Virus (CTV) and its main vector Toxoptera citricida, combined with abiotic constraints such as drought, salinity and alkalinity. The search for alternative CTV-resistant rootstocks that also withstand the other constraints is now considered an urgent priority for a sustainable citrus industry in the area. Complementary progenitors can be found in citrus germplasm to combine the desired traits, particularly between Poncirus and Citrus genera. The production of somatic hybrids allows cumulating all dominant traits irrespective of their heterozygosity level, and would appear to be an effective way to solve the rootstock challenge facing the Mediterranean citrus industry. This paper presents the results obtained during a regional collaborative effort between five countries, to develop new rootstocks by somatic hybridization. New embryogenic callus lines to be used for somatic hybridization have been created. Protoplast fusions have been performed at CIRAD and IVIA laboratories, focusing on intergeneric combinations. Analysis of ploidy level by flow cytometry and molecular markers confirmed the acquisition of new interesting tetraploid somatic hybrids for six combinations. Diploid cybrids with intergeneric (Citrus × Poncirus) nucleus and C. reticulata or C. aurantifolia mitochondria were also identified for four combinations. The agronomical performance of a pre-existing somatic hybrid between Poncirus trifoliata and Citrus reticulata was validated in calcareous soils in Morocco. Somatic hybridization is now integrated into the breeding programs of the five Mediterranean countries.

Improved response of triploid citrus varieties to water deficit is related to anatomical and cytological properties.

Polyploidy plays a major role in citrus plant breeding to improve the adaptation of polyploid rootstocks as well as scions to adverse conditions and to enhance agronomic characteristics. In Citrus breeding programs, triploidy could be a useful tool to react to environmental issues and consumer demands because the produced fruits are seedless. In this study, we compared the physiological, biochemical, morphological, and ultrastructural responses to water deficit of triploid and diploid citrus varieties obtained from 'Fortune' mandarin and 'Ellendale' tangor hybridization. One diploid clementine tree was included and used as a reference. All studied scions were grafted on C-35 citrange rootstock. Triploidy decreased stomatal density and increased stomata size. The number of chloroplasts increased in 3x varieties. These cytological properties may explain the greater photosynthetic capacity (Pnet, gs, Fv/Fm) and enhanced water-holding capacity (RWC, proline). In addition, reduced degradation of ultrastructural organelles (chloroplasts and mitochondria) and thylakoids accompanied by less photosynthetic activity and low oxidative damages were found in 3x varieties. Triploid varieties, especially T40-3x, had a better ability to limit water loss and dissipate excess energy (NPQ) to protect photosystems. Higher starch reserves in 3x varieties suggest a better carbon and energy supply and increases in plastoglobuli size suggest less oxidative damage (H2O2, MDA), especially in T40-3x, and preservation of photosynthetic apparatus. Taken together, our results suggest that desirable cytological and ultrastructural traits induced by triploidy improve water stress response and could be a useful stress marker during environmental constraints.

Triploidy in Citrus Genotypes Improves Leaf Gas Exchange and Antioxidant Recovery From Water Deficit.

The triploidy has proved to be a powerful approach breeding programs, especially in Citrus since seedlessness is one of the main consumer expectations. Citrus plants face numerous abiotic stresses including water deficit, which negatively impact growth and crop yield. In this study, we evaluated the physiological and biochemical responses to water deficit and recovery capacity of new triploid hybrids, in comparison with diploid hybrids, their parents ("Fortune" mandarin and "Ellendale" tangor) and one clementine tree used as reference. The water deficit significantly decreased the relative water content (RWC) and leaf gas exchange (P net and g s ) and it increased the levels of oxidative markers (H2O2 and MDA) and antioxidants. Compared to diploid varieties, triploid hybrids limited water loss by osmotic adjustment as reflected by higher RWC, intrinsic water use efficiency (iWUE Pnet/gs ) iWUE and leaf proline levels. These had been associated with an effective thermal dissipation of excess energy (NPQ) and lower oxidative damage. Our results showed that triploidy in citrus enhances the recovery capacity after a water deficit in comparison with diploids due to better carboxylation efficiency, restored water-related parameters and efficient antioxidant system.

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.

Triploid Citrus Genotypes Have a Better Tolerance to Natural Chilling Conditions of Photosynthetic Capacities and Specific Leaf Volatile Organic Compounds.

Low temperatures during winter are one of the main constraints for citrus crop. Polyploid rootstocks can be used for improving tolerance to abiotic stresses, such as cold stress. Because the produced fruit are seedless, using triploid scions is one of the most promising approaches to satisfy consumer expectations. In this study, we evaluated how the triploidy of new citrus varieties influences their sensitivity to natural chilling temperatures. We compared their behavior to that of diploid citrus, their parents (Fortune mandarin and Ellendale tangor), and one diploid clementine tree, as reference, focusing on photosynthesis parameters, oxidative metabolism, and volatile organic compounds (VOC) in leaves. Triploid varieties appeared to be more tolerant than diploid ones to natural low temperatures, as evidenced by better photosynthetic properties (Pnet, gs, Fv/Fm , ETR/P net ratio), without relying on a better antioxidant system. The VOC levels were not influenced by chilling temperatures; however, they were affected by the ploidy level and atypical chemotypes were found in triploid varieties, with the highest proportions of E-ß-ocimene and linalool. Such compounds may contribute to better stress adaptation.